!"
 

$
%"&

  
% 




 
$


%  
%


   
(!
$
R & 
$!
$"
$
 %



   
   
 ! "# !! #  # !
$     !  %

 !!

+
,!
-./.


  

01


% 2!%




 %!%
!%
3%(
4 !%5


 %!%
% 2!%
0 !
 "

6"!%
!%
3%(
  ! 

6"!%

!%
7
8! %



%!%
$
 
% 2!%
(%
$
"&%

%!%
$
 







 
/9.
M
"% 
$
  
%

$
%!
"% 
!
$
%
 !%

( "

(%
$

$%% 
!
$
$
$

" 
%;
%

&
& 

8!

""&%
$!
,!%5
% !(

R6% 
$
"
 %
%"%"


!%
'
( )"

*'
&)*
 !%
( %

$
R

!%
*'+ ,# !!

'(  ##!, !
 !%
( %

$R6"%

%(>




%"%
!;
,


%"%%
(("
*'
- .
 !%
"R( %
22%
R  %!
$
2%
!
$  %;

$
"R( %
 !!
,!
(%;
(!@
% !(%

R6% 
$
" 
 %
% >

%
!
'

)
 !%
 
 !


&  ">

0
%"%
"

$ !%
/# 0 !

+# )  
 !%
"R( %
2 %"


%%

 

$(%
"  $
$
%(
6 %">


0


6%"%
 !
"
% 

# *#! 
 !%
 
 !;
 
$
% !

!%


% 2 

!
"R
%"
$
%$%
!
 2

" 


 
% 
 !, !%
!
""

 !

 ""

 !, !%

 %$>




2;
,
%"%
 !
R

%
 

 


+


%

!#

%  % ;
 $;
;
% ;
!%;
% ;
 


2;
;
00







%$
%

"
2"


&# !#

%

( !
$!6
$
( %
;
( %
$;
( %
 2

$R%
 !, !%
% 

"
 >


2 ;
"  ;
 !%
"R( %
 !, !%
 !!

! %%
"
!
$R!"!%
 !
!
 
$

 ;
2
$
!

$
&>
%
$



 
" !%>

2;
"%

 $ 

 !
#)# >

 &&  

 

4  >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>/

    >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>C

'
# . # >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>D

'
#  # >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>+


   >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>+
•
((%!
%
!
% !
$
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>+
•
((%!
%
!
% !
$
B& % >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>9
•

B E
( %
2((%! >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>9

        >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>/F
•

(%!
$

 !>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>/C
•

(%!
$

 (%
0!>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>/C
•

(%!
G>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>/D

'
 #! 3 ! !4##    >>>>>>>>>>>>>>>>>>>>>>/H


    >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>/H

     >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>/9

    >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>-.

'
#!  # # >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>-F


      >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>-F
•
R( >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>-F
•

%  
%

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>-C
•

$>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>-C

       >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>-C
•
/ >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>-D
•
-

-4>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>-I
•
F >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>-I

•
C

C4>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>-H
•
D >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>-+

'
 $! $  #! !   >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>-9


!   R    >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>-9

!  R    >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>F.
•

 %  >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>F.
•

%""&%% >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>F.
•

5  >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>FD

     >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>F+
•

( 
$
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>F+
•

( 
% >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>F+
•

( 
>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>F9
%
    >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>C-
&
R     >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>CH
'
  R    >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>C+
(
       >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>D/


40 

   >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>DF

  >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>DH
  
/>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>D+
  
->>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>I.
  
F>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>+/

 
7 

  >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>/F/

44 7 3 >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>/FH

     

   


40 

  


 


 
7 


 


44 7 3

 

4 

/

 
$
$  65%& ! 

 

4 

-

 
!
%
%
&
"&%
& 
  $%
(%
 
& *
4
 $
%
% 
&


 

4 

F

=

M!" %
% 
2 %N
% % $
2 %
I
">8
5% 
B
-
 
(%!$!$
 
$ %








#

&% ( 
$
 "
$
(%!
 
 2 %"
!6
% ""$ 
$!
 "
% 
 !%

6  "
$
(%!>

 2%
E
2(%
(%! 
(1 (%!
B! !5
 
(%!
$

$ !
 
%
!% % 
(%!

%
(% % 
(%!
+
(%!
$
R 
, 
(

(%!
$
R 

M3N
(
(%!
$
R""! $ 2
!"
M3N

(%!
$
R 



 

4 


 
 


40 

  


 


 
7 


 


44 7 3

   

D

T #10 -4'031


O %& (%!O
!
O %%  $& %
(%!O
 
$
(%!
"!6
%"

$

(% &% 
%
$
(!%
%%  $
 "  
M" !



$  !
!
5
!
!
" 
 "6
%
% 
!%

(%!;
%%  $
(!%


(% &%
M!6;
-..FN>

(%!

"!
$
R % "
$
R%%  $

% (

2!
 %

R%%  $
2
%"
!

(%!
%
<%
!
" %!%
 %
$R!
%
 !
!%
!
(% &% >

$%%
$ ( 
 %
!
2 
 !
(
"2 
$
R 
2!6

% 
!&
R"&
$


!%

"
$!
(%!
$

!%


"!6
%(
 
$
% %( %;
 
$R % "
"2!%


R ""


!
 !(
!

$
M !&
S
7 %  !%& ;
-..-N>

%& (%!
%

!!%
2"
(%

 !
!
% 
% !
$
(%!


(  
M!
$
D..N
(
!
6 
 !%

(%!



M 2%
E
2(%
(%!N>
!
!

 
 !
 !%
!%
$
2 
5" "


%& (% 
 
$
"$
  %
$
@ 
% 
M

2 
%& (%
 
% %

2  
$
@ 
%
$

2 
 
% 
 !(
  
%
 %$

%$
", %
$
2 
%& (%
 
5" "

%& (% 
 
$
"$
$R! 

!!%
$R%

 
$
"% 
( %
% "% 
M ";
-..CT
G(%
S
;
-./.N>

  " 
$R "% 
 
 
!6
" $2 
$
R(% "


R(
!"

   

I

2( %
R% $! 


% 2 % 
$
(!%



% %( %
"
$
%
%& (%!
!
%
% 
$!


!
!

%%  $
(!%



2;
!
%$ 
 

!
%
( >
!";

%"
(%!6
 2

% 56
 %
$
%& (% 
% 
$ 
!%

!%(
$
2 5%
$ "  

   

H

# / ;$< *!) ! !!   )A ••• '
M ' N
 (%
,!
$
>
2%
2%
2%

•••
 

'
M ' N
 
)* 
$;

$!
!$;
!% ;
;
& %
•••
 
'
M ' N 2 (%
 " %%  •••  )!# A #
  M+  N
 
!  "
  M+  N
5$% "
2 &%
 ;

••• #   
M  ,  (%

 &  "
 &  "
4
" $%
•••  
M   N
 (%

 &  "
 &  "
4
" $%>;

•••
(
  -  M   N
 (%
 " %% 

.9* 4. #!) #R) ;  
 3877%< 

   

+

T #1* 4'4'031

    


%
'
2
%
$

2"
$
'
(

%
 

.   >

 "%$
!
$
H.
 
$ 
!
%

  

$
  
!"



%
'

$(

% 
% !
& 
!%

!%
$!
(!%
$
%" 




5!"
 
 $%"
!&$( 

% !

/N>

• ((%!
%
!
% !
$



 !
$
(%!
$ 

(!%
%

!
" !
!%
$%&! 
%

(%!
  
!6
" !

2 %"
%(
$
R2 
!"
 
"
 " %% 


(%!
  
!6
" !
%"
!6;

(%!
G

" %
!
$%&! 

!%
5
!%
"

!%
$

(%!
 
!% % 
(
!
 
",!%
$!
% !;

(%!
$

   

9

R 
, 
M0N>


!$


• ((%!
%
!
% !
$
B& %

% !

 %

% 


(%!
  
!6



% 
$

!

(%!
%
!
 %  "6
4
M B& %
N
 
 !



-N
(
!
%! 
$
R 
$
(
!

% (
 !%

!>
R2 
!"

2
(
!
" %!%
$


• 
B E
( %
2((%!

% !
$
L



(%!
  
!6
!(  !%


  
$
$22 %
 
M ;
2%

(%!
  
!6
%  !%
%!
6!("
$

 !(!
$


%
!6
!
 2 
$
$
@ 
&
% %>

2 
!"
 
 !(
$
(
!
$R2 
!%
M(%!

4%(

(%!
$ N

!!%

$
& % %
M ;
-..FN>

   

/.


B E
(%&%
 

&%$

4


B E
( %

9
## !)" !A#$  #.# ## A !   : ;#># ? 3877G< 

   

//

8
## !)" !A$  #.# # # A !#  #!!# !"!    
 %

   

/-

6 # ! !!  )A " 
    

 (%
,!
M1N

!

 
$
 !
M N

!

.8* 4$  #!) #R)

   

/F

        

@
R "";
R2 
%
!
2((%!

 !(
%
"
$
2 %"
5" "



5" "

&%(
%2 
$
$  ;
$
(% ;
! ;
( "";
$%% 
!
!
%
! >
R ( ! 

 %"
2( %&
(
% % 
$
 
% 
F

I
, !%

!!

FN>

%
;
!
$!6 "

2 &%

&%(

!
%
 " 
$
5""
!
 ( %


 %

"

($
$
R(
$
%
"  !

(%
M&(%GN;

R6

$
%"
2((% 
 2

!%
$
%"
 
2


"

!%
 %

$22 %
5"">
!
$
(
6
 %

(% 

!

(
 %

2 (%
,!;
R 
, 

R 



%
 !AR  $! . ##A

   

/C

• 
(%!
$

 !

$ !

!
"$
$

!&

(!%
",!%


" !

"$

% % 
%
$

2 (%;
$
  ;
$
"5 
"
!
( !%

2 %"
!
 ( %



6
!!%
5  
!%

2 
$ 

$ ( 

3

;
-...N;

!6
(
$
(%!
%!
$
R % "
!"
M!    ;
-...N


%$

R( 
"(
$!
5 "
""!%
!
!
%" &
(!%
%!
T
R!
!
 " %% 
" %


%"
6
 
!"
5" ">>N>
$;
$!6
(
((
 ! 
 

 !%
$
$ ( ">
R!


 !
%
!!%
 
!%
!
$


$%%
"&
% "!%;

$(%
%%



$R


• 
(%!
$

 (%
0!

R 
$!
(%!
%  5
$!
%
' ;
 
 !%

%" %
2 

/9-H
M4%%
S
3 ;
-..HN>

"$
 !
"
R 2%


%"

$

(% &% 
%
<%
$
" !

2%
%"
%

(!%

   ;

(%!
!
"% %
 %$

" %


   

/D

2 (%
,!
 !
!, !%$R!
 %
$
"%
$
% 
M,!

22;
$

%  
$R 2%


 %&!
$ 


%5%
!
"$
 !%
 %&
%

( >
!6
(
 %

2 (%
,!


$ (  
$!


 
/9F.
%
 ! 
$
 !
!( 
 ;

 !
%
(% % 
(%!
MN

&>

 !
%
!% % 
MN;
$ %( 
$!
;
2!

%"%
(
! ;
%"

2%
 %
 
22 ;
$
 " 
  (
! %
 
%%

/9+/>



%"
$!
%

(
/H
$ %(
$

 !
!( 
&
M4%%;
/99HN>

• 
(%!
G

(%!
2!
 

%" %
2 

/9FH

! $

%%
$R!
%  ("
 !
$R!
2""
2 
M"&     ;
-..-N>
R
!
%& (%!
% "
% $!
$

" $
(
$
%  
$ "

6
$!6
 5
",!%
$!
(%!
G

MGN


  


G

"!
(
!
5
@ 

!
!
2%
$
(% &% 


R2 
!"


!
 !(
!&
R!%
%;

% !&
!%  

" !
 ""

!%
$
2 

2((%!;

R6
!!
(
!"
 %

(%!
G
;

 %
(%
22;

% ( 

%

$ " ! 


 %
$
%  
$($!
M"&   ;
-..-N>

   

/I

WNV WNV
WNV WNV
YFV DENV , YF V, WNV DENV , WNV
DENV,
WNV YF V,
DENV DENV ,
WNV

B#!.!  )A#!#$  #R ! ! ; <3  C ;@<3?#! ;? < ;R #/!3877%< 

   

/H

T 0% ,'1 2'-,Q0M.*'! 2'-,#2#6.0#11'-,"3%M,-+#4'0 *

    


2((%!
 
$
%!
(%
 %
DN>

 
$
(%!
(  
 ! 
$R!
$

5" %
  $%


R( 
(%

 ! 
$R!
& !
$
!6
%  
(%
(
R%
$

& !


5 %  

M( N


% !%!%
%
$

%  
$
""&%
M N
M >
DN>

%!
(%
""!%
" %

%(%

( 
$
 %  
%  

R$2 
$!
 "%"
$!

% %% "
$
%  
$
!%2

%"

( 
$!
% !%!%
%

%  

%

%  
!%
2!%>
%
%

!
(% 
,!

 !%%
!
2%
!
!%
!>


%  


!
%
$ %"
$
R2 
$

%!
2((%
M     ;
-..+N>
7%
!
( 
$!
$
 ;

%"

2! 
$!
(% 
(

""&%
!%

R6 5 
$R!
(% 
"!%

2!6>


5 %  


% 
% 
$!
(%!
$

!
%
$ 5 ;
R( 

% % 
 !
R22
$
R$2 
$!
3

6 

$
2! 
%"

2! 
$

""&%
$

! $


& % 
$

5 "

!%
!

%$! 


%  
$
R >

   

/+

;<




Virion immature Virion mature





;
<

:!!#  #$  # ; < %  
% 
;< %  
 "
;
< %  
 "

   

/9

     


 "
$
2((%!

 " 
$R!

"
&%
2!6
M [N
$R(% 
//
B&
2
R6% "
DR
  $
!
 22
$
5

"H7 "-
$


% %

 
&
$R% %
%
$
 " "% 
$




%
DR
$!
 "
(%;

% !(


IN>

   

-.

     


(%!
R

$
% !%
!%



2&
3
$
R$  "
%
$
% %% "
%!!%!6
$
R( 

6 

$
2! >

2! 
$
R( 
(%
(

""&%
$
R$  "
%"
R% 
$!
(% 
$

5 "


& % 
$!
 "
(%
M"
S
$"&;
-..9N>
R 
(%
M [N

%

% 
%

"%
$
%$! 
!%

%$!

!
!
 5%  
% "
 
!
% !!"
$ "

 %
$
( 
 

 %$! 
%
$
%  
!%
!

 %
%  
(%
M-4FN
%"
$R &%

$6
%  
(%>

%  
 
%!!%
R"&
! !%
$
""&%
$!
% !!"
$ "
R 
 "
!
%"%

% $! 
$
"!
 
$!
&%
$
 %
 (>
%
$

"  !
 
! 
 !%

%$! 
!

%  
M %$
S
7"%B;
-..9N
T
$R!%
 
$ 
$


 !%
2 %"%
$
(% 
""!%


% 
"!% 
!
 !%
$!
% %
%  

R22!

( 
$
%
%

2!%
!%>

R!
!
 "
 2 %" 
$
R( 


2! 
$
( !
$
% %
(

""&%
"
HN>

   

-/

9'








          




8'








%'

=  #!  #$  # 9' !!    R 
$
 %
 (

2


8'!!"!   
( 
%  5
%'#4!  # #

%  



% $!
$
( 
$
 
% !%!%
%
%
!
%  
!%
$!
 "%"
%  ;

2!%
M2 
(%N>

   

--



H
" ! #$  # ;R #*!'877G<
'

(%!




!
%
$
% !%

5
  %
%
$ 5 >
'
! 
$
R( 
(%
(

""&%
$

( !
$R$ 5 ;
$  
$

! $

& % 
$
R 
(%
$

5 ">

'
%$! 
$!
 "
(%

 5%  ;

'
  
$
R 
 "
'
"& 
$
(% 
""!%

R %!%
$!
% !!"
$ "
' % %

"!% 
M( 
$
%

N
$
(% 
$
R%
$
 >
/'
 %  
%  


(% 
"!%
R( 
$

""&%
$!
>
%  
$
(% 
(  

R6 %!%
$

!>

M 
22%
$

   

-F

T #1.0-2M',#14'0 *#1


%  
 
 % 
%
( 
$

 5%  
%

$
%  
!%
!
(%;
!
(!
$

""&%
$!
% !!"
$ "


$
( 


$2 
%
"% 

      


% 
%  

%"
$

 5%  
 

%  
%!!%
;
%

>

( 
!
(!
$
,  
% ;
% 

/

!
$

!" %
$!

%
$
 
!%
M EB   ;
/9+9T
"
S
$"&;
-..9N>

• R( 

%  

$R(% 
D.
B 


%  
$R( 
6  


!%2
$
(% >
R
R 
",!%
$
%!
(%
"!%

R % 
$
 %
!%
@

!,
2 >

, !
!
%
2 $"
$

5
(%
M !B $55   ;
-..DN>


% &
$
" "
$R$  
$
(% 
!6
!
&

$

2! 
$
R( 
(%
(

""&%
!%
M3@
S
 ;
-...N>

%!!%

$ %" 
%

% 
$
5 
%"

2 %" 
$
6
 
$!2!%
MGB%     ;
/9+HN>

%  


&
$
 %
$R 
 " %


%% !(
 !
2 %"
$
%" %
$R  % $" %
% 


!%2
$
(% 
""!%;
2 %"
I.
% %! 
%  
$
2 
  $%

( 
$!
$

%

$  
$
  % $" %
% 


2 %" 
$
9.
 " $" %

$

(%!
"!%;
!
 2 %
!

!


"%  
% 

%  

 
"
$
 2 %" 

 " %" %

 %
$

2! 
(
!
""&%
!%
M\    ;
-..FN>

   

-C

•

%  
%



%  
%


% !%!%
5 5
$

%  
%!!%
>



$
!
2 ;

%!
2((%

""!%
 
% % 

%(%

( 
!%
$
 %  


%  
%
, !
!
%
$
% 
((
$

%  
$R( 

M %@   ;
-..-N>

22;

$

%  

(% 
""!%
 %
!
2! 
% "!% 
(

""&%
!%
M    ;
-..+T
\   ;
-..FN>

( 
$
%
R22!
%
R 
$R!
2!%
!%
% "

R6 5 
$!
(%!;
 !%
$ %



%  

"!%

• 
$

%  


!
"
"  !%
$
/-

/C
B >
!
2
$

2 %
%  % 

% $!
% 

5;


% 
 ("

R 
(
R 
 %
$
R$ 
 !%
2 %"%

! $
M $  ;
-..FN>

$ "
"
R %  


% 
"  !%
$

%  

$

! $
$!
(% 
 
 !>

% %! 
$R" 
$
"%  
% 

$ 
$

2&
 %( 
$



       


 
!
 "&%
$


/;
- ;
-4;
F;
C ;
C4;
D>

%  
/;
F;
D
 
 !&;
$%"
  
!6
""&%
$




%  
-4F

D


&! !
!$ 
$!
2
$
!%
( 
@5"
%"
;
D
 %

2  
" 5%2 %

 5" %

F
!
!
%
%
(

2  
 

%  

 >
R!
%;

%  
% 
(
D
 !%
2 %"%

 "6
$
%  ;
% &
$

5 
$
&%
$

 2

 2
 %
$
R 
$
%  
$!
 "
(%
M!  ;
/99+N>
R!%
%;

% 
(

   

-D

-4
 !%
2 %"%
!
 "6
%  

/;
- ;
C ;
C4

 %
!!
" 2
@5"

!$

 !2 
% !(
R" 
$
/;
$
-

C
$

" "
$
%  
$!
 "
(%
M $&
S
;
/99HT
B@     ;
/99+N


%
$
-
$

" %  
$
(% 
ML!""%%
S
;
-..-N>

• /

%  
/
$
C+
B 
 %
!;
$!6
!
% 

$
 5 5 
 

 >

R 
$

!
%  
 
%!!%
5 5 
 !
@

(%!>


%!!% 
%
/-
% $!
5 
 %( 
2 %"
$
 
$!2!%
M      ;
/9+9T
 ;
/9+9N>
%
$

5 ;
% 
( 
$

 5%  
%
!
%  
!%;
/

%
!
 !%
$

%(% 
$!


$
R%
$
 ;
/

5 5 


$" %
MGB%   ;
/9++N>

2 %"
$" % 

%"
  
!6
""&%
MGB%   ;
/9+9N
%
!
" "
 %
 !
M0 &   ;
-...T
G %     ;
/99.N>
%
$
R2 
$
!
$
"""2 %;

%  
/
6
"
 !
!
2 %"
 %  
M6" %


%% !( 
!
(!
$
R 
$ !&
&%

2  
 ""
!
 2!%
 %


%  
(%

% 
(

%  
C
M $&
S
;
/999N>
R" 
$
/
$

 
$
2((%!

% "
$ %
""


" "
"
/
 !%%
2%

2 %" 
$
 "6
""!
M (%!
   ;
-..IN;
% ( ;
-..-N;
 $%%
$
$ "" 
!6
!
$  
(

( 
$!
 " "
 %$ $
M 
   ;
-..DN;
"2%
$%"
R2 
M    $%
  ;
-..DN
!
 !%

( 
%(
$R( 
$!
 " "
%

26 
$!
2!%
3
M!   ;
-..IN>

 !%%
, !%
!
%
" $!!%
$
R""!
 

&

( 
$
  
F
$ $
MG 
   ;
-..+N
&


!%
2 %"
$
/;
!
  !;


$ %
 !%

2((%!
$!
 % % !
$
R 
, 
M0N

/R
M4(   ;
/999N>



   

-I

$

%$! 
$

%  


2 %" 
$R!
 !(!

$
( 
$

+
%"%
! $
$
- >


% !%
!
%  
/R
!
  !
M%
S
B;
-..9N

•
-

-4
-

!

%  
5$%  &
"!2  
$
--
B 

-


$
" %
 2

 !%%
% %%
$%"
R 
(%
$

$
%  
!6

$R"& 
(
$
%!!%
""&%%
(%  $!
M !
   ;
-..FN;

%%
!6
" "
$R"& 
$
%!
(%
M !
  ;
-..+N
%
!
% 
!  
(
F
ML!""%%
S
;
-..-N>
-4

"
!

%  
  
!6
""&%>

2 %"
!
 "6
(

%  
F

 
 ""
 2!%
$

%  
(%
-4F
M %
  ;
/99FT
 !
  ;
/99/T
$(
  ;
-..+T
1! 2
  ;
-...N>

•
F

%  
F
MH.
B N

!
%  
"!2  
$ 



 %
!
(
 K 

F
, !%
"
!
%
$
R"& 
$
(%!


$ $""
$

2  
5

22;
 !%

(%!
1;
R! 
$
%  

$R 
2!6
" %



$ "
 

 

!
$

%
%"
$

%  
F

%


%  
$!
 "
(%
M7 %&5
S
L ;
/9+9T
"
  ;
-...N>
R(
 
%"

$ % !"
$
%!!%

$!6
$
R 
(%>
R % 
 %


$  
$
 
5$% 


5$% 5

%  
%

$ "
 
 !
!
$ "
 
M%B
S
";
-..IT
G%%%

 ;
/99FN>
R K 
$
2((%!
%


!%2"

$
 



   

-H

ML$%
S
3;
/99HN
 !
 ""

$
(%!>

 " %

" 2
 %( 
!

$

!%2"


4
;
-..+N>

!$
% 
% %
!
 2
$
!&%

(%
$
R 
$!
(%!
G

R(

DR
% 

% &
$

%" %

$
 22 
$

(%!6
M4 
S
"5;
/99HN



$2 
!%

%
%"
$

%  
F
%

$
!$

(%
M4%"
S
$"&;
-..-T
G %;
/99FN>
R(
 %
%  

 % 
%

%  
%"
$
F>

%
5$% 
$
-4

 %
 !%
2 %"%

 "6
%  

2 
$ 

 2!%
-4
(

%  


!$ 
%

$
!$
% %
-4
R % 
!6
 
$
F

!
 2 %
!
 2 %" 
(
!
(
M 
  ;
-..HT
%&
  ;
-..IN>

• C

C4

%  
C

!

%  
5$%  &
$
/I
B 
$ 

2  

 %
"  !>

%
$%
$

%  
$
R 
(%
"&

 2%"%
!
C
, !
!
%
" %
$

%  
(%>

22;
 
% 
(
/


" % 
 %


%  
$!
2((%!
1
M $&
S
;
/999N>

!;

"&
"

%  
C4

!

%  
  $
$
-H
B 
!
!$ 

  
!
(!
$!

!%>

  
(
F



(%!6
$ !&
&%
!
(!
$
%!!%
""&%%
$ %( 
$!

!  
%


$
%  >



" %

!";

%!!%
$
C4

 !
"
$
5
& 2 %"

   

-+

% %
(
$
" 
$

%  
$!
%
$!
;
$
R % 
$
R6% 
$


$
R22
$

%  
MG
  ;
-..HN>

•
D

%  
D

!
%$
%  
$
/.F
B ;
"!2  

% "
 %( 
!

$!
%
' >

 %
!
(
" 5%2 %

%"
M  22
  ;
-..-T
5
  ;
-..IN

!
(

 5" %
$ $

 %"
M B%"
S
$"&;
-../N>


 "
2((%
  $
!
 22
$
5

M"H7 "N

DR
%";

D
 %
R(
H

-R
" 5%2 %
"
% 
 
$R!
!55%2 %
 
$2 ;
!
%" %
" 5 
 %

  
H
$

!
M"H7 N
!(
$R!
 $

  
-R3
$!
%& 
$!
%"%
! $
$
R 
(%
M"H7" N>

(
" 5%2 %
$

%  
D
$
2((%!

$&
!
5
(%>
R(
 5" %

$!

$
$ "
 %( 
!

$

2"
$

 5" %
$ $
ML ;
/99/N;
 ""
!
%
7
% %
R!%
" 2
 %( 
 
"


5 ";
D
2 %"
!
 "6
(
F

!

"!%
R(
 

 
MDR
% N
$
F
M!
   ;
/99+T
1 
   ;
-..DN>



R % 
$
R 
$

5 
$R 
 
%
 
!
 "
(%
%!%
 ""
$ " %
 !%

(%!

M " %
  ;
-..IN>
 %


% %
!
 5!

!%
%

R" %
K β
%
% 
$R!
 
$
  
! %
M N
!%
D
M0  
  ;
-../N>

%  
$
% %
" %
"&
%%

 "  
(

%  
(%
F
 !%

 

D
T


% %
!
 5!
$


2 %"
! %
$
D
%"%
$
" $!%
 ("

% $! 
$
5 B
%  2"" %
 +
!
$ &!
$
R2 

2( %%


%   
$!
(%!
M%5 %
   ;
-..HN>
%
$
R2 ;
D

$ 
&
$
%%
%
5 "

 5!
 !%
%%


2 


%  
(%



" $! 
%% 
$

!%
$
R>

   

-9

T
,$#!2'-,9$* 4'4'031#2'++3,'2M',,M#


(% &% 
 


 !

"
$R!
( 
"% &

 !%
;


$ ( 
$
5 "
$
$ 2
""!%
$ 

"%

 
2!6
$
R % ">
!%
5 "
""!%

$(

$!6

R""!
 

R""!
 
 

!
"&
$
$ 2
M&%% %
5

%" %
&%% %
 

&%% %
 "

& %
$
$ 2
& "
%

 !   R   

 !%

!%
$
2((%!;

!
$$%

(%!
  %

!
$$%
;

%"

"


$

%  
""!%
 
!
$(>

22;

!
$$%
;

 "&%
$
"  5

"%  
R% ;

 " "

( ;

!
!%
L%
MLN
%

,!


% !
$
%  
$R 
!6
!
$
R""!
$(

"


ML
S
" $;
-..+N;
%

%" %
( !
$
(% ">
 !
%

"  5
K !

!
$$%

   

F.


2((%!

$ "%
!
%

( 
 !

5"


2 %"
%(
$
$ !
!
$
2 (%
,!;

(%!
% (

 !  R   


5 "
""!%

!
" "
$
$ 2
$
R % "
&
$
$ !%

O O
$!
O 
 O
R$%
 !



O 
 O

% %
%

% 
$R 
6 %!%

R % "
 ""

%  
(%;
"
!
%

% 
$
O !6
$ %O
 
R 
$R2 
M0E5;
/9+9T
@ %;
/99+N>

$  
$
R % "
% %
R22!
%

$
" 2
"  !%
%!%
 
" 2
"  !%
  
!6
 
M !
 ;
  $
" !%
%N;
%% !( 
!

 
% !





6
!!%


• 
 %  


 %  


!6
!%2
"% &;
(

( 


• 
%""&%%


%""&%%
 
", %%"
%%  
%

2"
$
% !%
B
M B
% %
!
N>

% !%
 
$
%  
%""&%%
 " 
$R!
%  
6%!%
%

!
M !%
%

   

F/

!
N;
$R!
$ "
%""&%%

$R!
%  
5 "

!%
 
6%" 
%
$
!
$
R""!



26 
$!
 $
!%


R22!
%
!
$ "
6%!%

$

$2 


2
$
 
/99.
M B
  ;
/99+N;
$R& %$
@

$%  ;

 !
!
% !
$
//
%  
$ 
9
  $
!
2  
 !;

 
&
$
% %

 !6
$ %
 ! 
$
" 2
$
/;
-;
C;
D

I
 
" 
$

""&%
!%

% 
$
" 2
"% &
$%"
&


!%2
$

!>
F;
H;
+

9
 
  
% 2 %"
$

( !
%!%


$ 

$
! 
+N>
F

 !, !%
%% !(
$

$  "
"
!;
 

5
!%
!
 
(!
$R6% 
%
"
% 


""&%
"

"! 
$!

%
 
 $
 2

!%
$
 !6
$ $

%

%  
$%
5 ++
M( 
5 ++
$ $N
!2
 !%

 !6
$!
%

F
!

C
 %
9N>


 
$
%$! 
$ $
$
5 ++

% 
% 
(
!%
 $;


"

$%%
R 
!6
B
 L
M L/

C
!
 /
 $
LN



(
 %

B
L/


2 %"
( 
$RLL
%

  %5 
$R κ4


$ %$ 
%

%   ">
 κ4
R
!
%!
$

5 "
%
 κ4

!
 %
" %%
(%

 5!
MLE
S
B%;
-..HN>

!
%  
! %
$
 κ4

%"


$ "%% 
$

%% 
$

$

%  
%2 % >

22;

%

   

F-

% ! %5
2 %N>
;
R  "
2 %"
%
 κ4

R"&
$
2!%
$
%% 

&
$
"!%

%% 
$

 !

$ $
$!
% " !%





 
$
%$! 
$ $
$
5 ++

F
!
!
( 
$R( 
$ $
$
5 ++

%%!
!
%  
$%
$22 %

>
C;
"!
%

;

"
&

% 
$

( 
5 ++$ $
$R!%

( 
$
  
(
!
 


( 
F$ $;

M !
 /N
% 
$%"
(

$ "

$!
% !%
$

!6
( 
$(% 
 %

 !%
(%
 κ4;

% %
(
 I
!

B
/
 !%
(%

 "6
LL
!
 κ4
 ""
$ %
%  $"">
% ";
F
M%2% 
 
 %
FN

%

22;

  %5 
$RF

$

5 ";
% (

   

FF

; <


 




Toll-like receptor PAMP associé
 9 $ 5
$
& %
7%"[

 8  $
$
& %
7%"

 $ 5
M7%"[N

 B $ 5
   5%$
M N
$
& %
7%"

 :  
$
& %
7%"

 =  %  
$
& %
7%"




;<
 



Toll-like

receptor PAMP associé
 % $&
 H +C+




"
&%

 G 
"
&%

 I 
M%

7N

G#!.! #  # #! ### # 

$%&! 

 

6!(


F;
% 


!%2
$
$  ";
!
!;
 

5
!%


(!
$R6% ;
%
6 


!%2
$

""&%
"

   

FC

I## #! &"GG,   !!&"GG,    !  #  # 
( 
$
  
5 ++$ $
 $!
(

$!%
 L/

C

 I

R( 
$
 κ4


%  
!
 5!
 !%
(%


 !

$ $
$!
% " !%
>

( 
5 ++$ $
 $!
!

R( 
$
 κ4
(

$!%


/;
"
%
!

  %5 
$RF


" % 
$

 5!
 !%
%"%

5 
$R%2 % 
$
5
>

   

FD

• 
5  
/.N>
7

!
%  
$
9-D
$
" ;
 ! 
$R!
$ "
 

& 
6 K3

%"


 

& 
6 K3
% 


$ !&
&%
(%!6

 %

&%
 " "%
%

!
(

%
7
$ 
"


"
&%
DR
% 

 22;
% %
!6
(%!
M B!
S
B%;
-..+N>

R!
!
 "
$
 2 %" 
$
7

D
2
!
%
$

2"
$
 

& 
6 K3


%!!%"
"%

7>
!
 ""
7;

%"
R( 
$

$
R%2 % 
(

""
  
M 5
S
 ;
-..IT
"%
S


 !;
-..HN>
$;

D
"&
$ %
$
(%!

$
" 2

$22 %
M $&
!
  N
$
!6
$  
%
7
M"%
S


 !;
-..HT
1 5"
S
!,;
-..HN>
%
6";

%"56 (%!;

(%!
2!@
 
% !
%
7
$



2"
$
' ;

(%!
0

3
 
% !
%
7
 %

/.N>


""
2";
7-

   

FI

97  # !! #) !A /,3& :!/*8

;R # >)!'877I< 
%  
7

D
 
 ! 
$
$!6
$ "
 

%"
M,!N;
$R!
$ "
 

& 
6 K3
M% 
&!
N

!
$ "
% %!%

 %">
!
% 
$
(%!
$
5
$22 %>

%  
7-

!
% !!%
 2
$
$!6
( 
7

D>

   

FH

99# #!    /,& : ;R # >!'877=<  7
!
D
% 
R $&
(%
(
!%
$ "
 >

 "
$
 2 %" 

   

F+

      


5 "
$!
 " "

R!
$
22!%
$
R""!
 
M5
S
B ;
/9+9N>


 " 
$R!
%
$
%  
 !&
!
""&%%
 "%
$
(!%;
$
&!%

$
"  !
&
$
 $!%


% 
$
!
 %"
!
$
 


!%
" 
%
2 %" 
$R!
 %
$
!%
""&%

%!!%
" 

6
% 
( 
$R( 
$!
 " "

• 
( 
$


( 
$

"

,!

26 
$R!
%  
$!
 %!";

4
M 
4$
N;
!%

" 
% 
!%
$
 "&%!
"  !
""&%%
$
(%!
 ""

$ !
M3B%
  ;
-..9T
 %!
S
%$;
-..HN>

%(;

4

!

%
!6
%  
!%
$
R
$!
2
$
R  
$
!%
 "6
!%

 5" 
$

%  
 %
$
!%
"!% ;



 "6
2 %"
%

 
$
4

$
%  
  

4
M 4   $
%
% 
!
N
%"
$
(%

$R(%

 "  
-

C;

R 
2
$

">
R5$% 5
  
$!
F

F

F&
%"
$
2 %"%
!
D
 (%
MC&-F&N

R 
!
% $!
(
C&-
M !
F
 (%N>
 !
(%% 
!
%$

•

( 
%

( 
%

 
%
R5$% 5
  


$!
F
 %

$%%
!

%
$
2 
 (
!6
 
$
!%2
% 
!%

%!
(%
%
6">

5

"!
2
$!
2
$
!%
!6
2&

$


F&
% 


!%2
$R 
% %;
!
%

2!%
4
 %

   

F9

%
!
(
%

%  
 %

 "6
F&4&

2 %"
  $
!
(
F
 (%


"

R  
$R!
"  !
$
F&


F
 (%
2 %" 
MF&4&N
!
 2 %
!
(
D
 (%
MF&4&F&N
 !


$!
 "6
C&-F&
$

( 
$


$

( 


• 
( 



 
%

2 %" 
$
 "6
  %
!%


/
(
!
 %
(%
C

-
 !%
2 %"%

F
 (%
!

D
 (%
%
 
$!
F&
( ;
$
2 
"%


( 
$

M"&
  ;
-..+N>

% 
( 
$R( 
  
$!;
 (% 
(%

2 %" 
$R!
 "6
$R

22;

D
 (%
% $!;
(

D

D

D&

!
% 
!
 %
$

""&%
$
R 
% %
!
$

!
2 

%
!%
" %>

22;

 " "

22
 !%
5%

!%
$
(%!

( 




$
$!
 " "
"


5
!%

 !(
 $ % 
!
2

 ;
"
$22 %
$
 % 
 %
$

2 %" 
$!

, !
!
%
$ 2
$

$ ( "
$R!
%  
2"" %
22;
R   
$
R 


!% 
$!
(%!>



F


D;
 
5 6;
!

$ %! 
$
" 5

& 



& % 
$R"
( (


$!6
$
(

"  5

!% ;

   

C.



F&

R  
",!%
$!
5 "
$!
 " ">

& !
$R"2 


%("
%  

 
!6
!
 %!
$
% !%
$!

!
$
% !%
$!
 " "

2 

!
%
!(
$R!
 5 
%

!
 5%
( 

%%! 
!

$R2"" >

R!
!
$%! 
%!%
$

%!
(%
 % 
M (%!
  ;
-..+N>




$ 


" "
 %
$
% ! 
$
 !

( 
R22!
%


% 
$
 !
6%""
&


!;

 " "
 " %
!
 %
$
%  
% !%



 " "

!
 " 

$

%  
""!%
 
"
, !
"
!
%
" %
$
R 
$
R""!
$(
M!"" 
  ;
-..HT
!B&% %
S
  ;
-./.N>

   

C/

98  # !! #% #R! !   ! ;R # .)!' 877G<  
( 


2!%
C;
F

D;
% $!
!
 !%
$
!%
2 %" ;
%

R2"" 
$


R"&
$
( 
& !


2 %" 
$!
 "6
$R

   

C-

%     



%"%
"
$R!
2 
(%;
R""!
 
" $ 
%

5 "
%2 % 
 !

!
" %
" "
$
$ 2
(%
$
R
M(%%  @
  ;
-..DN>

%2 % 


$2 
 ""
$
%  
&
$R(%
!
(!
!%
!
% %""
 $!

!
& 
$

%  
(%
M

 ;
/9DHN>


%2 % 
$
5


% %



M/F
N

!
M/
N;
% $!
%

!%
$
!
M3 $
  ;
-..DN>
R
$
5
;
!

γ;

5 
!"
!


% 
$
!
2 
%
%

$
!
""!%
ML;

 C[;

 +[N
M4 ,%$";
-..DT
7 $& !%
  ;
-...N>

% " !%
$
R
β
 



$

K!
 
!6
$

!
 
2 

 
2 %"
$!

%  

!
 
$ %



 
",!%
$
R2 
(%

%% 
%
R 
$ !&
&%
M $&N
2 %"
 %
$

%  
$!
2((%!
M7 $& !%
  ;
-...N>

22;

%  
2((%

%

5 
$
&%
$R 
 2

R 
$

%  
%2 % 

!
 !
$R& %$
%

% 
$

 >
!6
5
$
% !%

 
 "
&
$

$ %


 
5  

%

  
!%;
$

$  "
!


!%2
$
%
!;
F
%!

 !6
$ %
$!
"!
6%!%
M %
  ;
-..+T
%$%B
S
7;
-..IT
%""
  ;
-..+N>

2 
R $&
% !;

 

%  
(
!
$
$
  
% %


F


!%

 "





$!
% " !%
$
R!
$

(
-K0!;
R"&
$

2!%
$
%% 
2 %"
R  "
!%

% " !%
$
R%2 % 
!

%"
R 
$

%% 
$!

!
M
   ;
-..IN>

   

CF


2 

5 
$!;
R%2 % 

 % 
%

!
2 

 
%
( 
! %

%%
 !%
% $!%
R
;
"2%

%  
%2 % 

$!%
!

(%
$

!
% $!%


!
$,
2
$
"%

%   
$!
(%!
M 
S
$@ (;
-..IN>





!



!
% !%
 ""!


!%2
!%;
 
M%2% 

 %N;
 !
$
$!6
 !! 
 /

 -
M 
  ;
/999N>

$ "
5 "
R"
$
R%2 % 
$
5

!%
 
% !%
%
R( 
%
  %5 
$
%  
5B-

0B/>
5B-
!
 %
  %5%

$ "
5 "
  %5
%
5B-;

%
$
2 %"
 !%

%%!"
$
 /

 
( 
M7 $& !%
   ;
-...T
%B
   ;
/99+N>

2!%
 /

 -
  %5 ;

$" %;
2 %"
!
 "6
%  ;
-..IN>

 "6
  % %" %
/FN>

   

CC

; <
















;<













9%# #! R !$   ;R # 3877I<  ; <  
$
R $&
(%
%
7
!
F

5 
$R!>
;<  %  
$R
$R!
$
2 
! %

%%;

$! 
$

5 
$

%2 % $ $
 %
 !%

"


$R!

(%>

   

CD

%"

%  
$!
%
R%2 % 
2 !%

L
M% 
L

$$N;

-RDR 
M  $5
5N


%  
6
M >
/CN>


L

!
%  
B
5 "
( 
%
R%" $%
(%;

$ !&
&%;

  %5

&
R( 
$!
2!%
-
M!B%5 
% 
 
2 %N;

"

R 
$

5 
%  



2 
( 
%
R $&;

-RDR
 
5

5 
$

 " %
$R$ 
"& 
$

 2 %" 
-RDR>
!6


!%
R$ %& ! 

M
N

R(;
!
%"
$
(%


"
&%
(%!6

!%;

$ 
$R"%

5 
%  


%  
6
%


!%2"
$
$5"

(
7 >

6
$!6
2 %"
$
%  
6
@

!"

6

64;
 !
$!6
5 "
 
" "
$R 
R

 "
!$ ;
"

 %


26
!%

 " 
(%!6



&
6
"&
R!"!%
$

!
% 

R%2 % 

&


R
, !
!
%
" %
 %

% 2 % 
(%

;

!!%
(!6>

"

%  
(%
$
$(%
2 
 ""
 !
R( 
$ %;
"
 %
!

$(%
$

!
2 

$!
!

%   


%"
"
$
2%


(
R""!
$(
%


2  
""! " $!%>

%"

% 2 % ;
R( 
$
!
5  6

! "
R6% 
$!
 "6
",!%
$R  "&
$

;
 3
;

%"
$ 

%  
 

, !
!
%
$

$22 % 
$

 +[

!

5  6
/DN>

   

CI

9B !  ! #!  # !#R !$   ;R # !'877H<

9:R #.#  ! !  ! ! !! 

   

CH

&  R    

R"&
$
% !
"


!
$ &!
$
R2 
(%

$ %
%  $"";
 %&!

R &"
$R!
%  
""!%
$(
 2


!
$$%


!
L
( ;
$ 

!
2 


& %
$
%!
5  6

!

$22 % 


 +[
5  6


 C[
% 

 
%  
%

 3
;
%"

%  
 


  % 
!%
R22!
!
%

 !
!
 %
$
5 B
% &
$
$(%
 !6
$R( ;
% 2 % 
!
 "! >



 "  
"
$

% $! 
$
!


4
" " %

   

C+

'   R   



$R!
(%!

!%((%


%  %
@
!

(% &%
$ $
$
 
!$

2%

!
&
 !




!%(
$!
5 "
""!%>

22;

 
$R!
(%!

 %%  

!
&
!&
%

(%!


" "
(%!6
$ (  
 !%
 %%
!
$  !%%
R""!
 

$(
$
R
2
ML@
  ;
-..-N>


(%!

$ ( 
!
 !%
$
!%
( ! 
$
 "&%!
% 
 !%
" $!%

%  
""!%
$
R


!!%
6 %
  

" %
!
% 
$
2((%!


%  
%2 % 
!
2 
R2 
&>

" "
",!%
$R& 
$
R22
(%
 
!

$22 %
(!6
$

%  
%2 % 

 

&

( 
$R$! 
$!

$
R!;
 

 
!%

  

(
$!
% !%
$
R!;
!
 %
!%

"  !
22%
$
R%2 % 
M " $;
-..9N>

Z
R& 
$

5 
$R!
%

2((%!
R22!

!

$22 %
$



"

R2 
%
G
$
!

!!%
" %
!
"! 
%%$ 
$!
2!%
!%
F

$ 
!
( 
%$(
$
R%2 % 

$

%  
(%>


%"%
" ";

(%!
%


%  
$
R

(

$  
%


!%>

!
 2 %
!
( 
$

 !%
 %
R
 !%
!
%"%
$R &%
!
2 
22
(



&

%% 
$!

$
R!
 ""

(%!
L!,>

%  
 
%!!%
-


" % 
 ""

% &
$
R& 
$!
% " !%
$
R!>

22;
!
2 

%  
"! ;
R2 

L
% 
!

   

C9

(
! " 
$!
% " !%
!

!
5 
$R%2 % 
%!>
$;

&


" "
$R 
$
-

 

 %
!$ 
M !
  ;
-..CT
!
  ;
-..IN>


&

  
F$ $
 ""

! %
$
6 %
!%
G>
% 
  "
$!
F
%
R $&
5 

%  
/


%   
 ""
, !
!
%
$

& 
MG 
  ;
-..+N>

Z

 "&%!
!$

$ " %
$
" $2 
$

  
! $ $
 %
$
R2 
%

2((%!>
R& 
$

( 
$
  
0 L 
 $!

"%

% $! 
$R7

(
(%>
!!%
%  
 
%!!%


"

!>
C4
$
(%!
;
G;
1
 %
!%

2!%
 /
M ! @0 %$
  ;
-..DT
! @0 %$
  ;
-..FN>
D
$


 %
!%
 -
(
 
$ "
 5" %
M @@ 
   ;
-..9N;
 %


(%!
$

$ !

G

%2 %%
"
(
R( 
$!
2!%
5B-

$ 
 /

 -
(
!%
%  
 
%!!%
M7!
  ;
-..DT
3
  ;
-..DT
0 
  ;
-..DT
!
  ;
-..DN>


$  
 
 !(
$ %$
( %
 


!;

!%
$
6 %
 
& 
!%
R6% 
$
%  
 
%!!%
$
2 
$($!
!
$
$
%  ;

 
$

 6
$R!
2 
(%
% %"
$;



" "
6
$R& 
$
R%2 % 
"

,!

 

!$ 

$R!%
!$
"&
 %
 !%
$ 2%

%  
(%

!%
 ""!

 !

2((%!;


Z

2((%!
!(
!
, !%
!
%
!%

22!%
$

%  
%2 % >

0
&%
R(
(%
$

( %;
"  !
$!
%
R%2 % ;

$!

$ %$ 
%  
%

%   "
M
  ;
-..+N>

(%!
L!,

%  %
$
R22
(%
$
%  
6


% 

(%!
G

% %



   

D.

%  
%2 % 
%
R 
$
F
!%
R7
 $5
5
M N
ML, !$B
  ;
-..IT
%
  ;
-./.N>
R"&
$

!$
%"
$
 %
!
!
"!6

 
$
2((%!

!%
" "
$R 
 !%
 %%
R""!
$
R>


%
$R!
%$
!
 !%
&%
$
%"
& 
%


% $! 
$
 !(
"  !
(%>

   

D/

(       


( 
$"!%

"!%
%"
 !%
!%
 %

(%!
%

!
 2 %%
!
%  
 ;
$
  !
$!% ;
(
 !
"2 

 !%

%& (% ;
R22
$
(
 %

2 (%
,!;
R 
, 
!

 


 " ;
 !%
$
2 
(%
 %$


"
!
 
$R!
(
!

!%%

$
$22! 
$R %$%

2;


!
% $!%
!, !%$R!
$
(
22

R6"
$

%
 %

% 
$ " %2

 %



(%!6
 !
$ 
!
%(
 !

2
 %;
!&
$R%
! 


$R2 
$ % ;
!
 !%
!
% ( 
& 

 !%
%"
M! 
$
"2!
 !%

% 
6 
!
(%!
%
3//;
$

%&(%


$R6  
$!
% 
" $
!
(%!
$

2 (%
N>

R(%
$
(;

!(
%
  !
 !%
%
22
!%
$
(%!
"!;
% 
$
!
""
% !
(%
M2"
!
%N>

$ ( "
$R(%!6

,!2
 !%
$
(% 
"% 


 !(
 "
$ "

$!6 "
! 


%"
%  


%  "
% ;

!
 (;


%"
!%2>

 

%%
!"

% 
5
$
 

 !%

%& (% 

 %;
% 
!
2 
% !;

5""
 
 
 2

   

D-


! 
$

 %
(%
 !%

(%!
 

$ 
%

$ ( "
$


"  %
%& (% 
 !%%
%
( 

% 56
$
$
! 
%! %
!
 !%
$R $ "
",!%

% 
$2 
$
R 
 ;
$
$
 ! 

%

 

4 


   


+
  

 


 
7 


 




44 7 3

40 

  

DC

9  *! 

 "&%!
%& (% 
" %
 !%
R ""
%!
"
$

%  
% 
!
!$
$!
 &
M1;
;
3LN>
$;
%
%& (% 
R $
$

%  
" % 
!6

R " %

$
 "&%!
!$
% %

$22 %

$
2((% 
!
4% ;
6


R" 
$R!
$
&%
$
 %
!

M ;
 
/9.;
"% 
$
  
(%N
$

5;
 !

%"
$
"%


!
 & % 
(

 
M%
 
$
2%"$$
% N
$

%!@>
7%

R 


 % 




$ !

!
"$
$
%$
" %

4 (
(
!!%
" %
%
>
!
$!6
;



$ % 
!
 
%

% $
& (>

2 (%
,!
%!
"
$
2 
 !!

4 (

;
" %
R6
$R!
(
22>

!!%
% 
!(
6

5


!
!(%
$
R " %
R!
 
$

 %
$
%  
$R!
2

"! 
$!
" !

" !("
$
% 
2( %

$% 
$
(%!
$

5>

&

!
"!(
 
$

"$
(%

$
" $
$
%" >


% !
!
  %
$
"  $
$
%  





&!
$

%(

$


40 

  

DD

8*!


2((%!
 
% &
$
 "&%!6

$R2 
 !(
%
,!

 ""
 !%
&! !
$R%& (% ;

6
% 
!
$
%"
 !%

"$
M !%

(
 %
1;
0

4N>
R&
$
%"
(%!6
 2
 !%
;


$ 
 %
$
 %
!
"!6

 
$

(%!

 "%$%

$52  "
$!
5 "
""!%
$
R;
$!
%
R2 
(%>



 $
%(;
 !
 !
 ""
$ 
 % 

!

!
2 $"
$
R2 

2((%!>

&!

$R !$%

%  
""!%
 
$
R
2

!
2 
2((%
%
G;

!
1

$
$ %"%


%  
 
%!!%
/
, !
!
%
$

" $! 
$

%  
%2 % >


  
$
/
$

!" %
$!
% !!"
$ "

 % 
6 %"


!
%"%
";
 !
( 
%%
!
(!
% 
%
F

/
%
$
6 %
$
!$ E
  
% (  

E%& >
 !%
;

%  
$R %


"%

%  


6%" 

!
!%5 
 !%
%"%
!
% 
$
$
 $ 
(


 "6


% ! % 
% 
5
!%
%
%  
$
 
 5$
!%
$
&
% 
$!
B>

%
!%;
 !
( 
!$
!
(!
   
!&!%
$

$!6
%  
%
""! 2! %

"%  
 2 

!
!
 2 

D>
2;
 !
( 

R$! 
$

%  
%2 % 
M5 
$R!
!
!%

  ;
7N
% 
  "
$!
% !%
F;

 !
( 
&%(
R22
% $!

40 

  

DI

%

%  
(%
/;
6%" 
$
2 
$ $
!
$

 6
$R!
2 
(%>
 !
( 
!
!
 !
5 

 

4 


   


40 

  


 

 
7 


 


44 7 3

 



D+



S    


E& "##$ $   $9IIG!877GF


(%!
$

$ !
M N;
!
%& (%!
$!
%
' ;
%% 
!
( %&
% & "
$

!&

(%!

%"

R ""
%
$
" !


!
$R2 %" 
 
$ &
!%

5 
$

$ !

4 (>
7%


 & % 
2

 !
( 


5
$


 !
( 
!
"%

($

 %! 
$
% 
 % 5
$
$ !
M  /;
-;
FN

R "% 
$
 !(!6
(%;
 %
$
$6
 
$
R !$>
(
R&
$
$  
%  $;

5
 

&
$
R $ "  
"  !%
$

$ !

4 (>

 



D9



 


!
%(%
$
 %
5
5  


!$
! "%
%"%
$
"!6
 "%$%

" !("
$

 ! 
(%>
 !
 !%% 
 %

$ %"
$ "  
 !
 !%% 
"%
!


% ;
!
" 
$
!%
$
" !

% !

$  
 "  


 !
( 
!
" %%
R "% 
$
 !(!6
(%


-..+;
 !
&%( 
R% $! 
$R!
 !(!
(%
$
/>


-..9;

4 (

 !
!
" %
$ "
$
$ !
M ∼D.
...
N
(
$
 "&%!6

$
$ !
 " %% 

 !%!
$
R !$
$ "  
 !
 !%% 
$2%

 % 5
%!

% %$%


 !(!
(%
%!

-..+


R % 
$
R $ ">
R5
$



%(
%
$
&
 !%
"!6
 "%$%

$ "
$
$ !

4 (

" %





"
% 
$

"$
$

5;
$
"!%
$
 %

$
!%(
$ (
%


 



I.





T 1-31.0#11#     


E&"$@L   $9III!877GF


(%!
$

2 (%
,!
M1N


(%!
%  5
$!
%
' ;
%"
%
$
" !
 %
R6
$R!
(
22
M/H N;

2 (%
,!
 !
$R%
!
"$
$
%$
" %

4 (>

 $ 
$
(



(%&
 
$;
!
$
$  
 
$ &
 %

4 (>


/999

-..+;

$ %"
$
"% &  
$!
 

 


!!%
 


 !
( 
!
5%
!
%
$

 2%" 
$
2 (%

,!>
 !
( 

7%

!
% %! 
5  




 %
!$
% ( 
!
%! 
 !!
$
1

4 (
(
!
$(%
 
 !%

%" %
2 ;
 !
( 
$2
$
 !
& (
$
1
%
!
 5
!$ " %
;

 " "
$
 !
$!
 5

$ 
R%

$ ,
 !>



 



I/

 

 

T 1-31.0#11#  $ 12 0     1


 




  
 




 
  
 
  
 
    

 

!   

!"   

 #
 # 
$  
%
& "
%
' (   )


* 
%
 
+ # 
,
*-!./
* " 
%

- %  
0
1   
%
! 
2 


3 " 22 
- 
$ 


++4!56
7
  
%
+ 8 % %
4 2 9
 : 
 "

4
  
; 
-
 <
! %
' % 
*=

)
+;+6
 
8
6(
;  
- 
$ 



0

 ( %
 


 %>

 



I-

  

 <
8"
7 $9


  
2(
  
2 (

 "
 

 

   
  >

 2  #
" 
 %
 8  
 
(
 %
"  (

% 
# % #

#  

    
%
2%  #
8
 "
$
" 
  >
; 
<
2 8 %

#  

    
8
<"

  
8
$
" 
  %

 "
( <
...
%
//?
(

  #
%
 #
<
# 
8

" 
# @

8 #
8
   
2  
A6 -A

72 ( 
%
 " 29
%

% 
#
A+-$A
2  #

 %
8

" 
# >
5
 %

" 

#
#  
%"  
8
$
  
   #

 "
%  #

 
% %@
<
% 8%


A6 " :A
# 2
11
" 
7  %

2 " 
   %
 "
  9
(
 
8


8

A  :A
# 2
1
" >
3  #

 2 
2  %
8

 %

 
8

AB #A
$
 %
(
%  %
7 
  
8
$
" 
"

" 
9
<  

 
8
 (

 >
; <" 

 "
2  %
8


 
 

 "
 
   

  

< 

 22 
8

 (
$
" 


%

 

  %

2%  # 
 " >

! "
 <
8"
M
+2%  #
:
D%

 



IF


#   

32 

"  ( 
8

 8
%
88 "
"  
7E39
 <
8"

 

2(
  

 

2 
 
8
D8 
%
  
D  >
4
" 
2 (
 <
8"
" 
7 $9


  :( 
8 "" 
 %
( <
2 (
" ( 
  
(
 8 %
  



# 

  

 
7- (

 
//9>
1
D8  
$

   %
 % 


  
 
8 
%
 " 
"
" 
" >
4  %
(

 (
"

  


 
2  % 
 #


2 2  
  #
" 
 (  

 (
 
8
D8  >
1

  
D  

8
 <
8"
" 

  
  #
  
"

" 

 " " #

8 
  
8
  
%
 
2
7 

 
// F
- (

 
//)9
%

2  (


2  


 
2>
 >
- 

//
 
8
B #
$


2 %
8 
  
D 
 

7 
0
- 
// 9>
- 
8


2 

 
6 

%
 "
%
 " "
 
8  
 
#%
G:)G
 
<

#   
%
8 
<  >

 <
8"
 
 
8 
 "(

- 
7 8 %



%
  9
%
2 
%  #


8 
 
 
8

 
<
2 2  
8
  
  

#
%  #

 

 
7 
0
- 
// 9>

' %
 8  
< 
"  (

% 
# % #

2%  #
8
 <
8"

 "
%


#  
    
8
 "
 <
8"
  >
; 
<
2 8 %

 2 "
 

    
8

  
8 
...

//?
  %
(

++4!56
 (  #

% #  
%2 
7
 
 " 9>
6 #  
    
%

8
"  (

2%  #
%
< 
 ( %

8 
   
 <
8"
% (  
"  
%

2%  #
%  #

 %
2  %>

 



IC


 

$


 
4
++4!56
 (  #
% #  
%2 
7
 
 " 9
"%
%  #


...://?
2  %

 #
(
8

  
 2
8
2 %
 
8
 <
8" >

4
< 
 %
8

2  
8
28
1#-

$
(

 % %
- 
+
 >
 

#
< 
2 2 %
8 

 
8


 8 %

#
 2 
(

 "
 <
8"

" 
 
! G,..>
D
# "
 
< 
2 2 %
 
 #

 
8

 8 %

>

D #
)E
2  
< 
1#-
( %

$

(
8
  
 %
(
 %
(
% 

  
8

 

H, H
;4

7! 

>
///9

+-+-
%

) F
  

2
< 
  %

% 
G
2 
 8  
 %
%
  "%

M?/ >

; 

 %
$
  
 %

++4!56
( <
...
%
//?
< 
 %%
74 (

9>
 
!D
< 
J  %
8 
//3'
8
8 
   
 #

L1D 2
" 
!D
 
7L # 9

 % #


 8    M
  %  >

  
%
2%  # 
%
# % #
 <
8"
 8  

 "
8 
...

//?

< 
(  %
8 

++4!56
% ( >

  
   
!" 
 2 
< 
2 8 %
 #
% 
J  
%

4 -
" 

 2 
 # 

7D22%
  9>
D2 
< 
#  %
 #

4 2  
6!

 

7+22 % 89
 %
 % %
6!
28 
 %  >
4
8 

8

 " "%

28 
8

EH
(2
8 #
 2  #

R
/?
 %
8

2 :( 
76 -9
2 

# 
%

GR
G.
 %
8

 " 2
7+9
2  : % #
# >
4
2 
< 
(  %

 #

# : 
2 
$:6 -:
O4DD4D!4DDD 44P
%

# :

 2 
2 
$:6 -:!
O4!DD 44!!DDDD4P
7 
 %9>
4
 %

8

 



ID


 " "%
28 
8

H.)
(2
8 #
 2  #

R
//
 %
8
G


 %

8

# 
5!$
%

8 
.)
(2
8

R
: % #
# >
4
2  
%

28


#
< 

# : 
2 
$:+-$:
O!D!D!D4D4DDP
7 
 %9

%

# : 2 
2 
3?:!
O4444DD44DP
7 
0
  
// 9>

D28%
6!
2 % 
< 
" %

 2 
#
%
2 8%
 #

L #
6!

J  
 >

D2 
< 
( 
 %
 #

28 
2  >
 
 %

 
8
 
 <
8"
  
< 
# %
 #
 &
74 2

 
..E9
# 
< 

"
 
 "%
8 
  >
6 #  

< 
2 8 %
 #
<
%88 

  %>
$  

< 
 % %
< 
-+D
" 
)>
74 

 
//E9
 #


   %
2:% 
8
% 
   
%
#( :   #
79
%
G//
(  2
2 
8

   
8
 %
>
 %
- J
 %
7-'9

< 
 %
 >
4
 %
8

 %
(   
%
2  
" 
< 
 %
"
- % 
76  %
0
 % 
..?9>
-'

2 #  

< 

  %

D!'1
7Q<
//H9
 #

4!R7#  9R1
 %
8

 %
(    >
4
(   
 J
( 
 2   
# 
% ( 
8
 #: 


"  
79
%

2 2 
8
 " 
 
719

"  (
8 

 2 % #
 

 >
D
(  2
 2 #

< 
 % %
 #
///
2 
#( :   #
79


( %


-'
(   
 J
% (%
( "

6D*6
7< 88 %
///9
722 
% 9>

$<
 "
$
 
< 
"  (

2(
% ( @
8 

6 -
%
<

+-$


( # #

  
D 
# 2
11
7 

6 -@
5EH?E
5EG).
5E. E
5?/H

%
 

+-$@
5EH?E
5?/H9>

 



II


% 

 
    
! 
 %
  #
 %
8
%88 
# 
# 
%  %
"
# 2

8
$
< %<%@
8"
# 2

D8 
%
<

  
D 
7 
0
  
// F
- (

 

//F
S #

 
..H9>
4
 
%
6 "
$
2 

<
 B
  
D 
$

# 2
1
%
11
2 ">

; 
<
J  %
<
# 
8
$
# 

8 #
8
   
2  
A6 -A

72 ( 
%
 " 2
# 9
%

% 
#
A+-$A
2  #

 %
8
G
%

2
8

R*4!
7 
0
  
// 9
8 
 "
  >

R
D 
8

6 -
#

6 #  

< 
2 8 %
( %


EH

 


6 -
#
%
" %

7$#
9


7 
8

<"
 "
  
 %%9
( #%

  
D 
# 2
11


# 
< 
2 " 
 %
7 

 
//G9>
S 

# 2
 "
  
< 

% ( %

<
%  
  
7#  
% 
( <
  @
T/>/)9@

#   #
  
8 

...
%
//H

# 2
D
%

 %

# 2

< 
  
8 
...
//
//G
%

//H
7$# 
9>
1
( 
# 2

<
2 %
  
< 
 
 %
72:% 
U/>//9
%



 "
 
2 " 
2 %
%
"  (

 ( 
72:% 
U///?9>
; <" 

< 
# 2
D
  
)E,/H
7//H9
< 
 
% 
 %


  
72:% 
T//G9>
1 

 
 %%



  
#
8

6 -
# 
" 
  
 %
(    



 "
  
8
# 2
>
4
  


2 

%  
"   
  #
 %

# 2
D



"  
8 
...
  
7.,..
! G,..
 ,..
?,..
5EG).
5EH?E

 



IH

5E. E9>
S 
# 2

  
 %
(
  %

...
//
//G
%
//H>
5"

"


2  %

#  
%" # 
( "%
< 
T/>V
%
 %%
  
  
>

$

8 

<
 "
  
( # #

  
D 
# 2
1
 %
(
% 8%>

5 
  
8 
//
< 
" 


 
  
72:% 
U//9>
5 
  
8 
//E

< 
" 


 
%
 
  
72:% 
// ://9>
4
<
 "
  


% 
 %
72:% 
T// H9>

 

 8 %
 
< 

6 -
8 # 
 
8

 
8
# 2
1

7J2
+;
/EE)
%
+;
G?) 9
  %

2  
E?

 
%

7(
   #
< 

D8 
 <
8"
  9
7 
0
  
// 9>
S 
# 2
11



;
(   


 

< 
( "%
< 
# 2
D
<  
# 2

  %

 

%
74 (
9>

1
< 
%


 

7//9
<
 "
  
( # #

<
%88 
# 2

7  
D 
# 2
1
%
119
 %
  %
  
(

%88 
 >

R
D 
8

+-$
#

6 #  

8
 "
$
  
( %

+-$
#
2 "%%

2  #



( %

6 -
# @
  
< 
% ( %


  
D 
# 2
11
# 2
D
7.,..
! G,..

 ,..
?,..
)E,/H
5EH?E9
%

7 ,..
/H,/
$G,/G
)G,/G
)/?,/H
5?/H9
%



  
D 
# 2
1
7??,/E
HH,/9>

  
   


  
D 
# 2
1

  
2  %
(
 
  
<  
# 2
11

  
2  2 
  
8 
6 >
 # 
  %
( <
 
%
6 
 "


 

 
< 
" 
8

<
# 2

 >
$# 

 <

% ( 
8


 "

  
 %%>

 



I+

!# % #
  
D 
# 2
11
7 
 %

 "
$
  
2 " 

% 8%9
8 
# 2
D
  
7 
8 
...9
# 2%
# 
# # 2 
< %

  
%

8

  


32 
8

 >
4
 
# 2
D
  
7)E,/H
//H9
< 
#   

% 
 %
8 


 "
# 2
D
  
%
# %
8 

#
  %
 
< %


<
732 
8
  ( ( 9>
 2

  
< 
  
  %


<
%
8


  


  ( (
%
'
6 
%2  
< 

J2 
,..
  #


 
7


32 
8

 9>

!# % #

<
  
D 
# 2
1
  
7HH,/
??,/E9
( 
< 
  %


8
# 2
11
  



 
%
'
6 
32  
2 ">

4 (

 

# # 2 
 8  
"  (
8
 
  
 %%>
1
 <

 

< 
( "%
8
2 
" #


( %
#
8
  %
7 
8 
//

?//
  9>

   

 

$# 

 <

% ( 
8
2  "
1#-

 <
8"
 %
(

++4!56

 (  #
% #  
%2 
7
 
 " 9
( <
  
...
%
3(
//?>

1

(
( "%


 B  
8
 
 %


 "(

- 
2  %
7 
%  #

%

  9
< 

2 
%  #

8 
 
8

 >

 



I9


& 
"

 
'$   

(

  "

hot and al humid
y y subtropical humid valley valley subtropical humid

Ecozone Climate
a

800 High plateau subtropical cold dry and

y Elevation eni eni 1200 Woodedplateau high Tropical hotand humid d d 400 plain Tropical hot and humid rno rno 580 Wooded plain Tropical hot and humid razama 2500 Wooded valley subtropical humid bezas bezas 450 mountainous valley subtropical humid rmejo 400 mountainous valley subtropicaldry
allegrande 2030 mountainous valley subtropicaldry Chipiriri 2500 Wooded valley subtropical humid








April 2005 M Cochabamba Eterazama 2500 Wooded valle June June 2002 M Santa Cruz San Mathias 100 plain Tropic February 2006 M Cochabamba Villa Tunari 300 Wooded
* *

*


Bolivia 2002 1266/02 PrM Bolivia 2007 2288/07 PrM Bolivia 28/99 PrM 1999 Bolivia 92/99 PrM 1999 March Bolivia1999 323/99 PrM January 2007 Bolivia JR35/99 PrM 1999 February Bolivia2006 471/06 PrM 1999 M May 1999 Bolivia1999 322/99 PrM March La Paz M Bolivia 2002 1026/02 PrM 1999 F Santa Cruz 1999 Bolivia2005 452/05 PrM Santa Cruz May Bolivia FBV115/05 PrM 2005 M F Alto B May Santa Cruz Ca Bolivia2006 408/06 PrM Santa Cruz March 1999 V 2002 2005 M February El To Be M Santa Cruz M 2006 La Paz Cochabamba M Ciuda Cochabamba Ete Viacha 3 Elevation, meters above sea level sea above meters Elevation, * Persons deceased following YFV infection infection YFV following deceased * Persons a



Genotype I Genotype Genotype name Isolate Month Year Sexe Department Cit South American American South Genotype group II, A Genotype group II, B





















 



H.

  

"

)$ 
 

(


$
*) *


* *
 
+ 

, -.

D

2  %
< 
(  %
 #
2:% 
2  
#  
8
% 
   

#( :   #
%
G//
(  2
2 
7" 
#  

G/V

 % %

 B
%

( 9>

;  
( 

2 2  

#  
% >

1%  
2  #
< 
(  %
 #


-'
  %  #
722 
% 9>

"
) 
 .
6 #  

   %
 #
$
6 -:+
# 
 
7
:HHH

8  #


(  #
8
 
  W GE/?W..?
  

(
D G)/)./9>4

2 "
#( 

   
8

  
D 
# 2
1
%
11

2  %
8 

 # 8  >

"

 .
6 #  

   %
 #
$
G: R*4!
# 
 
7

//.E:/H./
8  #


(  #
8
 
D(
.E
  

(
D H)/G?.9>4

2 "
#( 

   
8

  
D 
# 2
1
%
11

2  %
8 

 # 8  >

 



H/

99 South American genotype I

99

South American genotype II 99

57 Nigeria strain56205 1991 99 Ivory-Coast 85-82H 1982 71 Nigeria H117505 1987 83 Nigeria IB-AR-45244 1969 Nigeria BA-55 1986 V
``1H:
$VJQ 7]V


99 Nigeria H117491 1987 99 Senegal Y5 Bolivia PrM-1266/02 2002 Nigeria Nigeria46 1946 85 Nigeria Jimo 1970 Venezuela 35708 1998 99 Nigeria M.Adejumo 1970 99 97 Venezuela 35720 1998 Nigeria A.Adeoye 1970 78 Trinidad CAREC9515207 1995 Nigeria T.Adeoye 1970 Senegal Dar Ar 276 1965 72 Brazil 2001 Senegal SH-1339 1965 Brazil BeAr628124 2000 BurkinaFaso SH-28580 1983 Trinidad T790882 1979 Senegal SH-1464 1965 99 Senegal SH-1446 1965 60 Trinidad-and-Tobago CAREC891957 1989 Burkina-Faso HD-38564 1983 71 Trinidad CAREC890692 1989 61 Guinea-Bissau Jose-Cachatra 1965 50 Trinidad CAREC889920 1988 Senegal Senegal65 1965 51 66 Senegal Dar1279 1965 V
``1H:
Trinidad CAREC891954 1989 $VJQ 7]V

55 Ivory Coast 1999 Trinidad CAREC797984 1979 Q% .
IV`1H:J
Gambia 2001 80 Brazil BeH425381 1984 73 Guinea 2000 57 $VJQ 7]V

89 Senegal ArD76320/Senegal90 1990 Brazil BeAr350397 1978 99 85 Ivory-Coast ArA29436 1992 Trinidad-and-Tobago GML902621 1954 64 French neurotropic strain 1995 Venezuela PHO42H 1961 Trinidad Trinidad79A-788379 1979 80

75 Asibi-strain 1927 Panama Jimenez 1974 French viscerotropic strain 1995 99 64 Panama 614819 1974 66 Vaccine-strain-17DD Senegal 17D-vaccine 99 Ecuador 1345 1981 80 Brazil vaccine-strain17DD Ecuador Ecuador79 Vaccine-strain-17D-213 1995 99 Brazil BeH35010 1995 Pasteur 17D-204-YF-vaccine Brazil BeH379501 1980 84 USA vaccine-strain17D-204 HONG3 1998 South-Africa vaccine-strain17D-204 HONG8 95 89 Brazil BeAr527198 1994 USA vaccine-strain17D-204 HONG2 1998 66 Brazil BeAr527785 1994 YFV X03700 vaccine-strain17D 1985 54 86 Spain viscerotropic vaccine-strain YF-AV Brazil BeAn510268 1991 60 South-Africa vaccine-strain17D-204 HONG9 Brazil BeAr527547 1994 USA vaccine-strain17D-204 HONG1 1998 79 Brazil BeAr511437 1991 YFV NC002031 vaccine-strain17D 1985 South-Africa vaccine-strain17D-204 HONG1 Brazil BeH203410 1971 China 17D-Tiantan Brazil BeH233393 1973 99 Angola Angola71 1971 J$QC:
$VJQ 7]V
98 Brazil BeAr233436 1973 Angola 14FA 1971 86 Sudan SSUD03-05 Brazil BeAr233164 1973 99 Sudan SSUD03-04 Brazil BeH385780 1980 Sudan SSUD03-02 76 97 Bolivia PrM-2288/07 2007 Sudan SSUD03 03 :
``1H:
Venezuela P128MC 1959 99 Kenya BC-7914 1993 $VJQ 7]V
88 87 Kenya KE93-477 1993 Brazil BeAr512943 1992 Uganda Uganda48A 1948 Brazil BeAr513008 1992 99 Uganda A709-4-A2 1948 75 Uga48 Brazil BeAr513060 1992

99 88 Central-African-Republic HB1782/CAR86 19 Brazil BeAr513292 1992 55 83 Central-African-Republic ArB8883 1977 Brazil BeAr544276 1996 52 Car77-883 Central-African-Republic HB1504 1985 99 60 Brazil BeH512772 1992

97 Democratic-Republic-of-Congo LSF4 1958 Brazil Tennessee 1996 Sudan M112 1940 59 98 Brazil BeAr424083 1984 95 Sudan M-90-5 1940 : LVJ `:C
``1H:
Colombia INS382060 2000 51 Sudan M112-4 1940 $VJQ 7]V
50 53 Uganda SE 7445 1964 94 Brazil BeH511843 1991 Uganda Z19039 1972 99 Colombia INS347613 1985 Ethiopia ETH2777/Ethiopia61 1961 Ethiopia Couma-61b 1961 96 Colombia V528A 1979 51 Ethiopia serie227 1961 Brazil BeH107714 1966 Central-African-Republic ArB17239 1980 Brazil Brazil35 1935 Brazil BeAn23536 1960 0,02
Brazil BeAr142658 1968 99 85 Brazil BeAr44824 1962 79 Brazil BeAr46299 1962

96 Brazil BeH111 1954 Brazil BeAn142028 1968 Brazil BeAr162 1955

69 Brazil BeAN131 1955 0,01 96 Brazil BeAr189 1955 Ecuador OBS5041 1997 Bolivia PrM-452/05 2005 74 Bolivia PrM-FBV115/05 2005 80 Bolivia PrM-408/06 2006 `Q%]

95 Bolivia PrM-1026/02 2002 99 Bolivia PrM-322/99 1999 Bolivia OBS8026 1999 99 67 Peru ARV0548 1995 Peru OBS2250 1995 Peru OBS2243 1995 52 Peru ARVO544 1995 59 Peru HEB4224 1995 99 Peru OBS2240 1995 Q% .
IV`1H:J
Peru OBS7904 1999 Peru 1368 1977 $VJQ 7]V

Brazil BeH413820 1983

94 Peru HEB4240 1995 99 Peru HEB4246 1995 60 Peru HEB4245 1995 Peru 149 1995 99 Peru HEB4236-153 1995 Bolivia PrM-471/06 2006 Bolivia PrM-323/99 1999 87 Bolivia PrM-JR35/99 1999 `Q%]

Bolivia PrM-28/99 1999 53 Bolivia PrM-92/99 1999 77 Bolivia OBS7549 1999

54 Bolivia OBS7687 1999 52 Bolivia OBS7937 1999 Peru CepA2 1995

98 Peru CepA1 1995 

7

50 Peru IQT5591 1998 Trinidad79 Peru 1371 1977 `
`V$1QJ
Peru 1362/77 1977 Peru 287/78 1978 73 Brazil BeH141816 1968 Peru R35740 1979 Peru OBS6745 1998 Peru 03535098 1998 Peru OBS6530 1998 Peru strain1914 1981 Peru 1899/81 1981 Ecuador 1914-81 1981


 



H-

99 South American genotype I 99

South American genotype II 90

Nigeria H117505 1987
Nigeria BA-55 1986 Nigeria 56205 1991 V
``1H:
67 Ivory CoAst 85-82H 1982 $VJQ 7]V


96 Nigeria Nigeria46 1946 Nigeria IB-AR-45244 1969

54 Ivory coast 1999
Bolivia EMF-1266/02 2002
Mali ArA20628 1986 88 67 Senegal Senegal65 1965 Venezuela 35708 1998 99 GambiA 2001 99 Venezuela 35720 1998
51 French neurotropic strain 1995 V
``1H:
Brazil BeH613582 1999 YFV strainFNVYALE $VJQ 7]V

57 Brazil BeAr628124 2000 Senegal ArD76320 1990 91 Trinidad CAREC890692 1989 58
99 Ivory-Coast ArA29436 1992 Trinidad CAREC889920 1988 60 French neurotropic vaccine-strain 1995 98 Trinidad-and-Tobago CAREC891957 1989 French viscerotropic vaccine-strain 1995 Trinidad-and-Tobago GML902621 1954 Asibi 1927
56 BrAzil BeH425381 1984 French viscerotropic strain 1995 Brazil BeAr350397 1979 74 Spain viscerotropic vaccine-strain YF-AV 82 Brazil BeH233393 1973
South-Africa vaccine-strain17D-204 HONG8 95 63 Brazil BeAr233164 1973 South-Africa vaccine-strain17D-204 HONG9 78 99 Brazil BeAr233436 1973 South-Africa vaccine-strain17D-204 HONG1 YFV NC002031 vaccine-strain17D 1985 Bolivia EMF 2288/07 2007
66 58 YFV X03700 vaccine-strain17D 1985 Brazil BeH511843 1991 Brazil BeAr424083 1984 Brazil vaccine-strain17DD 67
USA vaccine-strain17D-204 HONG3 1998 Brazil BeAr513008 1992 Brazil BeAr544276 1996 USA vaccine-strain17D-204 HONG2 1998 77 USA vaccine-strain17D-204 HONG1 1998 Brazil BeAr513060 1992
Vaccine-strain17D-213 1995 Brazil BeH512772 1992 Angola Angola71 1971 J$QC:
$VJQ 7]V
Colombia INS347613 1985 Kenya BC-7914 1993 Q% .
IV`1H:J
:
``1H:
91 BrAzil BeH203410 1971 99 Uganda Uga48
99 $VJQ 7]V
66 BrAzil BeH350698 1978 $VJQ 7]V

99 Uganda Uganda48A 1948 BrAzil BeH379501 1980 97 Central-African-Republic Car77-883 51 Brazil BeH535010 1995 Central-African-Republic HB1782/CAR86 19 99
Brazil BeAn604552 1998 Republic-of-congo STA-LSF-4-4143 1958 74 Brazil BeAr527785 1994 Uganda Z19039 1972 95 Brazil BeAn510268 1991 Ethiopia Couma-61b 1961 91
73 : LVJ `:C
``1H:
Ethiopia Serie-227 1961 Brazil BeAr511437 1991 65 $VJQ 7]V
Brazil BeAr527198 1994 67 Ethiopia CoumA-61 1961
63 Ethiopia ETH277/Ethiopia61 1961 99 Brazil BeAr527547 1994 98 Venezuela PHO42H 1961 0.02
55 Venezuela P128MC 1959
Panama 614819 1974 97 Ecuador 1345 1980 Colombia V528A 1979
Brazil BeH385780 1980 Brazil BeAn23536 1960

99 Brazil BeAr142658 1968
99 Brazil BeAn142028 1968 57 Brazil BeAr44824 1962 84 Brazil BeAr46299 1962
66 Brazil BeAr189 1955 89 BrAzil BeH111 1954 0,01 68 Brazil BeAr162 1955
Bolivia EMF-1026/02 2002

99 60 Bolivia EMF-452/05 2005 63 Bolivia EMF-FBV115/05 2005 `Q%]


Bolivia EMF-408/06 2006 63 Bolivia EMF-322/99 1999 54 Bolivia OBS8026 1999
Peru ARVO544 1995 50 Peru 1371 1977
65 Peru ARV0548 1995 99 Peru OBS2240 1995 89 Peru HEB4224 1995
Peru OBS7904 1999 99 Peru 1368 1977 77 Peru CepA1 1995
Peru IQT5591 1998 Bolivia EMF-92/99 1999 90 Bolivia OBS7687 1999 99 Bolivia EMF-28/99 1999
`Q%]

Bolivia EMF-323/99 1999 96 Bolivia EMF-JR35/99 1999
Bolivia EMF-471/06 2006 61 Peru CepA2 1995

90 Peru R35740 1979
52 Peru OBS6745 1998 

7

51 Peru 149 1995 Q% .
IV`1H:J

Peru strain1914 1981 Peru HEB4236 153 1995 $VJQ 7]V

 
`V$1QJ
Peru HEB4240 1995 66 Peru HEB4245 1995
98 97 Peru HEB4246 1995 Peru 03535098 1998
Peru OBS6530 1998 Peru OBS2250 1995 97 Peru 287/78 1978 89
Peru 1362/77 1977

 



HF

& 

"
/ 
  
   

(
   "

Amino acid conservation of ancestral African residue (in PrM region), within South American genotype I and genotype II, of YFV strains in this study (Bolivian strains studied included).



I1JQ
H1R

Q1 1QJ
:


I1JQ
H1R

5
`Q%]


5
`Q%]



"
#
"

a Based on PrM coding region, 680 nt. GI: genotype I GII: genotype II

  

"
/  $ 
  

$
  

  "

 

 



HC



  
0
"
# 

$

 
 



  1
2


 R
 R
 R
 R
 R
 R
 R
 R
 R











 



HD

3  

  

   
 

1

 
 %

"% 
8
 (

8
 <
8"

 "
"

...:

//?
2  %
 %
(
% 8%>

# 8
  
8
 
< 
2 %
%  #

2  %
%

 B  
8
 8  
 %

  #
%
 
7J

,8
X
>?G
%
#
X
)9
2  (

8  #
#  
 %
J2  
8
( >

4
% "
8 

 


 
8
 <
8"

 "
7< 
2 
8

2 %
$
 (  

8
( 

?HE9
7"

 
.G 9
( 
 (
%
B #
2%


8
" 

 "
(
2 %>

 
< 

 B
"
   %

 (

2%

  
D  >
; <" 
(
.) 

 
 %
(
"  
 %
8 


 "
  
%

%


88 "
 
8
 (
2%
8
$
7"

 
.G F
( 

//9>
32 
 %
2 %
8
: 8 
(

 
7  
.)?
7"

 
.G 99
 <

8"
< 
 8 
   %


  
8 

 " " #

B #
  
   

7"

 
.G 9>

S 

 J 
8
 
  

 
 22 %


 
%




.?/
%
< 
2 %

2%
8
% #
8" >
4
J  
8
% #
8"


 #
2
8


 "
  
732  
8

 
'
6 
4 B 
  ( (
%
 
7! 

 
//.99

 % 


 
 
<
( %
2  %

 " >
32 

2  
8

 (
"

8
 <
8" 

 #
 (
$
 (  
 %


 >
* (

8

" 

  %
 %

2 
%

7
%
 8

 
..?9
%

" 


(
 8 %



" 
>

4 

 %


 %  %

( 
8
 (
 <
8"
 >
388 

# # 2 
2 2  
8
  
 "
(
 <

" 


2 ( 

$
7( 

.?9
%


#
(
2 %


 
2 2  

: 8 %

 "
 (

 

2 
% 2 %



8
 "
$
  
<
 "
(
 8 %
8

 



HI

% %


B #

 % #
%  
" 
  >
; <" 

2 
 
(
 %

(
 

 
%
 "
2 2 
8

 
  %
 
%

# 8

 2  
8
" 
2 
%

< 
( "%




 


 >

D 
2 
8 

 %
  >
1

 

 <
8"
"  
8

 (


2 2 
#
J2 

( 
8
 (
$


2  
8

 
 
//E
 

 "
 <%
 <
"  
  
 " #
7  > G:)/V9

  "
 %
2 8 %

..

%
..E
7
%
 8

 
...9>

  

 <
8"
"  
 2 #
< 
 % %


//E
%
 <%

 

"  
 " #
8
?HV
76 

 
//.9
<



(

  %
< 
# 8
2 " 
8
$
2  %


   >
6 



"  

 2 # 

 #
 (  
8
% #
8"
( "%
 

?/M
 
 "
2 %





 
8
 (
 <
8" >
;   # 
( %

% #
" 
 
 % 

2 ( 

$
 8 
7- (

 
//)9

2 " 
2 %


 
8
 2 
 2  
74
0

;  
.E)9>

1
%%  


2 (

2%
 #

   # 
( "  
%
    #
 (  

 
 "
 %
J #
( <
" 
%
 (
  
%
 8 

22   
8
$

 "

8
 
" 
>

  
   


  
!# % #
 
8
B #
 <
8"
2 %


 
 %

22

(
 %

"  
8
#  
"  
8

" >
 
2  #
  
D 
# 2
1
  
< 

( "%


D  
#
8


 
32 
%


2 

8

'
6 

32  >
 
2  #
  
D 
# 2
11
  
< 
( "%
  
8
  

D 
# 2
1
  
7$#
9>
1

%88
8 

%

%   

 
8
2   

%,
 # 
8

%88 
"  
 
 
8

< 
%  %



%  #

 

 >
"

" 
#
%

( 
8
% 8%
 

 
J2 
 #
% 

2 
8
" 


2 ( (

2   
 


 %
# # 2 
 
%
  "

 



HH

 % 
8
<
"  
8 
( %

 >
1
# 
< 

2 
  
D 

# 2
11
# 2

  
< 
( "%


 %

  2  #

< 
2
8


  ( (
32 
%

  
2
8

'
6 
32 

...
//
//G
%
//H>

1

28
 





" 
< 
   %
  


" 

8

E:
2  %

%

2   
< 
  %
< 
 %
#  
% 8
7T/>V


6 -
#
"

 2 

2  %
<  

:
  
  9>
4

## "
8

J  
8
  
8 
%

 

 " "

$

 
"  
   %
< 
 2 %
2 2  

 ""


" ( 
 
(


 % 8%
 >

388 
" 
" 
 %
(
% 8%

 
 @

...
( 
  
D 
# 2
11

# 2
D
%

  
< 
 %>
 2
D
" 
< 
  %



 
#
%

# 2


" 
< 
  %


 
 
 #

2 ( 

( 
"  
 :  %


 

  >
; <" 
#  

" %


# 2

  
< 
 
 %

  

 %


< 
#
8

  
%

 
 2 %


2 %
 



 

# >
1
//
  
8 
( 
  
D 
# 2
1
%
# 2
11
< 
 %
(


 %

%
# # 2 
 >

1
  

 %
" %


#  
%"  
8
$
  
   #

 "


 
2

2 " 
("%>
1
2  
# 2
1
" 
 %
(
% 8%
8

8 



%% 

# 2
11
  
2 " 
%  %>
4
%88 
"  
8

" 


(

  %

 
J 
< 

# # 2 
#
%

 #
"% 
8

 :  
8

%88 
"  


 

< 
% 8%>
$ 


 
% 8%
(

++4!56

 (  #
% #  
%2 
< 
  %
< 

2%  # 
 J
8
B #


%

"% 
8
 (
" 
  
 %
(
%  %>
!
 

8

 2
  %
8 
8( 
 "
2  
8 

  ( (
#
< 

  
% # 
8

 



H+

3 #
8"
 
" %

 
8

 
< 
 <
8"
7   
2  
% 9>
4
 :


8  <
2
8
 <
8"
  # 
 "
7+'1D
%  
8
28
1#-
( %9
(


++4!56
## 

  %
  
8
 <
8"

 "
< 

 
2  #



8 
 
8

 >
32 
 
 
 

B #
 
 <
8"
  

  

%
2 ( (
 %   %
2(
  
2 (

 "
%

 
8

 22 
8

 (
" 

(

 
  
% "

  %
 " 


J
8  >

3   
4


 2  #
8  
   
J >

% 

- (
6
S #
;
'
'
 
+
 
D3>
6 #  
%
"   
  2
 #
 <
8"
" 
  

D8  >

 
//FEG@H...:E//?>
 

S #
;
 ( 

!  

S 
3
!
=
 
D>
+  

8
 <
8"
" 

6 >
+ #
1 8
3
// F.@.H: >
- (
6
 
D
4 "  
%
! 
D
4
!
 
D3
;##
>
 <
8"
" 
 8 " 
8
 "
D%
#2 
  >
+ #
1 8
3
//)F/@HGE:H/>
 
D3
- 
46>
+2%  #
%
  #
8
 <
8"
" >
D%"
 
!
// FH@.: G>
 
+
 
D3>
 2 "
2 # 
8
 <
8"
  
8 
6 
%
 >
$+-
1 
-%
- ( 
// F .@/ :?>
4 2
3
(
4
6<  
$
  #
$
;## 
3>
4
'*4D'W&
< % <
  8 @
8J(
 #
8
 2
 
# 
%%
(
  

>

D%
!
..EFG@)?EH:?>
4 
=
3%


-
=
>
-+D)@
- 
+"   
  
D 
7-+D9
 8 < 
" 
)>/>
- 
 
+" 
//EF)@G.H:.>
6  %
3
 % 
=D>
-53+'4+4@
  #

 %
8
3D
(    >
  8  
..?F)@?E: ?>
Q<
3>
  
#  
22 
8

2 #  

8
 #
(  # 
 
%  
 %

 J
 %
    >
6>3>
% 
//HFD  @
4
* "  
8
4J 

D  >
< 88 %
3'>
6D*6>
6 # 

 #
2  
7 %

  %9>
) 
%>
 %  %
///F-D@
 
D  >
S #
+
S "

 2
!+
4
!
S 
3-
 
D3>
  
"  

 <
8"
" @
%2 


M
 % #
#
8
  
8 
D8  >
  #
..HFG@E):?>
 
+
   
6$
!B ( %
!
- (
6
;##

 
D3>

  #  


M
 % #
#
8
  
D 
  
8
 <
8"
" >

 
//GFE.@ ?/E:>

 



H9

"

4 :- 

3  :-%
>
 <
8"

 " 
 
 
%
2%  #>
D

4 2
-%
;#
.G F@)H):?>
(
3>
;
( " 
 8  
< %<%>
D


D %

//F.G@ :)>
! 

 

!" 
!
 

' 
!
 "
'
$ %
!4
$ 

; "
6
%
' (  
&>
- 
2%  # 

8
% #
8"

 "
8 
..?

//?>

 
Q 
3
//.F.@ E:))>

%
 8
6
 
D
6 %
-
; 
D
6 %

  
-
 % 
>

    @
% #
 2

( 2
111
7M   M9
2%


 
 " >
4 2
-%
1
;  
..?F @?GE:?>
(
3
 " 
!>
- 
>>

>
D  2 %
"
 2  :2%  # 
#  
%
(  # 
 %  >
6 % #
8

   
 8  

#  
8
 
% 
" 
.?F # 
1 @ ) : E?>
  
S
 ( 
4
 ( 
-
$22
D-
   
64
! %
-
-  
-%
'
-
!>

 2  
8
 
D%
#2 
%
D>
( 2 
8

 
 <
8"
" 
  >
4 
!
 
4 2
-%
;#
//F.H@H: >

%
 8
6
 
D
6 %
-
2%

'

6 %

6# 
'
 % 

  
->
* (  
8
 <
8"


 
 " >
' 
...F G @GG?:H>
6 
'
6 %

; 
6
 2

D % <

! " J
5>
  : 2
 "
%

  
 
 2 #

"  
 <
8"
  
 " #
8  < #

 
 2 # 
 " 
//E>
4 2
-%
1
;  
//.F)@ GG:H>
4

; 
S->
 :2  
( <
# 2

( " @
 



 2 

 2  
%
 >
' 
 2  
" 
 %%
(
3 #
" 
   >
1 8
1
.E)F.@./.:G>

 



+.



 


 %
R6
$R!
(
22;

2 (%
,!
 !
$R%
!
% & "
$

",!%
$

%  
% 
$R 2%


5;

 !(%!%
(

2&

 !
( 
"

($
!
%! 
 !!
$!
(%!
 %
$
$% %
 >


R5
$



 %
!$;
 !
R( 
!!
$ 
$R!
%" 
!%&
$!
(%!
" %

% 
$!
(!%

  
M      ;
-..9N>
1
"&
!
"!
$
!
5
(

!$
 " "%

 "  
%"%
$
 "%$%

 " %"
$
(!%

4 (


%" 
( %
$
1>

 !%%
 !
%%






%
5
5  
 !
R( 


!$
  
$

 
!(
$
!%



%(
%

!
"!%
 "%  
$
R $ "  
"  !%
$

2 (%
,!

4 (>

" %

 



+/




U !!#.2M     


E  #!!! # 9$# #A!,. $  # ! ).!  %#  F


2((%!



!6

$
" 
$

$R2 

2((%!
$
%

" $;

$ ( "
$
%"
(%!6
 %

%& (% 

$(!
!"
!
,!
" %
$

!&


R""!
 


%" %
%  
$
R
"


2
!6


 "&%!6
(%!

$ ( 
$
% 
 !%
5
%

 !( %
&%
!
2 
22>

 "%  
$
" "
"


%

2((%!
 !%
 %%

%  
 
 %
$R!
2 
%"%
$R$2%
$
&
 %
 !%
!
% 
(%>


%  
%2 % 
%% 

%  
",!%
$
R""!
 >
!!%
(%!
5
% 
%

R 
$
!%
%  
 
%!!%
!%
$(%
22!%
$

%  
M " $;
-..9T
   ;
-..DT

S
7;
-..HT
 " 
S
%;
-..HN>


%  
/

 !(

%   
 ""
!%
$
R& 
$
R""!
 
M (%!   ;
-./.T
!   ;
-..IT
G    ;
-..+T
G 22
S
!$E ;
-..9N>


!$
%  $;
&

!
(!
$

$(%
% !;
 !
 !
 ""
%  
$R !$%
R 
$
/
$
% 
2((%!;
$ !;
2 (%
,!

G
;
!%

%  
%2 % 
$!
% 
"! 
$!
F>

 



+-

 !
( 
!$

  
!&!%
$!
F

$

%  
2((%
/


R% 
$

$!6
%  >
 !
( 
!
5
R$! 
$

%  
%2 % 
M5 
$R!
!
!%

  ;
7N
% 
  "
$!
% !%
F;

 !
( 
&%(
R22
% $!
%

%  
(%
/;
6%" 
$
2 
$ $
!
$

 6
$R!
2 
(%>

 



+F





U !!#.2M " ,1
'0-*-%7

Nonstructural proteins NS1 of several mosquito-borne flavivirus do not inhibit TLR3 signaling

Cécile Baronti, Joséphine Sire, Xavier de Lamballerie and Gilles Quérat §

UMR IRD 190, Emergence des Pathologies Virales, Université de la Méditerranée, Faculté de Médecine de la Timone, 27 Bd Jean Moulin, 13005 Marseille, France.

§ Corresponding author: Gilles Quérat

E-mail: [email protected]

Phone: (33) 4 91 32 44 20

Fax: (33) 4 91 32 44 21

 



+C

Abstract

Flaviviruses are single-stranded positive RNA viruses that replicate through double stranded RNA (dsRNA) intermediates. These dsRNA may be recognized as pathogen- associated molecular patterns by cellular receptors including membrane-bound Toll-like receptor 3 (TLR3) and cytosolic helicases RIG-I and MDA5. dsRNA stimulation results in signaling cascades converging to activation of interferon (IFN) regulatory factor 3 (IRF3) and to transcriptional activation of several Interferons stimulated genes, including IFNß and inflammatory cytokines. There are conflicting reports concerning the ability of West Nile virus to counteract TLR3 signaling. In our analyses, transiently or stably expressed NS1 proteins from two West Nile viruses, two dengue 2 viruses and a yellow fever virus failed to inhibit TLR3 signaling in two different mammalian cell lines. Moreover, using siRNA inhibiting the helicase signalization pathway, we show that viral infection did not impede TLR3 responses to poly(I:C).

We conclude that NS1 from distinct mosquito-borne flaviviruses do not inhibit TLR3 signaling.

Keywords: Flavivirus; West Nile virus; TLR3; innate immunity; NS1; interferon;

 



+D

Introduction

Members of the Flaviviridae family, including dengue virus (DENV-2), yellow fever virus

(YFV), Japanese encephalitis virus (JEV), West Nile virus (WNV) and others, can induce neurological disorders (encephalitis, meningitis) and hemorrhagic fevers that may result in fatal infections. In the absence of effective treatment or vaccine (with the exception of YFV vaccine), flavivirus infections remain a global public health problem. This is particularly evident for dengue virus infections that cause at least 500,000 new cases of dengue fever/hemorrhagic fever/dengue shock syndrome (DF/DHF/DSS) each year with an estimated 25,000 fatal cases.

Flaviviruses are enveloped, single-stranded, positive-sense RNA viruses that are transmitted via arthropod vectors. Their genome is a single RNA molecule, approximately 11 Kb in length, 5’ capped, encoding one open reading frame that is immediately translated, producing a polyprotein precursor tightly associated with endoplasmic reticulum (ER) membranes. The precursor is then post-translationally processed by virus and host-cell proteases to yield the individual viral proteins, three structural proteins, i.e. capsid (C), pre-membrane (prM), envelope

(E), and seven nonstructural (NS) proteins, i.e. glycoprotein NS1, NS2A, protease cofactor

NS2B, protease and helicase NS3, NS4A, NS4B and the polymerase NS5, that associate with viral RNA to form the replication complex (for a review, see (Brinton, 2002)).

During viral RNA replication, a negative strand RNA is generated that is found in association with the genomic positive strand RNA. These dsRNA intermediates, interact with

NS proteins, and generate new positive strand genomic RNA. These replicative complexes assemble in small membrane structures named vesicle packets, localized in the lumen of the endoplasmic reticulum (ER) in the perinuclear region. Viral dsRNAs may be identified as foreign by two types of cellular receptors activating the first lines of cellular defense, triggering the synthesis of interferons (IFNs) and ß (for a review, see (Meylan & Tschopp, 2006); (Saito &

Gale, 2007)).

The cytoplasmic DEx(D/H) box helicases RIG-I (Retinoic-acid inducible gene1)

(Yoneyama et al., 2004) and MDA5 (Melanoma differentiation-associated gene 5) (Kawai et al.,

 



+I

2005) contain a helicase domain which binds to viral RNA and a caspase recruitment domain

(CARD) that recruits an adapter, IPS-1/Cardif/MAVS, which relays the signal through nuclear factor Kappa B (NF- B), interferon regulatory factor-3 (IRF-3) and factor-7 (IRF-7). These factors in turn contribute to transcriptional induction of various pro-inflammatory cytokines, apoptotic responses and type I IFNs. Among flaviviruses, DENV-2 has been shown to trigger both RIG-I and MDA5 (Loo et al., 2008). Moreover, these helicases were decisive for IFNß induction following DENV-2 infection, as cells lacking the adaptator IPS-1 nearly failed to activate IFNß production (Loo et al., 2008). However, infection of mice with knock-down IPS-

1/Cardif showed only a delayed activation of IFN responses (Perry et al., 2009) suggesting that other viral sensors were able to confront dengue virus infection. Similarly, WNV infection recruits both RIG-I and MDA5 signaling through the IPS-1 pathway (Fredericksen et al., 2008) and, at least for in vitro infection, these cytoplasmic helicases appeared to be the main controllers of WNV infection. Yet, another signaling pathway was also involved in the control of

WNV infection in mice as TLR3 -/- mice were more vulnerable to WNV infection (Daffis et al.,

2008).

Amongst RNA viruses that initiate innate responses through RIG-I and MDA5 signaling, some have been shown to encode inhibitors of IFN induction. The V protein of paramyxoviruses inhibits MDA5 signaling (Andrejeva et al., 2004), and the protease of hepatitis C viruses, another genus within the family Flaviviridae , encodes an inhibitor of RIG-I signaling (Li et al.,

2005). To date, however no such inhibitors has been characterized for any flavivirus.

The TLR3 receptor (toll-like receptor 3) is an integral type I glycoprotein that is expressed in membranes of the ER and multivesicular bodies in most cells including myeloid cells, and at the plasma membrane in the case of some fibroblastic cells (for a review see

(Vercammen et al., 2008)). The extracellular domain exhibits a leucine rich motif that binds dsRNA. As such, TLR3 senses extracellular dsRNAs which have been internalized by endocytosis and reached a late endosomal compartment close to phagosomes (Nishiya et al.,

2005); (Sanjuan et al., 2007) where acidic pH facilitates multimerization of TLR3 and the ensuing signaling (Leonard et al., 2008). The most potent activator of TLR3 is dsRNA that is

 



+H

cell-associated such as those of infected dying cells (Schulz et al., 2005). The synthetic dsRNA poly (I:C) is also a strong inducer of TLR3 signaling, even when added, free, in the culture medium. The sensing of dsRNAs by these receptors initiates the downstream activation of transcription factors such as IRF-3 and NF- B promoting the expression of IFN /ß and proinflammatory cytokines (Vercammen et al., 2008).

Therefore, TLR3, RIG-I and MDA5 sensing of dsRNA converge to the IRF-3 and NF- B pathways for activation of innate immune responses. The role of TLR3 as a sensor and controller of flavivirus infection is unclear. In one report, dengue virus appears to recruit TLR3 sensing in vitro both in myeloid cell lines and in TLR3 expressing fibroblasts (Tsai et al., 2009) as the presence of TLR3 in HEK cells greatly increases cytokines and IFN production following infection. In another report, Dengue was unable to induced IFNß synthesis in fibroblast cells devoid of the helicases adaptor IPS-1, suggesting that RIG-I and Mda-5 were the only receptors of innate immunity in dengue infection (Loo et al., 2008). WNV infection of TLR3 knockout mice showed conflicting results regarding the survival of infected mice and the viral burden in the brain (Daffis et al., 2008); (Wang et al., 2004). Both studies nevertheless revealed only a modest increase of viral replication in peripheral tissues associated with either unchanged or a slight decrease of cytokine responses. In cell culture, knockout of the TLR3 adaptor TRIF did not significantly affect WNV replication whereas RIG-I null cells produced higher replication

(Fredericksen & Gale, 2006). Contrasting results were obtained using established replicon- bearing cell lines. TLR3 signaling was greatly reduced in WNV replicon-bearing HeLa cells

(Scholle & Mason, 2005). The decrease in TLR3 signaling was reported to be due to blocking of

IRF-3 and NF- B nuclear translocation that was TLR3 specific, as MDA5-controlled Sendai virus infection still induced IRF3 nuclear translocation and IFNß activation. NS1 appeared as the viral protein responsible for this inhibition of TLR3 signaling in replicon-cured HeLa cells

(Scholle & Mason, 2005).

NS1, a glycoprotein of ca 48-KDa, is expressed in the lumen of the ER by translocation from a signal peptide located at the C-terminus of the structural envelope protein E. NS1 dimerizes (Winkler et al., 1988) and associates with membrane components through unknown

 



++

mechanisms (Winkler et al., 1989) where it interacts with the NS4A protein (Lindenbach & Rice,

1999) from the replication complex. NS1 is also exported via the secretory pathway to the cell surface (Pryor & Wright, 1993) and can dissociate from the plasma membrane of mammalian cells and accumulate in the extracellular medium as a hexamer (Gritsun et al., 1990) (Flamand et al., 1999). In vivo , WNV and DENV NS1 are found in high amounts in sera of infected patients (Young et al., 2000); (Macdonald et al., 2005). Soluble DENV NS1 associates with liver cells (Alcon-LePoder et al., 2005) in vivo and can be endocytosed to late endosomes of cultured normal liver cells (Alcon-LePoder et al., 2005). NS1 is indispensable for viral replication as it is required for early dsRNA synthesis by unknown mechanisms (Lindenbach & Rice, 1997). Large deletions in NS1 are readily trans complementable (Lindenbach & Rice, 1997); (Khromykh et al., 2000), except when deletions encompass conserved residues in the C-terminus which appears to be required in cis (Khromykh et al., 2000). NS1 interacts with NS4A as demonstrated by mutation in YFV NS4A proteins that enabled DENV NS1 trans complementation of an NS1-defective YFV. In addition to its role in viral RNA replication, NS1 has also been described as an immuno-modulatory factor through its binding to the complement regulatory factor H (Chung et al., 2006a) and as an inhibitor of TLR3 signaling (Wilson et al.,

2008). Lastly, NS1’, an elongated NS1 protein encompassing the 5’ region of NS2A through a -

1 ribosomal frameshift has recently been described for WNV and Japanese encephalitis viruses serogroup (Firth & Atkins, 2009), (Melian et al., 2010) and may be involved in neuroinvasiveness (Melian et al., 2010).

In this report, we analyzed TLR3 and the NS1 of WNV, YFV or DENV-2 through protein interactions, sub-cellular localizations and functional studies of the regulation of interferon- related genes. Our results show that in HeLa and HEK 293 cells the TLR3-induced levels of translation of several interferons related genes are not altered by expression of either ectopic

NS1 or virally encoded NS1, indicating that TLR3 signaling is not a target of NS1 .

 



+9

Materials and Methods

Cell lines and viruses.

HeLa cells were purchased from ATCC and cultured in Dulbecco’s modified Eagle medium (DMEM) containing 1 g/l of D-glucose, 1 mM of sodium pyruvate and 2 mM of L- glutamine, supplemented with 10% decomplemented fetal calf serum (FCS) and antibiotics.

HEK 293 derivative cell lines were cultured in the same medium except that the DMEM contained 4,5 g/l of D-glucose. HEK_TLR3+, a HEK 293 cell line stably expressing the human

TLR3 tagged with the HA epitope, was purchased from Invivogen (293-hTLR3-HA). West Nile virus (Uganda strain B956) was amplified by transfection and replication in HEK 293T cells of the pWNII-Not molecular clone (kindly provided by R.W. Doms) in which the viral genome is under the control of a CMV promoter (Pierson et al., 2005). WNV-NY strain 385-99 virus

(Genbank AY842931.3, (Ding et al., 2005)), YFV 17 D vaccine strain (Genbank X03700.1,

(Rice et al., 1985)) and DENV-2 Martinique strain (Genbank AF208496.1, (Tolou et al., 2000)) were amplified on HEK 293 cells. All working pool stocks, stored at -80°C, were titrated on HEK

293 cells.

Plasmids and transfections

pUNO-hTLR3-HA was purchased from Invivogen. pUNO-TLR3-His was identical except for the HA epitope tag which was replaced by a 6xhistidine tag. pUNO-TLR3-ecd, a construct expressing the ectodomain of TLR3 was constructed by deleting the transmembrane domain

(beginning position 2112 of the coding cDNA) and the intracytoplasmic C-terminal end of TLR3. pGFP-IRF3 was a kind gift from D. Gerlier (VirPatH, CNRS, Université Lyon 1). IRF3 was amplified from this construct and inserted downstream to an in-frame HA epitope tag in pBC13-

HA vector. A pCEP4 plasmid (Invitrogen) was modified to contain either an HA epitope tag

(GYPYDVPDYA), a 6xHis tag, a modified Flag-M2 (GDYKDHDDIGDYKDDDDK) epitope tag or a modified Glu-Glu (GEYMPMEGEYMPME) epitope tag downstream of the multiple cloning site.

 



9.

These constructs were used to clone and express the C-terminal tagged NS1 from flaviviruses.

NS1 from molecular clones of WNV Uganda (pWNII-Not, (Pierson et al., 2005)), YFV17D vaccine strain (pACNR-FLY17Dx, kindly provided by P.J. Bredenbeek, Genbank X03700.1,

(Rice et al., 1985)), Dengue-2 virulent strain 16681 from Thailand (clone pD2/IC-30P-A kindly provided by R. M Kinney ((Kinney et al., 1997)), WNV-NY strain 385-99 virus (Genbank

AY842931.3), and DENV-2 Martinique strain (Genbank AF208496.1, (Tolou et al., 2000)) were

PCR or RT-PCR amplified with primers incorporating appropriate restriction sites and cloned into pCEP4 derivatives. The last 30 residues of the C-terminal end of E genes were incorporated into the NS1 construct to provide a signal peptide allowing insertion and cleavage of NS1 in the ER. All constructs were verified by sequencing and western blot analysis.

DNS1_WNV, a WNV-UG derivative of the pWNII-Not plasmid was rendered defective for NS1 by introducing, through PCR-mediated recombination, a deletion of 155 residues (from genome nucleotides 2467 to 2931 or NS1 amino acids 4 to 158, inclusive). 293T cells were transfected with JetPEI (Polyplus transfection) using a ratio a 1µg of DNA per 2 µl of JetPEI. HEK-293 derivatives and HeLa cells were transfected with Lipofectamine 2000 (Invitrogen) using a ratio of 1µg of DNA per 1.5 µl of reagent. During transfection (4 to 5 hours), HeLa cells were maintained in OPTIMEM (Invitrogen) medium supplemented with 3% FCS.

Generation of the HEK_TLR3+-NS1F cell line expressing NS1 .

The TLR3 expressing cell line HEK_TLR3+ from Invivogen was transfected with a pCEP4 derivative vector expressing a Flag M2-tagged NS1 from WNV-UG or with a control empty pCEP4 vector. pCEP4 derivatives encode a resistance gene for hygromycin. Forty-eight hours post-transfection, cells were selected using both blasticidin (10 µg/ml) and hygromycin

(80 µg/ml) antibiotics. A pool of resistant cells was established without cloning and tested for the expression of NS1 by Western blot analysis. HeLa cells were transfected with the pCEP4 derivative vector expressing a HA-tagged NS1 from WNV-UG and pool of resistant cells were selected with hygromycin (200 µg/ml).

 



9/

TLR3 and RIG-I/MDA5 stimulation .

TLR3 was stimulated by addition of poly (I:C) (Invivogen or SIGMA) in the culture medium for 6 to 8 hours. Helicases were stimulated by transfection, using Lipofectamine 2000 reagent, of a mix of 200 ng of poly(I:C) and 200 ng of viral dsRNA, plus 1.6 µg of plasmid DNA as carrier. A 504 bp fragment of WNV-UG corresponding to the 5’ end of the genome was amplified with either the sense or antisense primers fused to a T7 promoter sequence. Plus and minus strand of viral RNAs were synthesized using the MEGAshortscript T7 kit of Ambion. DNA was then digested with TurboDNAse from the kit and the RNA was purified on MEGAclear column (Ambion). RNAs were buffered by addition of 1/20 volume of 10 fold concentrate phosphate buffer saline and equal amounts of plus and minus strand RNAs were annealed by incubation at 80 °C for 5 minutes and allowed to co ol to room temperature.

siRNA transfection .

Silencer FAM-labeled Negative control#1 siRNA and three VISA/IPS-1 Silencer Select

Pre-designed siRNA (33178, 33179 and 33180) were purchased from Ambion. They were reverse transfected in HEK_TLR3+ and HeLa cells using RNAiMax from Invitrogen at a final concentration of 12.5 nM, using the recommended procedure for these cell lines and seeded in

24 wells plate. Transfection medium was replaced 24 hours after plating.

RNA extraction and analysis.

Intracellular RNA: At 24 h post-transfection, cells in 12 or 24 wells plates were treated with addition of poly(I :C) in the culture medium during 6 hours to 8 hours, before being lysed by addition of 300 µl of RLT buffer (Rneasy mini Kit from Qiagen) supplemented with 1% of ß- mercaptoethanol. Lysates were forced through QIAshredder mini-columns (Qiagen) by centrifugation and total RNA was purified using the RNeasy recommended protocol, and the recommended DNase step to eliminate cellular DNA. Alternatively, total intracellular RNA were extracted and purified using the Nucleospin RNA II kit from Macherey Nagel.

 



9-

RNA from virus particles: Supernatant medium from infected cells was recovered, clarified by centrifugation at 3000 g for 5 minutes and filtered trough 0,45 µm pore filters. Viral

RNA was purified by the EZ1 virus mini kit on an EZ1 BioRobot workstation (Qiagen).

Real-time RT-PCR comparative analysis (Applied ABI-Prism 7000 and 7900 HT or Mx-

3000 Q-PCR of Stratagene) was performed with SuperScript III Platinium one-step qRT-PCR kit

(Invitrogen) using either Sybr green chemistry (IFNß, ISG56, Viperin, VISA and YFV) or hydrolysis probe chemistry (actin, WNV-UG, WNV-NY and DENV-2). Actin was used as a normalizer to account for differences in cells number and/or quality of extracted RNA.

Sequences of primers are listed in Supplementary materials.

Pull-down and Western blot analysis.

HEK 293T transfected cells in a 6-well plate were lysed 36 h post-transfection with 500

µl of cell lysis buffer (20 mM Hepes pH 7.3, 150 mM NaCl, 1% Triton X100, 7.5 mM imidazole and Complete, EDTA-free anti-protease cocktail (Roche Diagnostics) . Following clarification, lysates were incubated with 30 µl of Ni ProBond (Invitrogen) equilibrated beads for 20 mn under agitation. Beads were collected by gentle centrifugation (200 g) and washed three times with

900 µl of washing buffer (20 mM Hepes pH 7.3, 150 mM NaCl, 0.5% Triton X100, 15 mM imidazole). Bound proteins were eluted with 60 µl of elution buffer (20 mM Hepes pH 7.3, 150 mM NaCl, 0.5% Triton X100, 0.5M imidazole). Aliquots from clarified lysates, washed Ni

Probond beads and eluates were resolved on 8 or 10% polyacrylamide gels containing SDS and transferred to PVDF membrane. Anti-Flag M2 mouse mAb (SIGMA), HRP labeled anti- penta-His mouse mAb (Qiagen) rat anti-HA mAb (3F10 from Roche), mouse anti-actin C-2 mAb

(Santa Cruz Biotechnology) and the corresponding HRP-conjugated secondary antibody were used. Protein bands were visualized using either ECL (Amersham) or Immobilon (Millipore) followed by exposure of the blot to film.

 



9F

Confocal immunofluorescence microscopy .

HeLa cells were seeded onto coverslips that had previously been placed in 24 well plates. Twenty-four hours post-transfection, cells were fixed by the addition of 3% paraformaldeyde pH 7.4, 0.1 mM MgCl2 and 0.1 mM CaCl2. Cells were then treated with 0.5 M of NH4Cl and permeabilized with PBS containing 0.1% of saponin and 10% of FCS. Primary antibodies (rat monoclonal anti-HA 3F10 and rabbit anti-Glu-Glu) and secondary conjugated antibodies (goat anti-rat Alexa-Fluor 488 and goat anti-rabbit Alexa-Fluor 555 (Molecular

Probes)) were used at a 1/500 dilution in PBS solution containing 0.1% saponin and 5% FCS.

After each step, coverslips were washed in PBS-1%saponin. Finally, coverslips were washed in

PBS and dipped rapidly in water, before being mounted on sideglass with mowiol and visualized under a Leica SP5 confocal laser-scanning microscope.

 



9C

Results

Ex-vivo interaction between NS1 and TLR3 proteins

It has been reported that WNV NS1 has the ability to inhibit TLR3 signal transduction

(Wilson et al., 2008) suggesting that NS1 may interfere with steps involved in this activation cascade. Except for TLR3, which is located at the membrane boundary between extra and intracellular spaces, all other components of the TLR3 signaling pathway are located inside the cell. Because both TLR3 and NS1 are present in the lumen of RE and endosomes, it is tempting to speculate that the only way for NS1 to impact on the TLR3 signaling pathway might be to target TLR3.

We thus looked for putative ex-vivo NS1-TLR3 interactions by co-expressing each of the partners in HEK 293T cells. It has been reported that flaviviruses NS1 proteins are able to homodimerize ((Winkler et al., 1989); (Chung et al., 2006b)). We therefore verified that NS1 dimerization was detectable by pull-down of the dimer on an immobilized resin. Cell lysate containing Flag M2 epitope-tagged NS1 and 6xHis-tagged NS1 of WNV-UG was incubated with

Ni beads, and the ability of Flag M2 epitope-tagged NS1 to bind to Ni-immobilized 6xHis-tagged

NS1 was analyzed by pull-down experiments and Western blot analysis under native conditions.

As shown in Fig. 1A, Flag M2-tagged NS1 was detected only when 6xHis-tagged NS1 was co- expressed in 293T cells and immobilized onto Ni beads. Upon increase of the imidazole competitor, the 6xHis-tagged NS1 and Flag M2-tagged NS1 complexes were co-eluted from the

Ni Probond beads, demonstrating the specificity of the interaction. These data indicate that co- expressed NS1 proteins efficiently dimerize ex vivo and that the dimerization was not due to direct binding of Flag M2-tagged NS1 onto Ni beads.

We next analyzed the NS1-TLR3 interaction in 293T cells co-expressing the 6xHis- tagged TLR3 and Flag M2 epitope-tagged NS1 from WNV-UG, YFV and WNV-NY. As controls,

6xHis-tagged NS1 from WNV-UG and YFV were included and bound proteins were analyzed by pull-down experiments (Fig.1B). DENV-2 NS1 from either the Martinique strain or Thailand

 



9D

pD2/IC-30P-A clone was expressed at much lower steady state level (see Fig.S1) and was excluded from the analysis. As expected, NS1 from WNV-UG and YFV readily dimerized onto their His-tagged counterparts (Fig. 1B, lanes 3 and 7). It is noteworthy that WNV-NY was also able to hetero-dimerize onto His-tagged WNV-UG, suggesting that the general structure of dimer is well conserved among WNV NS1 and that most residues involved in dimérisation are conserved between the West Nile strains of lineage I and II. Neither WNV-UG nor WNV-NY

NS1 was retained by TLR3 (lane 4 and 12). YFV NS1 was repeatedly retained in low amounts onto Ni Probond resin loaded with His-tagged TLR3 (Fig. 1B, lane 8) but also by empty Ni-

Probond resin in absence of His-Tagged co-expressed proteins (lane 6) suggesting that the retention of YFV onto TLR3 was non specific. It is noteworthy that the steady-state level of the

6xHis-tagged TLR3 (lane 4, 8 and 12, lower panel, see the band at ca 130 KDa) was much lower than that of 6xHis-tagged WNV NS1 (lane 3, 7 and 11, lower panel). We also made the reverse pull-down experiment, using histidine-tagged NS1 from the three viruses to retained co- expressed HA-tagged TLR3. Unfortunately, full length TLR3-HA was found to be retained non- specifically onto Probond resins even in the absence of co-expressed NS1 (data not shown).

Addition of Poly(I:C) in the culture medium of transfected cells and in the lysis and interacting buffers did not affect the results. In conclusion, although our conditions of interaction were clearly compatible with the demonstration of NS1 dimerization, we could not detect specific interactions between TLR3 and NS1 from WNV-UG, WNV-NY and YFV.

Co-localization of NS1 and TLR3 in HeLa cells

Although our results did not demonstrate any direct interaction between NS1 and TLR3 using pull-down assays, the possibility did exist that they may nevertheless be in very close contact in the physiological context of cellular expression. We thus looked for co-localization between TLR3 and NS1 transiently expressed in HeLa cells by confocal immunofluorescence microscopy. HeLa cells were co-transfected with pUNO-TLR3-HA vector and expression vectors encoding Glu-Glu epitope-tagged NS1 from WNV-UG and YFV. Representative images are shown in Fig. 2. WNV-UG NS1 and TLR3 did not co-localize except at the margins of their

 



9I

respective distribution. WNV NS1 was markedly absent from the perinuclear region, whereas

TLR3 was mostly absent from the sub-surface region. In contrast, there was much more yellow merging coloration between TLR3 and YFV NS1 since YFV NS1 was more evident in the perinuclear region. The low level of expression of DENV-2 NS1 did not allow meaningful co- localization studies. In conclusion we found no evidence of protein co-localisation for WNV-UG

NS1 and TLR3, but TLR3 and YFV NS1 were found to partially colocalize suggesting that they share, at least in part, a similar sub-cellular compartment.

Cells expressing NS1 still stimulate TLR3 signaling

Contradictory data have been reported concerning the impact of WNV infection on TLR3 signal transduction. In one study, using WNV replicon bearing cell lines and IFN-cured derivative cells, Wilson and collaborators (Wilson et al., 2008) reported that TLR3 signal transduction was negatively affected by the expression of NS1. In contrast, another study had reported that WNV did not antagonize signal transduction mediated by TLR3 in either WNV replicon-bearing Huh cells or in the context of a native infection (Fredericksen & Gale, 2006).

We thus decided to revisit and extend these experiments to other members of the Flavivirus genus.

It has been well documented that addition of poly(I:C), a synthetic analogue of dsRNA, in culture medium of epithelial or fibroblastic cells, leads to its recognition by the specialized TLR3 receptor triggering IFNß secretion. In a first series of experiments, we evaluated levels of IFNß mRNA in HEK fibroblastic cell line stably expressing TLR3 (HEK_TLR3+ cells) in the absence or in the presence of NS1 from three distinct flavivirus groups, namely WNV, YFV and DENV-2.

TLR3 signaling was induced by addition of poly(I:C) (20 µg/ml) in cell supernatant medium 24h post-transfection leading to a stimulated production of IFNß mRNA up to 153 fold in cells transfected with empty vector (Fig. 3A) demonstrating the functional expression of TLR3.

However, IFNß mRNA levels were at least equal or superior to levels of empty vector when we transiently expressed NS1 of two different WNV strains, two different DENV-2 strains and YFV followed by poly(I:C) treatment suggesting that NS1 of these flaviviruses do not down-regulate

 



9H

TLR3 signaling in HEK_TLR3+ cells. Using Western blot analysis, NS1 from both WNV and

YFV strains were found to be expressed at high and similar levels in HEK 293 cells whereas the steady state level of DENV-2 NS1 were much lower (Fig.S1). In this experiment, poly(I:C) stimulation for each NS1 constructs was assayed and calibrated against background level of

IFNß synthesis in absence of the dsRNA analogue. As there were no significant differences between background levels of IFNß synthesis between various NS1 constructs (data not shown), the background levels of IFNß were thereafter calibrated against that of empty vector.

In the previous experiment, TLR3 was stimulated by addition of 20µg/ml of poly(I:C) in the culture medium.

Subcellular localization of TLR3 has been shown to be different in fibroblastic and epithelial or myeloid–derived cells, so we were concerned that the cell-surface expression of

TLR3 in fibroblasts might be responsible for the lack of signal modulation by NS1. To confirm these data we performed experiments in epithelial HeLa cells transiently expressing TLR3 (Fig.

3C). Unexpectedly, a barely detectable stimulation of IFNß mRNA was observed when poly(I:C) was added in cell supernatant medium. However, co-expression of IRF3 in addition to TLR3 in

HeLa cells led to more than 30 fold induction of IFNß mRNA when a dose of 10 µg/ml of poly(I:C) was added in cell supernatant medium and more than 50 fold when cells were treated with a dose of 100 µg/ml of poly(I:C). These data indicate that endogenous IRF3, at least in

HeLa cells, was expressed at suboptimal levels to induce a strong IFNß response. It must be noted that the overall level of stimulation of TLR3 signaling varied from experiment to experiment but was always strongly stimulated by the addition of IRF3. To demonstrate that

IFNß stimulation, as observed in the presence of overexpressed IRF3, was still TLR3- dependent, we analyzed the IFNß mRNA stimulation in poly(I:C)-treated HeLa cells co- transfected with IRF3, wild type TLR3 and the TLR3 ectodomain (TLR3-ecd) constructs. This

 



9+

latter construct encoded a TLR3 protein deleted from the cytoplasmic tail that is necessary to transmit downstream signaling. Results showed that the co-expression of TLR3-ecd led to an approximate 50% decrease of IFNß mRNA synthesis indicating that TLR3-ecd acts as a competitive inhibitor of signaling in cells over-expressing IRF3.

We next analyzed the function of NS1 from WNV-UG, WNV-NY, YFV and DENV-2,

Thailand and Martinique strains, in HeLa cells that overexpressed both TLR3 and IRF3 on TLR3 signaling upon poly(I:C) stimulation (30µg/ml) (Fig. 3D). Although in this specific experiment, we observed a modest increase of IFNß synthesis, there was no significant downregulation of IFNß mRNA synthesis in presence of any NS1 confirming our data obtained with HEK_TLR3+ cells.

We next verified that this lack of down-regulation by NS1 protein was not due to a saturation of

TLR3 by excessive dose of poly(I:C) by setting a dose response study with poly(I:C) from

1µg/ml up to 100 µg/ml. As shown in Fig.3E, increasing dose of dsRNA analogue led to parallel increase of IFNß synthesis, for both empty vector and WNV-NY NS1 suggesting that the lack of down-regulation of TLR3 signaling by NS1 in HeLa cells is not due to saturation of TLR3 stimulation.

Poly(I:C) stimulation of TLR3 signaling is also known to activate other cellular genes including ISG 56K (Guo et al., 2000) and Viperin (Severa et al., 2006).

We thus analyzed the effect of some NS1 on the mRNA synthesis of those genes.

Results (Fig.3F) showed that both ISG56 and Viperin mRNA synthesis were increase upon

TLR3 stimulation but, NSI from WNV-UG, YFV and DENV-2 Thailand did not inhibit TLR3 stimulation of these genes as compared to that of an empty vector. Taken together, our data demonstrate that flaviviruses NS1 proteins from three mosquito-borne flaviviruses expressed in cells by transient expression vectors were not involved in the down-regulation of TLR3- mediated signaling in either epithelial (HeLa) or fibroblastic (HEK 293) cells.

Stably expressed NS1 does not down-regulate TLR3 signaling .

It has recently been reported that the mere introduction in cells of plasmid DNA by transfection results in its sensing by the cellular stimulator of interferon genes (STING) and

 



99

leads to subsequent activation of IRF3 and increased synthesis of IFNß mRNA (Ishikawa et al.,

2009) (see also FigS2-B). We were thus concerned that the transient transfection used in previous experiments may have induced a background synthesis of IFNß mRNA, thus masking or perturbing the NS1 effects on TLR3 signaling. We thus constructed, in the context of

HEK_TLR3+ cell line, a cell line that stably expressed, in addition to TLR3, the Flag-M2-tagged

NS1 of WNV-UG, namely the HEK_T3-NS1F cell line. We verified by Western blot analysis that these cells readily expressed NS1 (Fig.S1A). To verify that the stably expressed NS1 was functional we took advantage of the known property of NS1 to complement in trans the replication of an NS1-defective genome (Lindenbach and Rice, 1997); (Khromykh et al., 2000).

An in frame deletion of 155 residues was introduced in the NS1 gene of the WNV-encoding pWNII-Not plasmid generating the DNS1-WNV. This NS1 defective genome was transfected in normal HEK_TLR3+ and in the NS1-expressing HEK_T3-NS1F cell line. As shown in Fig. 4A, replication of the NS1 defective WNV was partially restored in HEK_T3-NS1F cells demonstrating that the stably expressed flagged M2-tagged NS1 was functional. Moreover, transient transfection of ectopic WNV-UG NS1 in HEK_TLR3+ cells was also able to trans- complement NS1 defective virus. Using RT-PCR with a reverse primer in the NS2A region and sense primers either upstream of the deletion or inside the deleted sequence, we verified that the produced viral RNA was still deleted for NS1 and not regenerated via recombination between the incoming plasmid and the stably or transiently transfected NS1 gene (data not shown).

HEK_TLR3+_NS1F cells and control HEK_TLR3+_vect cells (which were stably transfected with an empty vector) were stimulated by the addition of poly(I:C) in the culture medium and the IFN related genes response was examined by comparative real time RT-PCR.

Similar induction of IFNß mRNA (Fig.4B) was observed indicating that the stable expression of

WNV-UG NS1 did not down-regulate TLR3 signaling and that the transfection-induced IFN responses did not interfere with the NS1 activity. Similar results were found regarding the induction of ISG56 mRNA (see Fig.S2).

 



/..

We next looked at whether NS1 would inhibit the establishment of an antiviral state induced by TLR3 signaling. HEK_T3-NS1F and its control cell line HEK_T3-vect were primed through TLR3 by addition of poly(I:C) in culture medium or RIG-I (Fig. 4C). Six hours after the addition of poly(I:C) in the culture medium cells were infected by WNV-NY at an MOI of 0.5 for 2 hours and then washed. Viral replication was assessed by measurement of the number of viral

RNA copies in the clarified and filtered cell supernatant 16 hours post-infection. As shown in figure 4C, upon TLR3 priming, there was no significant differences in viral replication in NS1 expressing HEK cells compared to control cells, suggesting that NS1 does not counteract TLR3 signaling. However, there was no obvious antiviral state induced by poly(I:C) stimulation of

TLR3 despite that TLR3 was very efficiently stimulated (see Fig. 4B) suggesting that HEK derivative are somewhat defective for IFN secondary responses (see discussion).

We therefore constructed another NS1 stably expressing cell line in the context of HeLa.

The HeLa_NS1 cell line stably expressed an NS1-HA from WNV-UG. We verified that the HA- tagged NS1 was expressed (see Fig.S1 A) although at lower level that in transiently transfected

HeLa cells. Upon poly(I:C) stimulation at both 10 µg/ml and 100 µg/ml IFNß stimulation was not down-regulate in NS1 expressing cell line (Fig.4D) . We therefore looked at the effect of NS1 in modulating the establishment of antiviral state in HeLa_NS1 upon stimulation of TLR3. HeLa was found totally refractory to infection by either WNV-UG or YFV 17D (data not shown). We thus went on to infect HeLa cells with the NY99 strain of West Nile virus. Two independent experiments in triplicate were done and results of one representative experiment are shown in

Fig.4E. Poly(I:C) stimulation in HeLa and HeLa_NS1 stably expressing cells was very efficient for the establishment of an antiviral state as viral RNA in the cell supernatant was reduced by more than 2 log in TLR3 stimulated cells relative to non induced cells. However, the stably expressed WNV NS1 did not counteract the TLR3 mediated antiviral effect. Because the level of expression of the stably transfected NS1 was much lower than that observed with transient transfection (see Fig.S1 A), we also realized a similar experiment using transient transfection.

We allowed transfected cells to recover for three days from the IFNß stimulation due to plasmid transfection, verified that the NS1 was still highly expressed (see Fig.S1 A), and proceed to

 



/./

poly(I:C) stimulation and then WNV-NY infection. As shown in Fig.4F poly(I:C) stimulation of

TLR3 did not induced a strong antiviral state relative to non stimulated cells, probably because the transient transfection 3 days earlier had already induced IFN secondary responses in both

TLR3 stimulated and non stimulated cells), suggesting that transfection alone led to inhibition of viral replication. Nevertheless, the presence of NS1 did not translate in higher replication of

WNV-NY. In conclusion, neither transient nor stable transfection of WNV-UG NS1 in HEK 293 and HeLa cells inhibited TLR3 signaling as judged by its capacity to induce an antiviral state.

Flavivirus infected cultures remain responsive to TLR3-mediated signaling

The TLR3 signaling inhibition by NS1 has been reported to be dependent on the levels of NS1 expression (Wilson et al., 2008). Although we could not fully appreciate whether the levels of NS1 obtained by transient or stable transfection is sufficient to modulate TLR3 signaling, we reasoned that NS1 expressed in the context of viral infection represented the bona fide level of expression to down-regulate efficiently the TLR3 signaling. Moreover, replication of flaviviruses takes place in replication complexes that involve numerous interactions between viral nonstructural proteins. NS1 is known to interact with NS4A during replication and we were concerned that expressing NS1 in the absence of other NS proteins could bias its activity regarding the modulation of TLR3 signaling. Therefore, HEK_TLR3+ cells were infected at high MOI by WNV or YFV for 16 hours to ensure that every cell expressed NS1 and other viral proteins at high levels. Viral infection alone led to high levels of IFNß mRNA synthesis, however, masking the effect of further stimulation of TLR3 by poly(I:C) addition (data not shown and see below). These response reflected ongoing viral replication and probably resulted from sensing of viral dsRNA intermediates by RIG-I and MDA5 helicases. We therefore pretreated the cells for two days with siRNAs directed against VISA/IPS-1, the adaptator involved in the signaling pathway of these helicases in order to lower the IFNß responses due to infection alone and to allow more precise measurement of IFNß synthesis in response to TLR3 signaling.

 



/.-

As shown in Fig.5A we first verified that a pool of three VISA siRNAs was able to lower the level of VISA mRNA by an order of magnitude relative to control negative siRNA, and consequently was able to diminished IFNß synthesis in response to dsRNA transfection by the same order (Fig.5B). RIG-I helicase was stimulated by transfection of viral dsRNA (made by annealing of T7 polymerase-synthesized minus and plus strands of a 504 bp fragment of viral

RNAs) and MDA5 was stimulated by transfection of poly(I:C). These results demonstrated that our VISA siRNA treatment of HEK_TLR3+ cells was efficient at lowering RIG-I and Mda-5 dependant IFNß responses.

Negative control and VISA siRNAs treated HEK_TLR3+ were infected 48 hour after siRNA transfection at an MOI of 2 with WNV-NY (Fig. 5 C), YFV-17D (Fig.5D), WNV-UG

(Fig.5E) and DENV-2 Martinique strain (Fig.5F) for 16 hours to allowed high level of viral protein expression and then treated or untreated with poly(I:C) to stimulate TLR3 signaling pathway. As shown in Fig.5C, VISA siRNA lowered the IFNß stimulation due to WNV-NY infection by two orders of magnitude relative to control Negative siRNA as expected (IFNß of siNeg, WNV-NY infected 1.2 10 3 fold stimulation versus IFNß of VISA siRNA and WNV-NY infected 13 fold stimulation). The lowering of IFNß synthesis upon pretreatment with VISA siRNA was observed for all viruses, although infection with DENV-2 Martinique strain led to very low level of IFNß synthesis (Fig.5F), comparable to levels obtained with VISA siRNA treated cells infected with other viruses. We don’t know whether this reflect DENV-2 lower level of replication in these cells, relative to WNV, or if it might suggest that this DENV-2 strain is equipped with activity lowering the IFNß responses to RIG-I and/or Mda-5 helicases.

Poly(I:C) stimulation of WNV-NY infected cells (MOI 2) pretreated with VISA siRNA led to IFNß synthesis at level (mean RQ = 118 fold) higher than that of poly(I:C) stimulated but non- infected cells (mean RQ = 94). In another experiment in which cells were infected by WNV-NY at MOI 20, poly(I:C) stimulation of infected cells led to an induction of IFNß of 315 versus 28.8 in non-infected cells (data not shown). Similar results were obtained with YFV infection (Fig.5D),

WNV-UG infection (Fig.5E) and DENV-2 (Fig.5F). In every instances, IFNß synthesis in responses to TLR3 stimulation in presence of ongoing viral replication was at least equal to, or

 



/.F

superior to, levels obtained in absence of viral proteins, including NS1. These results demonstrate that TLR3 signaling was not inhibited by NS1 nor any other viral proteins expressed at high level in the context of viral replication.

 



/.C

Figure 1. Ex-vivo NS1 interactions. (A) WNV NS1 homodimer ex-vivo . 293T cells were transfected by a vector expressing Flag M2 epitope-tagged NS1 from WNV-UG in the presence or absence of a vector expressing the homologous 6xHis-tagged NS1. Thirty hours post- transfection, cells were lysed and the clarified lysate was loaded onto Ni Probond affinity beads.

Lanes 1 and 4 show input lysate, lanes 2 and 5 show bound proteins to Ni-affinity resins after extensive washes, lanes 3 and 6 show proteins eluted from affinity resins with the 0.5 M imidazole-containing buffer. The Western blot analysis was visualized with anti-Flag M2 epitope primary antibodies. (B) Flag M2 epitope-tagged NS1 from WNV-UG, YFV and WNV-NY99 were expressed alone or together with either their homologous 6xHis-tagged NS1 or 6xHis-tagged

TLR3 in 293T cells. Lanes 1, 5 and 9 show cleared lysate from transfected cells with Flag M2 epitope-tagged NS1. Lanes 2, 6 and 10 show proteins from Flag M2 epitope-tagged NS1 lysates retained on Ni-Probond resins in absence of co-expressed 6xHis-tagged proteins. Lanes

3, 7 and 11: Flag M2 epitope-tagged NS1 bound to Ni Probond when co-expressed with His- tagged WNV-UG (lanes 3 and 11) and His-tagged YFV NS1 (lane 7). Lanes 4, 8 and 12: Flag

M2 epitope-tagged NS1 bound to Ni Probond when co-expressed with 6xHis-tagged TLR3.

Bound proteins were visualized with anti Flag M2 primary mouse monoclonal antibodies (upper panel) and HRP-labeled penta-His antibody (lower panel).

 



/.D

 



/.I

Figure 2. Co-localization of NS1 and TLR3 . Glu-Glu epitope-tagged NS1 from WNV-UG or

YFV were expressed together with HA-epitope tagged TLR3 in HeLa cells. Cells were dually stained for TLR3 protein subcellular localization using an anti-HA specific antibody (TLR3-HA) and NS1 protein expression using an anti-Glu-Glu epitope-specific antibody (NS1-EE). Cells were stained with fluorescent-conjugated secondary antibodies and observed by confocal microscopy. Green indicates TLR3 and red indicates NS1 expression. Two representative sets of pictures for each virus are shown.

 



/.H

Figure 2

 



/.+


Figure 3. Transiently expressed NS1 from three mosquito-borne flaviviruses do not inhibit IFNß production mediated by TLR3 signaling.

(A) HEK_TLR3+ stably expressing TLR3 were transiently transfected with empty pCEP4 vector or vector expressing NS1 of either WNV-UG, WNV-NY, YFV, DENV-2 Thailand 16681 strain

(pD2 clone), and Martinique strain. Twenty-four hours post-transfection, TLR3 signaling was induced by addition of poly(I:C) (20 µg/ml) in the culture medium for 7.5 hours. Total RNAs were extracted and the level of IFNß mRNA was quantified by real time comparative RT-PCR using actin as normalizer and uninduced cells with each construct as calibrators. (B) HEK_TLR3+ stably expressing TLR3 were transiently transfected with empty pCEP4 vector or vector expressing NS1 of WNV-NY. Twenty-four hours post-transfection, TLR3 signaling was induced by addition of increasing concentration of poly(I:C) in the culture medium for 7.5 hours. Total

RNAs were extracted and the level of IFNß mRNA was quantified by real time comparative RT-

PCR using actin as normalizer and empty vector as calibrator (C) Coexpression of TLR3 and

IRF3 results in strong TLR3 mediated IFNß mRNA synthesis in HeLa cells. HeLa cells in a 12 well- plate were transfected by either TLR3 alone (0.5 µg) or in combination with IRF3 (0.5 µg) and/or the ectodomain (ecd) of TLR3 (1 µg). The total amount of DNA was kept constant at 2 µg by adding empty vector. Twenty-four hours post transfection, TLR3 signaling was induced by addition of poly(I:C) in the culture medium (100 µg/ml or 10 µg/ml) for 6 hours. Total RNA was extracted and the level of IFNß mRNA was quantified by real time comparative RT-PCR. (D)

Transiently expressed NS1 does not inhibit TLR3 signaling in HeLa cells co-expressing TLR3 and IRF3. HeLa cells were cotransfected with vectors expressing TLR3 (0,5 µg), IRF3 (0.25 µg) and either empty vector or vector expressing NS1 (1.25 µg) of either WNV-UG, WNV-NY, YFV,

DENV-2 Thailand (pD2) or DENV-2 Martinique. Twenty-four hours post transfection, TLR3 signaling was induced by addition of poly(I:C) in the culture medium (30 µg/ml) for 7.5 hours.

Total RNA was extracted and the levels of mRNA from IFNß were quantified by real time comparative RT-PCR using actin as a normalizer. (E) HeLa cells were cotransfected with vectors expressing TLR3 (0,5 µg), IRF3 (0.25 µg) and either empty vector or vector expressing

 



/.9

NS1 (1.25 µg) of WNV-NY. Twenty-four hours post transfection, TLR3 signaling was induced by addition of increasing concentration of poly(I:C) in the culture medium. (F) HeLa cells were transfected with a combination of TLR3 (0.5 µg), IRF3 (0.5 µg) and either empty vector or constructs expressing NS1 from WNV-UG, YFV and DENV-2 Thailand (pD2) (1 µg). Twenty- four hours post transfection, TLR3 signaling was induced by addition of poly(I:C) in the culture medium (100 µg/ml) for 6 hours. Total RNA was extracted and the level of ISG56 and Viperin mRNA were quantified by real time comparative RT-PCR, using actin mRNA as normalizer.

 



//.


Figure 3

 



///


Figure 4 . Stably expressed NS1 is functional for viral replication but fails to inhibit TLR3 signaling.

(A) WNV-UG NS1 stably expressing HEK_T3-NS1F cells and control HEK_TLR3+ cells were transfected with plasmids encoding either NS1 deleted WNV or wild type WNV. Two days post transfection, the supernatant medium was recovered, clarified and filtered through a 0,45 µm filter. Viral RNA was purified and analyzed by real time absolute quantitative RT-PCR. Results are the mean and standard deviations of two independent experiments. (B) NS1 stably expressing HEK_T3-NS1F and control HEK_T3-vect were either untreated or treated with 100

µg/ml of poly (I:C) during 7 hours. Intracellular RNA were purified and the levels of IFNß were quantified by real time comparative RT-PCR using actin mRNA as a normalizer, and untreated cells as calibrator. Results are the mean and standard deviation from two experiments (C)

HEK_T3-NS1F and control HEK_T3-vect were either untreated or treated by addition of poly

(I:C) in the culture medium in triplicate experiments. Cells were infected with WNV-NY (MOI of

0,5) for two hours. Virus-infected cell monolayers were washed twice in PBS, and the infection was left to proceed for a further 16 hours. Viral RNA from supernatant and intracellular RNA were extracted and the level of viral dsRNA was quantified by quantitative real time RT-PCR

(Mx3005P) using intracellular actin mRNA as a normalizer. (D) HeLa and HeLa_NS1, a stably transfected HeLa cell line expressing WNV-UG NS1-HA were either untreated or treated with 10 and 100 µg/ml of poly (I:C) during 7 hours. Intracellular RNA was purified and the levels of IFNß were quantified by real time comparative RT-PCR (Mx3005P) using actin as a normalizer, and untreated cells as calibrator. ( E) stably expressing HeLa_NS1 and control HeLa cells were either untreated or treated by addition of 100 µg/ml of poly (I:C) in the culture medium (triplicate experiments). Cells were infected with WNV-NY (MOI of 0,5) for two hours. Virus-infected cell monolayers were washed twice in PBS, and the infection was left to proceed for a further 16 hours. Viral RNA in the supernatant and intracellular RNA were extracted and the level of viral dsRNA in the supernatant was quantified by relative quantitative real time RT-PCR (Mx3005P), using intracellular actin as a normalizer. (F ) HeLa cells (25 cm 2 plate) were transfected with

 



//-

either empty vector or WNV-UG NS1-HA. Cells were allowed two days in culture before being trypsinized and splitted in six wells of P12 plate. The day after, three wells of each transfection were either treated or untreated with 100 µg/ml of poly(I:C) for 7 hours before being infected with WNV-NY (MOI 0.5) for two hours. The viral inoculum was washed and cells were allowed

16 hours in culture. Intracellular RNA was extracted and the level of intracellular viral dsRNA was quantified by relative quantitative real time RT-PCR (ABI 7900HT), using actin mRNA as a normalizer. In panel ( C) , ( D), ( E) and ( F) Results are the mean relative quantity (RQ) and errors bars are RQ max and RQ min values generated by either the Stratagene Mx3005P software or the RQ Manager software of Applied Biosystems, at 99% confidence level.

 



//F

Figure 4

 



//C

Figure 5 . Viral infections do not inhibit TLR3 signaling.

HEK_TLR3+ cells were reverse transfected with either control negative siRNA or a mix of three

VISA/IPS-I siRNAs in 24 well plates (in duplicates).

(A) and (B) 60 hours post transfection, cells were either untreated or transfected for 7 hours with a mix of dsRNA to stimulate the helicase pathway. Intracellular RNA was extracted and the level of VISA mRNA (A) and IFNß (B) were quantified by relative quantitative real time RT-PCR

(ABI 7900HT), using actin as a normalizer. Alternatively, siRNA treated cells were infected 48 hours post transfection at an MOI of 2 with WNV-NY (C), YFV (D), WNV-UG (E) and DENV-2

Martinique strain (F). Sixteen hours post infection, cells were either untreated or treated with 50

µg/ml of poly(I:C) for 7 hours. Intracellular RNA was extracted and the level of IFNß mRNA was quantified by relative quantitative real time RT-PCR (ABI 7900HT), using actin mRNA of uninfected cells treated with the negative control siRNA as a normalizer. Results are the mean relative quantity (RQ) and errors bars are RQ max and RQ min values generated by the RQ

Manager software of Applied Biosystems, at 95% confidence level.

 



//D


Figure 5

 



//I


Discussion

In infected cells, the NS1 viral glycoprotein is found in the lumen of the ER, closely associated with membranes. Some NS1 proteins are transported to the cell surface by the endosomal secretory pathway (Fan and Mason, 1990); (Pryor and Wright, 1993);(Flamand et al., 1992). TLR3 is also expressed in the ER with its dsRNA-binding domain facing the lumen of the ER (Liu et al., 2008). TLR3 traffics from ER through endolysosomes via its interaction with the UNC93B targeting protein (Brinkmann et al., 2007) ; (Kim et al., 2008). In HEK 293 cells,

TLR3 has also been reported to be present at the cell surface (Matsumoto et al., 2002). As NS1 has been reported to inhibit TLR3 signaling (Wilson et al., 2008), it was tempting to speculate that NS1 and TLR3 might occasionally colocalize and even interact. We therefore looked for in situ colocalization by confocal fluorescence microscopy and protein interactions using pull– down assays. DENV-2 NS1 from both pD2/IC-30P-A clone and Martinique strain were not expressed at a sufficient level to draw firm conclusions in pull-down and colocalization studies.

Because the DENV-2 NS1 expression vectors were constructed similarly to expression vectors of WNV and YFV proteins we suspect that it is a specific property of DENV-2 NS1 that either could be more unstable or might be exported and secreted in extracellular medium much more readily than its WNV and YFV counterparts.

There were good concordances between localization and pull-down studies for WNV

NS1. WNV-UG NS1 and TLR3 barely colocalized and we could not find any direct interaction between the 6xHis-tagged TLR3 and WNV NS1 from Uganda and NY99 strains. It is noteworthy that NS1 from the two divergent West Nile strains were able to interact and dimerize suggesting that the general structure of dimer is well conserved among WNV NS1 and that most residues involved in the dimérisation might be conserved between the West Nile strains of lineage I and

II.

We found substantial merging between TLR3 and YFV NS1 localizations but we were unable to detect a specific and significant interaction between YFV NS1 and His-tagged TLR3.

 



//H

YFV NS1 was indeed retained on TLR3 loaded resin but to the same extant than onto Ni-

Probond resin without any other expressed His-Tagged protein, strongly suggesting that the retention of YFV NS1 was non specific and resulted from protein precipitation. Either TLR3 and

YFV NS1 share a common sub-cellular localization but do not interact in vivo, or our pull-down assay is not specific enough to detect this interaction.

It must be noticed that, as judged by western blot analysis, there was much less His- tagged TLR3 transferred to the blot than His-tagged NS1. However, transfer of large glycoprotein such as TLR3 to PVDF membrane is notoriously inefficient and a faint band of His-

Tagged TLR3 on the blot may not reflect a low level of TLR3 protein in the cell extract or on the

Ni-Probond resin. Indeed TLR3 expression level was sufficient to allow good poly(I:C) stimulation in HEK_TLR3+ and transiently transfected HeLa cells.

Poly(I:C) stimulation of TLR3 stably expressing HEK cells led to a robust increase of IFNß mRNA synthesis that was not affected by the co-expression of NS1s. In contrast, HeLa cells required co-expression of IRF3 for detectable stimulation of the IFNß natural promoter.

This observation is reminiscent of Wilson and collaborators’ work (Wilson et al., 2008). They reported that in HeLa replicon-cured cells, they found barely detectable level of IFNß synthesis from its natural promoter upon poly (I:C) stimulation. This may reflect a low level of constitutive

IRF3 expression in HeLa cells and/or a saturation of IRF3 signaling by the transient transfection of the TLR3, NS1 and control vector plasmids. Transfected DNA plasmid has been shown to be recognized by the STING/MITA pathway (Ishikawa and Barber, 2008), leading to IFNß stimulation through IRF3 (Zhong et al., 2008). Indeed, we observed that co-transfection of TLR3 and IRF3 without further poly(I:C) stimulation in HeLa cells led to ca 10 fold stimulation in IFNß mRNA synthesis relative to that of TLR3 alone (data not shown) suggesting that the IFNß synthesis, that was triggered by the incoming plasmid DNA, was already limited by the low amount of IRF3. Over-expression of IRF3 in HeLa cells may provide enough IRF3 to engage both the STING/MITA signaling and the TLR3 stimulation by poly(I:C). In addition to the increased synthesis of IFNß mRNA, poly(I:C) stimulation of TLR3 has been shown to trigger

 



//+

expression of ISG56 (Guo et al., 2000), and Viperin (Severa et al., 2006). We therefore looked at mRNA synthesis from these two other natural promoters after TLR3 stimulation in the three cell types. Results were very similar to those obtained with the IFNß indicator, showing that none of the NS1 proteins affected the TLR3-dependent mRNA synthesis from ISG56 and

Viperin promoters.

Poly(I:C) stimulation of TLR3 is known to induce an antiviral state limiting viral replication. To overcome induction of a background antiviral state by the incoming transiently transfected plasmid we constructed a HEK derivative cell line stably expressing both TLR3 and

NS1. NS1 is known to be indispensable for viral replication and this defect could be complement in trans (Lindenbach and Rice, 1997); (Khromykh et al., 2000). Using a molecular clone of

WNV-UG in which we introduce a deletion in NS1, we first verified that NS1, in both stably and transiently HEK 293 derivative cells, was expressed at levels sufficient to allow partial recovery of replication of this NS1 defective WNV. This may also suggest that transiently and stably expressed NS1 in HEK 293 cells were expressed at levels sufficient to interfere with TLR3 signaling. Nevertheless, using the stably expressing HEK_T3-NS1F and a control cell line we were unable to detect any down-regulation of TLR3 signaling as measured by either IFNß or

ISG56 mRNA synthesis. We therefore turned to another consequence of TLR3 stimulation, the establishment of an antiviral state. HEK_T3-NS1F and a control cell line were subjected to infection by WNV-NY. Both cell lines allowed high levels of WNV replication but were defective for the establishment of an antiviral state induced by poly (I:C) stimulation despite allowing high levels of IFNß and ISG56 mRNA synthesis. This was probably due to a defect of these cells in the IFN secondary response pathway. In support to this conclusion it must be noticed that

Invivogen, the commercial supplier of the type I IFN sensor cell line HEK 293-derivative “HEK- blue IFN- /! cells » had to stably expressed both STAT2 and IRF9 genes « to obtain a fully active type I IFN signaling pathway »

(http://www.invivogen.com/family.php?ID=171&ID_cat=11&ID_sscat=84 ).

 



//9

In contrast to HEK 293 cells, establishment of an antiviral state was readily achieved in

HeLa stably expressing WNV-UG NS1. Viral replication was inhibited by more than two logs in

TLR3 stimulated cells, versus unstimulated cells. Nevertheless, stable expression of NS1 did not led to increase of viral replication upon poly(I:C) stimulation. The level of NS1 stably expressed in HeLa_NS1 was quite low, much below that of transiently expressed NS1. The reason for this discrepancy relative to what is observed in HEK 293 cells is unclear. We next looked at the effect of transiently expressed NS1 in HeLa cells upon the establishment of antiviral state. Because the transfection alone led to non-specific background antiviral environment, poly(I:C) induced only a small, and of questionable significance, lowering of viral replication. Transient expression of NS1 in HeLa cells did not restore any viral replication, however. Lastly we went on to measure TLR3 signaling in cells infected at high multiplicity of infection to ensure that nearly every cell expressed NS1 at high level and in the context of other viral proteins. To achieve this we choose to lower the IFNß responses to infection by inhibiting the RIG-I and mda-5 pathway. TLR3 stimulation of infected cells led to high level of IFNß responses, however, comparable or even higher than that of non-infected cells, demonstrating that the presence of NS1 in the context of infection did not inhibit TLR3 signaling. Moreover, these results strongly suggested that no other viral proteins were able to inhibit TLR3 signal transduction.

These results are in stark contrast with previous work that reported the inhibition of TLR3 signaling by NS1 from West Nile virus (Wilson et al., 2008). There are numerous differences in experimental approaches that may explain these discrepancies. First, most of the experiments in the previous work were performed in either replicon-bearing or replicon-cured cells.

Establishment of flavivirus replicon-bearing cells has been reported to be associated with selection of favorable mutations in the viral genome (Rossi et al., 2005) and may also reflect the selection of favorable cell subpopulations as shown for the Huh7.5 sub-clone bearing HCV replicon (Blight et al., 2002) ; (Sumpter et al., 2005), which exhibited impaired dsRNA signaling.

It is therefore possible that the replicon-bearing cell line and its cured HeLa C derivative may

 



/-.

exhibit differences in some elements of the TLR3 signaling pathway making it more susceptible to NS1 inhibition. Alternatively, there may be differences related to the WNV strain, as Wilson and collaborators used a human isolate from the Texas strain (Rossi et al., 2005) belonging to

WNV lineage I. Our work has been performed with the WNV belonging to either type II lineage

(WN 956 D117) Uganda strain, type I lineage NY 99, the vaccine strain FYV 17D and two virulent DENV-2 strains, Thailand strain 16681 (clone pD2/IC-30P-A ) ((Kinney et al., 1997)), and Martinique strain ((Tolou et al., 2000)). Lastly, TLR3 was either transiently or stably expressed in our experimental design. We relied on ectopic expression of TLR3 because, in our first set of poly(I:C) stimulation of endogenously expressed TLR3 in HeLa, we failed to detect mRNA synthesis from natural promoters of IFNß and ISG56. Co-transfection of TLR3 allowed easy measurement of the activity of these promoters by real time PCR suggesting that TLR3 endogenous expression was a limiting factor. Alternatively, the feeble increase in promoter activity could be due to competition for limited amounts of IRF3 by both TLR3 signaling and

STING/MITA signaling of incoming NS1 and control DNA expression vectors. As a result the ratio between TLR3 and NS1 proteins in our study and that of Wilson and collaborators may be different.

NS1 inhibition of TLR3 signaling would make sense if it provides an evolutionary advantage in viral replication in the host resulting in enhanced transmission. High transmission efficiency to mosquito is likely to depend on high viral load in the blood of the host. However,

WNV infection of TLR3 deficient (TLR3-/-) mice show only modest increase of viral loads in the blood and lymph nodes compared to those in wild type (WT) mice (Daffis et al., 2007); (Wang et al., 2004), suggesting that TLR3 does not efficiently control viral replication in cells responsible for virus shedding in the blood. Consistent with this, viral replication in primary fibroblasts, and myeloid cells were similar in TLR3 deficient and WT cells (Daffis et al., 2008). Moreover, IFN /ß synthesis in sera and draining lymph nodes and from in vitro infected cells showed little differences (Daffis et al., 2008) respective to the presence of TLR3. Lastly, WNV replicates similarly in mouse embryo fibroblast proficient or deficient for the TLR3 adaptor TRIF

(Fredericksen and Gale, 2006) demonstrating that the TLR3 pathway does not control WNV

 



/-/

replication, at least in fibroblasts and myeloid cells, although this may not be true in some brain cells (Daffis et al., 2008). The lack of control of WNV replication by TLR3 may reflect an absence of sensing of WNV dsRNA by TLR3. In agreement with these observations, we demonstrate that when IPS-1/VISA signal transmission was strongly inhibited by siRNA treatment, flavivirus infections led to very low level of IFNß synthesis, suggesting that the helicases pathway was the main innate control of flavivirus infection. In agreement with these observations, others had shown that cells in which the IPS-1 adaptor for both RIG-I and MDA5 pathway was cleaved by the HCV protease were unable to relocate IRF3 toward the nucleus following WNV infection, demonstrating that TLR3 alone did not detected WNV infection and initiated innate immune responses (Fredericksen et al., 2008). Similarly, dengue 2 virus does not appear to elicit TLR3 signal transduction in mouse embryo fibroblasts which have been depleted of both RIG-I and MDA5 or of the IPS-1 adaptor of cytosolic helicases (Loo et al.,

2008), mimicking the situation of WNV. From an evolutionary point of view it is therefore difficult to imagine that flaviviruses would have evolved and selected NS1 variants that inhibit signaling of a cellular sensor, which does not recognize its dsRNA intermediates as foreign. In conclusion flavivirus NS1 proteins do not appear to inhibit TLR3 signal transduction.

 



/--

Supplementary material

Primers used in quantitative RT-PCR were as follows:

IFNß forward CAGCAATTTTCAGTGTCAGAAGCT , IFNß reverse TCATCCTGTCCTTGAGGCAGT,

ISG56 forward GAAACACCTGAAAGGCCAGA , ISG56 reverse TCCTCACATTTGCTTGGTTG ;

Viperin forward CCTGCTTGGTGCCTGAATCT ; Viperin reverse GCGCATATATTCATCCAGAATAAGG;

Actin forward ATCATTGCTCCTCCTGAGCG, Actin reverse CAGCGAGGCCAGGATGG,

Actin probe Vic-AGTACTCCGTGTGGATCGGCGGC-TAMRA

West Nile-UG forward GCCACGCTTCAGGCAATATC ; WNV-UG reverse : CCATCCTCCCCAGAAGCAC

WNV-UG probe FAM-TCCCACTCCGTCAACATGACAAGCCA-TAMRA

WNV NY99 forward CAGACCACGCTACGGCG , WNV-NY99 reverse CTAGGGCCGCGTGGG ,

WNV-NY99 probe FAM-TCTGCGGAGAGTGCAGTCTGCGAT-TAMRA

YFV forward TCCACTCATGAAATGTACTACGTGTCT ; YFV reverse GGAGGCGGGATGTTTGGT;

DENV-2 forward TGGACCGACAAAGACAGATTCTT , DENV-2 reverse CGYCCYTGCAGCATTCCAA

DENV-2 probe FAM-CGCGAGAGAAACCGCGTGTCRACTGT-TAMRA .

VISA forward GGCAGGGACTGTGAAATGTT, VISA reverse CCTCCAGTTAGTCCGCTCTG ;

 



/-F

Legends to supplementary figures  FigS1: Steady state level of expression of various NS1 constructs A: HEK 293 cells: Western blot analysis with anti-Flag M2 and anti-actin antibodies of lysates non transfected cells (lane 1 and 6), of HEK_T3-NS1F, stably expressing Flag M2 tagged NS1 from WNV-UG (lane 2) and of transiently transfected HEK293 cells expressing Flag M2 tagged NS1 from WNV-UG (lane 3), WNV-NY (lane 4), YFV (lane 5) and DV-2 (Martinique strain) lane 7. Lower panel is a longer exposure of the blot presented in the upper panel. B: Western blot analysis with anti-HA and anti-actin antibodies of lysate from HeLa cells (lane 4) together with lysates from HeLa_NS1 cells stably expressing HA-tagged NS1 from WNV-UG (lane1) and HeLa cells transiently transfect with HA-tagged NS1 from WNV-UG at day 2 (lane 2) and day 3 (lane 3). Please note that, depending on cell types, the NS1 protein appears either as a single band (in HeLa) or as a doublet (HEK293 and derivatives) and that the stably expressing NS1 from HEK_T3-NS1F exhibited a modified banding pattern from the transiently expressed protein. We don’t know whether this relate to different glycosylation patterns of NS1 in different cell lines, and whether the stable expression of NS1 in HEK 293 lead to modification of the trafficking of the protein trough the vesicular compartment. Nevertheless, stably expressed NS1 in HEK 293 was still exported into cell supernatant (data not shown) and fully functional for trans complementation of NS1 defective WNV-UG (Fig 4A).

 



/-C

FigS2: NS1 stably expressing HEK_T3-NS1F and control HEK_T3-vect were either untreated or treated with 100 µg/ml of poly (I:C) during 7 hours. Intracellular RNA were purified and the levels of ISG56 mRNA were quantified by real time comparative RT-PCR using actin mRNA as a normalizer, and untreated cells as calibrator. Results are the mean and standard deviation from two experiments.

 



/-D

Acknowledgments We want to thank Ernest A. Gould for critical reading of the manuscript and invaluable help in English writing. We want to thank Eric Ghigo and Lionel Pretat for invaluable help in confocal immunofluorescence microscopy experiments. This work was supported by Université de la Méditerranée and Institut de Recherche pour le Développement.

References

   $%;
>;
% !;
>
>;
 !6;
>;
%B;
>
>;
%& % ;
>;
!%$$%;
>
>;
!%;
>;

4;
>;
7%!&% ;
0>;
$
"$;
>
M-..DN>

%$
2 %"
2
$ !
(%!
 %!!%
% 
/

$ 5 $
&5
 5
$
!"!


$  "
" 
2 %
(%
2(5>
32 
HI M/HN 3
//C.F //>
$%,(;
0>;
$;
L>
>;
1 ! ;
>
>;
% ;
>
>;
 B;
>;
7 $& !%;
>;
$
$;
>
>
M-..CN>


% 
2
%"56 (%!
&$

$!&

;
"$D;
$
&

( 
2

&
% " %>
 - 
 4

979 MC9N 3
/H-IC9>
4 ;
L>
0>;
L ;
0>
>;
$
;
>
>
M-..-N>
3 5
%"(


2 %
!&  "
$
 "


(%!

% >
32 
H= M-CN 3
/F..//C>
4%B";
>
>;
 %;
>;
3 &;
L>;
4!%;
4>;
  ;
3>
>;
$
L";
1>
>
M-..HN>

% 
&E


""&%
% 
9F4
$
F;
H;
$
9

%!
2 %

  >
3  
9HH M-N 3
-IDHD>
4% ;
>
>
M-..-N>

" !%
&  5
2
G

%!

E
($%
2

E%
"%>

!5   
:=3
FH/C.->
! ;
L>
>;
@EB;
>
L>;
5&BB;
7>;
(;
>
>;
E$;
>
>;
%" ;
>
3>;
B ;
0>
>;
$
" $;
>
>
M-..IN>
G

(%!
 %!!%
% 
/
&
 ""
( 
&5
&$

% ! %5
% 
2 %
3>
  - 
 4

97% MD.N 3
/9///I>
! ;
L>
>;
5&BB;
7>
>;
 " ;
4>
>;
 ;
>
0>;
%%;
>;
%" ;
>
3>;
$
" $;
>
>
M-..I&N>
& $
 
G

%!
 %!!%
% 
/
%(

2 
% ! 

""
% %$$
$
$$
"">
32 
G7 MFN 3
/FC.D/>
22;
>;
"!;
>
>;
L%;
4>
>;
7;
>;
0%>;
$
" $;
>
>
M-..HN>
2
F
% 
% 
 
G

(%!
2 
&5
%2% $$
$
 $$
"">
   
%MHN 3
/.I>
22;
>;
"!;
>
>;
!%;
>
>;
7;
>;
0%>;
$
" $;
>
>
M-..+N>
B
% %
F


% (
% 
 
G

(%!
2 >
32 
G8 M-/N 3
/.FC9 D+>
 ;
>;
G!;
>;
!;
>;
%;
>;
7!@";
3>;
1 ;
\>;
;
>
4>;
$
 ;
>
1>
M-..DN>
! $
$
"
$
 

G

(%!
%
6&
%
% "

"%>

3+ 5.
H% MCN 3
+.FH>

 



/-I

;
G>
>;
$
 ;
>
G>
M/99.N>
"&%
  
$
% 
2

0

(%!
/
% 
2% "

6%
/
  >
2  
9HH M-N 3
CH.I>
%;
>
>;
$
B;
0>
>
M-..9N>

 %($
%$$
!$ B 


-  $

2 3
=3
/C>
"$;
>;
!&;
>;
$
7%%$;
>
M/99-N>
6% 
$
% 
2
0

(%!
 %!!%
% 
/
&5


!

% "&
&! (%!>
2  
9I9 M-N 3
+-IFI>
"$;
>;
 %;
>;
!;
>;
!;
0>;
5;
>
>;
$
!&;
>
M/999N>
 !
(%!
5
/
 %!!%
5 % 
/

%$
2% "
"""



 !&
6"%


5 5 $$
2 >
32 
H% MHN 3
I/.C/.>
%$%B;
4>
>;
$
7;
>;
0%>
M-..IN>
G

(%!
($
( 
2
%2% 
% ! %5
2 %
F
% ! 
7$$
$
$$
E5
E !
 @
 
$2
  >
32 
G7 MIN 3
-9/F-F>
%$%B;
4>
>;
L%;
4>
>;
 %B;
0>;
L@;
>
7>;
$
7;
>;
0%>
M-..+N>
&"
$
"
2


(%
% 

G

%!
( (
& 
7
$
D
 
% ! 
/>
32 
G8 M-N 3
I.9/I>
7%!;
>
>;
!;
>
>;
 (;
>
7>;
$
B(;
>
>
M/99.N>
` !
2 %"
2

/
% 


"
 " 
2

 (% 
MO !&ON
 
2

B& %

(%!a>
2 2 
%: MIN 3
CH/C>
7! ;
0>;
%;
L>
>;
$
;
7>
>
M-...N>
$! 
2

!"
% 
DI
&5
%2% ;
$ !&%$$
 ;
%
(%!
2 >
2  
8=H M-N 3
-.9/9>
BE;
3>;
$
4%&%;
7>
>
M-..+N>
 7


$ "
%!!"
$ %

2

""!
  >
- 
B:: MH-/FN 3
IHC+>
BE;
3>;
;
\>;
$
4%&%;
7>
>
M-..9N>
 7
% !
%!%
 "$$;
5

%2% $$

""!5>
- 
B=9 MH-IDN 3
H++9->
LE;
>;
B;
L>;
 ;
>;
 &;
>;
L!"%;
3>;
L ;
3>;
;
L>
0>;
B!;
>;
$
B%;
>
M-..DN>
/;

$ %
% %
7
$
$D"$$
5

%2% 
$! >
- ) 
=M/.N 3
9+/+>
L% "5B;
>
>;
$B;
>
>;
$
GE5;
>
7>
M-...N>

$
%
"

2((%!

% >
32 
HB MHN 3
F-DFIF>
L";
1>
>;
4%B";
>
>;

>;
$
  ;
3>
>
M-..+N>
9F4/
$(%
! $
 B
% %

$ 5  ">
-  >
L5;
>
>;
4!%;
>;
 ;
7>
0>;
!5;
L>
>;
 % ;
0>
>;
4"%%(;
>;
$
7!&%;
>
0>
M/99HN>
 %! 
2
2 !
 
 
2 %
$ !
-
(%!
%
/II+/
$

!$
(
$%((;
%
 LDF>
2  
8%7 M-N 3
F.. +>
 %$;
0>
>;
7%$ ;
>;
B;
0>;
4;
0>
L>;
% !;
>
3>;
(;
>
>;
$
 ;
>
>
M-..+N>

F
 
 "6
2 %"
&5
 %(
% %
$"%@ >
 - 
 4

97: M/N 3
-D+IF>
;
L>;
 5;
>;
%% ;
0>
>;
B"!%;
>;
%% ;
>
>;
B$;
>;
5;
>
>;
7;
>;
0%>;
$
" ;
>
>
M-..DN>
""!
( 
&5


(%!
FKC
%  "$$
( 
2

B
% %
F
$ %
% 
>
 - 
 4 

978 M+N 3
-99-H>
$&;
4>
>;
$
;
>
>
M/99HN>
% "" 
2
5 E
2(%
(%!
/
%(

% 

%5

% >
32 
H9 M/-N 3
9I.+/H>
$&;
4>
>;
$
;
>
>
M/999N>
7
% 
2
2((%!
 %!!%
% 
/
$
C


$%"
2
%
2! >
32 
H% MIN 3
CI//-/>

 



/-H

!;
>;
4  ;
>;
G ;
1>;
 %$;
0>
>;
 ;
0>;
 ;
>
>;
$
(;
>
>
M-..+N>
%!!%
&
2
 B
% %
F
 
E
$ !&%$$
 >
  
%87 MD+HCN 3
FH9+/>
;
1>
>;
 %B;
0>;
% ;
>;
4,E;
7>;
%E%;
>;
%@ &%$ ;
>;
B%;
>;
7;
>
>;
7%%;
>;
L@;
>
7>;
$
7;
>;
0%>
M-..+N>

7
$

D
 
&5

(%!


""!5>
32 
G8 M/N 3
FFDCD>
$ $;
0>;
%5;
0>;
3;
>
>;
G";
4>;
 ;
7>;
 B;
>
>;
0&$ ;
>;
%B;
>;
;
>
4>;
4%;
>;
$
B;
G>
>
M-..DN>
/
% 
% 
$!%

!

2
G

(%!
2 >
32 
HI M--N 3
/F9-CFF>
!"  ;
>;
LBBE;
>;
L ;
>;
5B;
L>;
$
5;
>
M-..-N>
&"
2

"   
& $5
 
!"
B
% %
F

& B
$ !&%$$
 "$$
  >
   ! 
8I% MDN 3
/FIC9>
;
>
4>;
3@";
>;
 B;
>;
%;
>
>;
G;
>
>;
 !E;
>
>;
4(;
4>
0>;
! ;
0>;
!B;
>;
B;
0>
>;
3;
>;
$
L% "5B;
>
>
M-./.N>
/_
2
2((%!


0

(%!
% % !


% $!
2
%&  "
2%"2
$
5

% 

(%
!% ((>
32 
GB MFN 3
/IC/H>
5;
>;
$
 ;
0>
M-..IN>
B
% %
$


E
%
E5

% %
(%
% >
5 
88 MDN 3
DI/9>
5;
>;
L,;
>;
E;
>;
$
2% ;
>
>
M-..DN>
F
$
H
%
% $


"
%!%
 "%"
&5
$
% ! %5
">
3   
8G7 MCCN 3
FH/.H/H>
%%5;
>
>;
%E $;
>
>;
$;
>
>;
4$;
>
>;
$
%;
>
M-..9N>
%$2 "$$
 
 % 



% 

$ !
(%!

(( >
3 2 
G% M/IN 3
+-HI+/>
% ;
>
>;
" $;
>
>;
"$;
>
>;
;
>
>;
(;
>
G>;
"!;
>
>;
3;
>
>;
!22%;
4>
>;
$
";
>
G>
M-..DN>

2 !
G

(%!

6%

7
% %%
>
2  
%%B M/N 3
-+C.>
%5 %;
>
0>;
$
G% ;
>
0>
M/99FN>

22
2
$%$
"! 


$"%@ 
$
% 
2

/
% 
2$
&5
$ !
(%!>
2  
9IB M-N 3
HI9+.>
;
>
>;
;
>
>;
$$5;
>
>;
;
>
0>;
;
>
>;
$
%!;
0>
3>
M/9+DN>
! $

  
88I MCH/DN 3
H-IFF>
 ;
>
>;
\ ;
8>;
_ ;
>
L>;
$
 ;
>
G>
M-..DN>
$ 
2
G

(%!
% 



!!%
$
!
2
% &%


% &
(%
 >
2  
%%9 M-N 3
CDHH.>
 ;
>;
$
7;
>;
0%>
M-..HN>
%
2
%!%
(%
%  >
    ) 
9I M/N 3
/H-F>
,!;
>
>;
 ;
>
>;
;
>
G>;
;
>;
%5;
>;
 ;
>;
L "!;
>;
B;
L>;
($;
0>
>;
G 22;
>;
$
7%;
>
>
M-..HN>
B
% %
 

"%  
B

!  5
E5

 5 >
- 
B:7 MH/HFN 3
/-DFH>
 ;
>;
$
 ;
>
G>
M-..DN>
G

(%!
% 
%2%
E
& 
 5MN $!$
%2% 

%% 
$
% 

%2% 
%">
2  
%B8 M/N 3
HH+H>
!@;
>;
& $;
>
>;
;
>;
!$;
>
>;
 ;
>
>;
6  ! !;
>;
@!";
1>
>;
(;
>
>;
,% ";
>;
$


 !;
>
M-..DN>
B
% %
F
% " 
% %"

(%!2$
>
- 
B%% MH.-+N 3
++H9->

 



/-+

(%;
>;
 ;
>
>;
$
@ %$;
L>
>
M-..IN>
B
% %$$
$
 $$
(%

6% 
$
 !%% ! 
&5
 &$
2 % /K4  />
3  
8G9 MFIN 3
-I/++9D>
!"%;
>;
0%>;
;
1>
>;
 5;
>;
;
L>;
1 5";
>;
!,;
>;
" ;
>
>;
$
7;
>;
0%>
M-..DN>
 !
%!%
(%
$2
$
%"(



(%!

% 
% ! 

!%

;
7>
32 
HI MDN 3
-I+999>
 !;
3>;
 !%%;
>;
%%;
>;
 ;
>
>;
$
"&%;
>;
$
 ;
>;
$
!%$;
0>
>
M-...N>
 "
 "

   ! 
8HH M/N 3
+99->
;
1>
>;
 ;
>
1>;
;
>
>;
$
L ;
>
>
M-..9N>
3!"
F
% @
$ !
(%!
$
" $!
(%
% 

(% >
5   
99 MCN 3
I.C/D>
%"";
>;
;
0>;
$
45%;
>
M-..+N>

2
(%
2 
$
( 
2

""!5
&5
 B
% %
F>
 5   !
89 M/N 3
/F-D>
G ;
>;
E;
>;
6  ! !;
>;
$% ;
0>
>;
B% ;
>;
$
(;
>
>
M-..CN>
 B
% %
F
"$
G

(%!
%5


&%
!

>
- 5
97 M/-N 3
/FIIHF>
G ;
0>
>;
$
 ;
>
>;
 ;
>
>;
$
 ;
>
M-..+N>
G

(%!
 %!!%
% 
/
&
F
 
%$! >
32 
G8 M/HN 3
+-I-H/>
GB%;
7>;
6E;
>
>;
!""%;
>;
$
 %;
>
M/9+9N>
E5
5@$
$ !-
(%!
 %!!%
% 
/


 !&
% 
&!
& "
%5
5$%  &
$
""&% $
2%
$"%@ >
2  
9H9 M/N 3
F.-D>
GB%;
7>;
$ ;
>
4>;
(;
7>
>;
5;
>
>;
$
 %;
>
M/9++N>
($


"!%
2 %"
2

2((%!
 %!!%
% 
/


$"%>
2  
9=8 M/N 3
/+H9I>
1 5";
>;
LB!;
>;
!BE;
>;
 &!;
>;
"@!";
>;
5 ;
>;
%;
L>;
B%;
>;
$
!,;
>
M-..CN>



7



2! 

$ !&%$$
 $!$

(%
% >
- ) 
:MHN 3
HF.H>
1 ! ;
>
>;
3$;
>
>;
45;
>;
$
3 %;
G>
M-...N>

 
!%
@5" B$
""!  %&
5
%(
 
(
2

$ !
(%!
% 
/


%
2
2$
>
3 5   
%G MFN 3
/.DFH>
\  ;
4>;
1 ;
1>;
;
>;
G ;
1>
1>;
;
1>;
 ;
>;
;
>;
3;
>;
\ ;
>;
;
>;
$
!;
3>
4>
M-..+N>

$ %
% 

B
(%!
% %

F
%% 
2 %
( >
)  
8I MCN 3
DF+D.>

 



/-9


 


(

 "&%
% 
$R2 

2((%!
$
%

" $;

$ ( "
$
" 5
 %!


$ 
$(!
%" %$
$
 "%$%
R"&
$
" "
"


%

%& (%!
 !%
&%
!
2 
22
@
R

;
2
$R$2%
$
&
 

(!
$R!
% 
(%>

 !
( 
$ 
!$

 %"!%
(%
"


%

2((%!
 !%
 %%
R 
$

%  
%2 % 
$
R>
!
(!
$R !$
%  $;
 !
 !
 ""
 % 

R 
$

%  
(%
/
!%

%  
%2 % 
$!
% 
"! 
$!
% !%
F>

 !
R( 
$ 
!!
22
$
/
!%

F


  

!
(!
  

% 


%  
2((%
/
"&;

22;

, !%
!!
%
!%

% $! 
$R%2 % 

$

  
% 
  "
$!
F>


% !
 %
(
R !$
%  $
$
6    
"
!(
R6


!%
$
6 %
" 
!%(


22! 
$
$
!
6%"
&"
!
%  
G
>
%



$ " %


  
!%
" 5 
%

6    !%%
% %
$
" $2 
$

  
F;


 !
( 
" % ;
$

%(;

 ""
$ " %
%
$(%

 



/F.



 "&%!
%  
 
%!!%
$
2((%!


%   



 !
 !%% 
%


R!%
!$
"&
$ 
 %
 !%
$ 2%

%  
(%

!%
 ""!

 !

2((%!;

 

4 


   


40 

  


 




 /    * *


44 7 3

 
7 

 
/F-

!
 !%
$

%(;
 !
( 
& %$
$!6

$
R2 

2((%!

!

$ "  



R!
%;
 !
( 
" %


4 (
R


%
(
!
$
" %
$

$ !

$

2 (%
,!
M!
$
D.
...

$
$ !

-..9N>


% 2
$ "  
!
$
$  
 
$ &
!%
R $ "  
$

$ !

4 (


!(
%
$ $!
$
$  
$ "  

" $
$
%" 
$!
(%!;

% $! 
$
 !(!6
(%
 
$22 %

$ $
$
" $
$
(
$

 ! >


$ 
" %
$
% %%

" $
$
%! 
$

$ !

4 (>

%(%
R5
$
R $ "  
"  !%
$

$ !;
 !
( 
!$

%! 
$

5
$!%
$6
>

/99+

-..+;
 !
( 
!
&%(%
!
%! 
 !!
$
% 
 % 5
$
$ !
M /;
-;
FN
(
R "% 
$
 !(!6
(%


 % 5
C
R


%% !(

4 (
$

$6
 
$R !$
&

 !
( 
!
" %%
!
 %! 
$
% 
 % 5

""
 
M-..FN



$!% 
$
%! 
$
""
 !

@
 

!;

$ !
"&
!(%
!
5
 %
(
"% 
$
;
", %%"
$
 ("&%

,!>
(
R&
$
$  
% % % 

4 (;

!$
%
 ""
!
&
$ "  
 %
%(
R
"
 " 
$R!
%2%
  

22;

5

!%(
 "
$ $
$
R6 %!%
 !%
 
$  
"  !%>
7%

 %
 %&! 
(
R!
$
%
 !%

( "

 
7 

 
/FF

M N;


!, !%$R!
$(!
!  "
 !%

$  


5 
"  !%
$

$ !;
(

$ ( "
$!
%
 
$
 2 %
!
 >

"%
$
$22 %



(

!
$
$  
!%

2 (%
,!

4 (;
 !
( 
"
!
!$
"%
2
$
"!6
 %
R $ "  
"  !%
$

(%!
$

5>
 !
( 
%(

 & % 
(

 
$
4 (

 %
!$
R
$!
!%
(% 
$6



%  $
!
%$
" 
$
( 
 %

2 (%
,!
M-..HN
M@@    ;
-..9N>

 !
( 
"

($
!
%! 
 !!
$!
(%!
1


 !
( 
$2
$22 %
(%
2 %"
$!6
% !
$

R %!%
$!
 5
!$ " %


 !%

%" %
2 ;
$
(%
%
!
 5
!$ " %
>

&%( 
 

%!(
$R!
"!(
%  
$

 ! 
& (
 %

2 (%
,!
$
R !$>

" 
$
( 
" 
%

!;

 
 %
R $ "  
$

"$
$

5>
 %
%(
 !%%
$ 
%(%
$
% 2 %
 %
$R!
!$
 "%(
(K% 

( 
!
 !%
$

!
 !%
$
 %
!$;
 !
R( 
!
"%

($
!
%" 
!%&
$

2 (%
,!

 !
  % ;


, !%;



4 (

$ 
 !

"
$R!
!%& 
$

2 (%
,!



% ( 

$

2 (%
,!
!%&

%
R $! 
%;

$%! 
$

%(%;

!
 %

  



$%%
$ 
%
" $!

2  
$
@ 
$ "  
!2 ;

"
$!
(%!
$
!
%
$


 
7 

 
/FC

@ 
$ "

  " 
2%
 %
!
"2 
(%
 ;
%
!
(!
%$ "%% 
$
R @ 
$ 
R
%
% 
!6
!6
$R""!
$

!( ;

 >
 !
 !%% 
5%

%" 
( %
$!
(%!>

 !%%
 !
%%




 %
5

%"
!
!(
$ "  
R !$
$
 !
(%
%!
$
2 (%
,!
 !

2
%$%
 
$

(%&
$!
(%!>
R6"
% 2 $
$
 "
(%!6
%"%
$R"  %%



22;
!
($
",!%
$

 
$

"$
% $
$

2 
$
%%

%%


;
(
$
( "
$ 2( %&
 %!6>

!;

 "%  
$
%
$
R ( ! 
(%
%"%
$
%( %
R2!
$
" $2 
$
R(% "
!%

%   
$!
(%!
1;







$!6
!$
%

!
"!%
 "%  
$
R $ "  
"  !%
$

$ !

$

2 (%
,!

4 (>

" %

 
7 

 
/FD



!
 $
%(;
 !
 !
 ""
 % 

R""!
 ;
!
%  "

%  
%2 % ;
"


%
R
2

!
2 
2((%;


% 
$ (  
%

(%!
 !%
5
%>

 !
( 
!$
R22
$

%  
(%
/
$
% 
2((%!
M ;
1;
GN
!%

%  
%2 % 
$!
% 
"! 
$!
% !%
!%
F>
 %
5
R
$ 
!!
(
(%
$
/
!%

5 
$R%2 % 
F$ $

$
7
% $!>

R""!
 


%" %
%  
$
R
"


2

!
2 >
$;

(%!

$ ( 
$
% 
 !%
5
%>


22;

(%!
 
$
%


!(

% 

 & 
%

(%!

R
""!  " 

$ 

% !
$R!


 6
" 
!
% 
!%
R ( ! 
$
R

$!
(%!
M %
R!
 ;
R

$ ( 
!
5 "
""!%
&
$
 %%

(%!
$


 
(%



%

2&
2$ 
$
!%
 5" %;

%  
$
(%!

;
 ""

2((%!;
 $%
$
 "&%!
%%!%>
%
$

"! 

!
!
%
   
!
 !%
$
R ( ! 
 !%
R( 


R$2 
$
 !(
% 
(%
$R ( 
2

R""!
$
R

R5
$
!%
2  
$
R2 
(%
%"%
$
"!6
 "%$%

5 "
""!%



2%

  
$
 !(!6
%  $ 
(%!6
2
$
%%

  
  


%& (%!>

 
7 

 
/FI


 "&%!
!$

$ 

%%
 !%
$2%

%  
% &
$

 %"!%
(%;


% %%

&
 
 !%

$ ( "
$R!
%"
(%>
!2 ;
!!%
%  
 
%!!%
$
2((%!


%   



 !
 !%% 
%


R!%
!$
"&
$ 
 %
 !%
$ 2%

%  
(%

!%
 ""!

 !

2((%!;

 

4 


   


40 

  


 


 
7 


 


 / *(

44 7 3

/F+


> 3&'M* .) 3 ';8779<'

 (
5
2

&5

$ !
(%!
 $$

 5"%
6&
"%!%
$$


 
&!
 
   
>
3  
8H= ;
F99-IF99FH>
3 ' M 1#N L#>3 ' (' ;8777<'
%
""
2
""!
( >
+  5   
G;
C/.C/+>
 ,*3 '3 !3 &' '3 43 *'3  >3 &' *'3 .3 &'3  , ) 3 '&'3&3&'3 3'3/ .3+'M 3&';877:<'

%$
2 %"
2
$ !
(%!
 %!!%
% 
/

$ 5 $
&5
 5
$
!"!


$  "
" 
2 %
(%
2(5>
32 
HI ;
//C.F//C//>
#) 3 ' '3 )" 3 ' '3 !  3 ' @' M  ! 3 '
' ;877H<'
%!!%
($
2 %
% ! 
$
25
2
2((%
% 
$
( ! 
2

((%$
2 $>
   
9= ;
H9D+.I>
 N3''3N3 ''3!3'M/ >3 '';877:<'
 
2

%!!%
%


F_ 
2
$ !
(%!

% ;

5;
$
(%
% >
2  
%%I ;
-..-/->
 N3''3!3
''M/ >3 '';877G<'
! 
5
2
$ !
(%!
5@ 

2  
%H: ;
--F-FD>
C 3 +'3
)#3 1' '3 @ 3 ' '3
#3 ' '3 !>3 '3 /. 3 ' M  3 '';877B<'


% 
2
%"56 (%!
&$

$!&

;
"$D;
$
&

( 
2

&
% " %>
 - 
 4 

979 ;
/H-IC/H-I9>
#3
''3*#)!3'M!##3+'(';9II%<'
 !
-
(%!
-4
$
F
2 %"

&
 "6


(
F
E


$ ">
2  
9I% ;
++++99>
!  3 *'3 )#3 '3 ()!3 ' '3  >3 *'3 @ 3 '3  3 &' ' M !> # 3 +' *' ;8797<'
 "
2

 ""
 " 
C
&5
2((%!
 %!!%
% 
/>
3&* 5 >
!  3*'3&))3'M 3&'';877G<'
 ""
$

% 

%  
$
 
2
2((%!
2 >
2  
8=G ;
/../.H>
!  3*'3* "3 '3 #> 3'31!3
'3 )3'3 !)    3 1'3 + #3 '3 1 "3 '3  !)L )>3 '3 *!!>) !3
'3 *!! >!#>3' '3@ )!# #3*' '3& >#"3+'31# >3?'3 !!#.!3 '3 (# 3 &'3 !! 3 &'3 # L!) 3 '3 )>3 ' M &#!3*';877=<'
!%
B 

(%
$ !
(%!
2 

 
% 
2 %

 %!!%
(%
% 
/
$
 "">
3)  %
9I% ;
/.H+/.++>
!!3 '';9IIH<'
1 E
2(%
(>
   
8: ;
/H-D>
!!3 ''M(#3';877H<'
1 E
2(%

$


5

&
 

 !&  
:8 ;
-.9--9>
!!3 ''M& !)3 '*';877%<'
$"  5
$
  5
2
5 E
2(%
(%!>

 2!
=9 ;
-9/F/D>
!3/'M* .) 3 ';8778<'
6% ;
!%2 ;
$
%%@ 
2


D_% 
(5
2
$ !
(%!
5
-
 %!!%
% 
F>
2  
8II ;
/--/F->
 3/'3 #,& N3 'M,& 3+';9I:%<'
1 E
2(%

4 (;

 %5
$
$"  5>

3+ 5.
8;
CICC+->
3'3
)##3 '3 3('3&3*'3/3' 'M.3-';8777<'
5 5
2

!
2((%!
!
 "
 $

3( 2 
G9*!I ;
-FF9>

44 7 3

/F9

# 3&'M"3/';9IIH<'
%
$
!%
@5"
( ($

5
2
"

%!!%>
2  
8%= ;
/H>
)!3 1' +'3 &1! 3 +' ' M 3
' &' ;8778<'
3 5
%"(


2 %
!&  "
$
 "


(%!

% >
32 
H= ;
/F..//F./C>
! )3'+'3  3'3)3'+'3&> N3+''M(3 ' ';9III<'
$2 
$
5
2
%!$
$
  $

2

2((%!
/
% >
2!
=7 ;
IHH9>
 C3
' ' ;877:<'
%2% 
MN
%
B5
5 B

& 

$
$(
(%
""!
% $
(%!
 !%

 >
5  ) 
H;
DI9 DH+>
"3 &' ;877G<'
3 5
 

(%
2 
 
2 %
(%
%%>

!
HG ;
/+>
 > 3&'&'3 3'3(.31'3!3'3*)3(''M13@'&';877H<'

% 
&E


""&%
% 
9F4
$
F;
H;
$
9

%!
2 %

  >
3  
9HH ;
-ID-HD>
 ! 3&' ';8778<'

" !%
&  5
2
G

%!

E
($%
2

E%
"%>

!5   
:= ;
FH/C.->
" !3+'3? 3('3
.N#3
'3 N3/'3?!!#3'3 ##31'M!!3 ';877%<'
@ 
%" 
2
5 E
2(%
(%!

%!>
&)  %
I;
9-I9FF>
" !3+''M!!3 '';877%<'
 "%(
5 
2
5 E
2(%
 
2% "
%!
$
4%@>
'&5) 55   
%I ;
/.F//+>
" !3+''3# #3*''3 C . 3 '
'3&!.3+'*'3(#3'M!!3 '' ;877:<'
@
% 5


F_
  $
%  
2
 !
"%
 
2
5 E
2(%
(%!>
32 
HI ;
F+.HF+-/>

.3/''3&$ 3 ' '3 !!3 ''M /.3 ' +' ;8778<'
G

(%!>
  )  %
8;
D/9D-9>

) 3@''3
) 3 '('33
''M 3@'' ;877G<'

!%
(%
% 
(%

!$
&5
%  ""$$
% 
$ %$ 

0

(%!2$
>
32 
G8 ;
/.CDD/.CIC>

) 3('('3)"3(''3? 3@'&'3 3''3)"3 '('3  3''M( 3@'' ;8778<'
 
2

$ 
&5
"" &@$
$ !
(%
 %!!%
% 
/
M/N
%  5%
$
%!!%
""%5
E

$ !
(%
/
 >
3)  %
9G= ;
HCFHD/>

) 3 @'3 1 3 ' +' M 1## 3 ' &' ;877B<'
, %
  "&5
 "6


M 3N
$! 
&5
G

(%!
( ("
2
-
 
E5

3
!% ! >
3)  %
9GI ;
ID+II+>

)  3 ''3)" 3' '3 #) 3 ''3 ! > 3''3!)3+'?'3 ! 3 '
'M!  3 '@';877G<'

E  " 
-4F
% 
%%

!E$
(5
2

G

(%!
F
>
3  
8G% ;
/H-H./H-H+>

)43 ';877%<'
(%(E
2
%& (%!
$
%& (% >
5       ;
FHHF+C>

) 31'&'3#NL#>3&'1'3 ".>> 3/'3 #3 ''3 L # 3 ' '3 !3' ('3 !> # 3+'*'M 3&'';877=<'
G

(%!
 %!!%
% 
/
&
 ""
( 
&5
&$

% ! %5
% 
2 %
3>
 - 
 4

97% ;
/9////9//I>

) 31'&'3 ".>> 3/''3 )# 3''3 3&'+'3&3 '3 !3'(' M 3&'';877=.<'
& $
 
G

%!
 %!!%
% 
/
%(

2 
% ! 

""
% %$$
$
$$
"">
32 
G7 ;
/FC./FD/>

44 7 3

/C.


) 3'3
##3 '3!3&' '3/N 3&'/'33&'3 # 3'M.3' ;9II:<'
!%
$"  5
2
$ !/
$
$ !C
(%!>
3( 2 
H=;*! H< ;
/+HH/++C>

3 '33 '+'3-30'33 '1'3
) 3@'
'M3+';9IIG<'
 "&
$ !
(%!
5
/
F
% 
6&
2
(%

&$
$
 
(5
% !$
&5

D
% >
2  
8B= ;
C.9C/H>

 #31''3? 3' '3 >3 'M() 3@'';877H<'
 
2
 ""

""!
% ! 
$

6  
&5
(%!>
2) 
87 ;
D.DD-C>
$$#3'33&' '313'
'3/3&'3+'M 3&'';877H<'
2
F
% 
% 
 
G

(%!
2 
&5
%2% $$
$
 $$
"">
   
%;
/.I>
$$#3'33&' '3!)3&''3/3&'3+'M 3&'';877G<'
B
% %
F


% (
% 
 
G

(%!
2 >
3 2 
G8 ;
/.FC9 /.FD+>
3
'''313/'M/N 3&'/';877H<'


%B
2 %
2 %
$ !
" %% 
2(%>

 2 
9:8 ;
DFFDC->
#43 '3
,3 ' &'3 )3
' M  .3 -' ;877I<'
 !
/
$(%5
$
"% ( ! ;
%
 5
-../-..I
  
E
$"  5
$
>
 -+ %
%;
C9F>
.3 ' M /#,
.!3 &' ;8778<'

%& (%!


(%!
@ 
!   
%8: ;
+DD+I/>
 3&'';877%<'
( 
2

$
$(
""!5
&5
2((%!>
)   
G9 ;
/9I-.I>
 3&'';877I<'
"
2
( 
2

5

%2% 
(%
% 
&5
2((%!>
3)   7 !
8I ;
D-/DF.>
 3&''33'3 .!#3 '/'33'M(#3';8777<'
2 
2
!"

&5
$ !
(%!

" $!$
&5
$22%

5
$
(%
%>
32 
HB ;
H+/CH+-F>
 3 &' '3 *# 3 '
' M  !3 ' (' ;877G<'

%!!%
""!  5
2
& $5
%  
 
G

(%!>
) !
88: ;
-/---D>
 3 &' '3 )#!)3 '3 &))3 '3 !!3 ' M  3 &' ;877%<'

$
$(
""!
% 
$%"
%  
 
$"$
2 
&5
G


(%!>
2) 
9= ;
-D9-H+>
 3-'3?3-'3 3 '3 3&'3/N 3('3@ 3O'3 #)3 ''M-3'@' ;877:<'
! $
$
"
$
 

G

(%!
%
6&
%
% "

"%>

3+ 5.
H% ;
+.F+.H>
#!!3
'3&" 3'M #)3+';877G<'
%!%
%%  
% %>

 %%!
=7 ;
+F.+C.>
 >.3+' 'M 3?'
';8797<'
 ""
$

% 


$
$(
""!
% >
!
87 ;
FCD.>
$$3&'*'3 )3'3#>3'3 !!3+''M
 3';8778<'



M! $-_N"5%2%


2((%!

 5"%
D
%5
%!!%
$
2! 
%%@ >
&5 3
89 ;
-HDH-HI+>
.3*'3) 3 '3P "3 '3  !#3&'313&'33'*'3@ 3O'313 '('3#  3'/'M(3';877=<'
%!!%
&
2 %

( 
2
2((%
F
% 
2% "
$ !
$
G

(%!>
-   5   
9% ;
FH- FHF>
  #3+''M3
';877H<'
22%
22
2
"! 

C4

G

(%!
% 
$
& 
2
%2% 
  >
32 
G9 ;
//+.9//+/I>

44 7 3

/C/

!3'3
) >3 'M3
'+';9IGI<'
% %
% 
2
$ !
(%!
 %!!%
5 % 
/
%
32 
=% ;
/+D-/+I.>
!3'3*!)3&'3O) 3@'&'M3
'+';9II9<'
4 
 %!!%
% 
-4
$
F
%
%
32 
=: ;
-CIH-CHD>
 3 ?' ' M &# 3 *' ?' ;9II7<'
"&%
  
$
% 
2

0

(%!
/
% 
2% "

6%
/
  >
2  
9HH ;
CH.CHI>
!3&'+'3/N,/!N3+'3 "3'3  #3+'
'3*!# 3'M/  #3' ;877G<'
 


 "%@ 

(5
% 
2
G

(%!
F
 K>
2!
9%: ;
/II/HC>
  N,/3 &' '3 &NN 3 &'3 +.#3 &' M 3 ' ;877I<'
 
2
2((%!
2 
!
$
&!

 
>
.  5  
:;
F/+F-+>
3 '&'3  3'/'3#!#3&''3&3&' '3 3''3&3&'*'3
#!3
' 'M#3'*';877G<'
 !
(%!
5
C;
!;
4%@>
&)   %
9B ;
IIHII9>
!3
' '3 3 &' '3  N3 ' '3 #3 &' &'3 *!# !3 ' M / >3 '';877=<'

D_

"
% " 
$ !
(%!

5


%!%
 ">
( %
87 ;
--F+--C9>
!)3 ''M !> #3+'';877I<'

 %($
%$$
!$ B 


-  $

2 3
=;
/C>
 3 &'3 .3 ' M /3 &' ;9II8<'
6% 
$
% 
2
0

(%!
 %!!%
% 
/
&5


!

% "&
&! (%!>
2  
9I9 ;
+-I+FI>
 3&'3&!3'3&!)3&'3!3+'3 "3' 'M.3';9III<'
 !
(%!
5
/
 %!!%
5 % 
/

%$
2% "
"""



 !&
6"%


5 5 $$
2 >
32 
H% ;
I/.CI//.>
#)"3 ' &'3 &# 3 '
'3
N3
'3 )3
'3 3 '3 / 3
'3
)3''3  3 '3&3
'3 3'3 N3 '3
)3 /' M 1)3 '+';877I<'
 !
(%!
% 5
C;
 %%
%!;
-..+>
&)  %
9: ;
/+/D/+/+>
># 3''M/3&'3+';877=<'
G

(%!
($
( 
2
%2% 
% ! %5
2 %
F
% ! 
7$$
$
$$
E5
E !
 @
 
$2
  >
32 
G7 ;
-9/F-9-F>
># 3 ' '3 13 '
'3  >3 +'3 1!N3 &' /' M /3 &'3 +' ;877G<'
&"
$
"
2


(%
% 

G

%!
( (
& 
7
$
D
 
% ! 
/>
32 
G8 ;
I.9I/I>
>3' 'M3 '&';877=<'
$%$



"
2
(%!
 % >
 %
98 ;
/F/D/FF+>
/,#!3 ';8778<'
"
2
& 
2

 
%2% 
K&"$$
(%
% 
&5
(%!>
5  ) 
B;
ICHIDD>
/ !3&'?'33 ' '3.3-'3 3 '1'3N)   3 ''M/3 ' ' ;8779<'
5 
% 
2
2((%!
 %%
E
%
$"  5;
$
  
$
&  %5>
3( 2 
G8 ;
/+IH/+HI>
/. 3 '3 >3 ' M  3 ' ' ;8777<'
%2% 

  ;
""!
" $! ;
(%
% 
$
(%!
 !%"!%>
3( 2 
G9 ;
-FC/-FIC>
/. "3 ''M1  3'';9IGI<'
%
% 
 

!%
 &$

-  
!
9H ;
+C/F+CC.>

44 7 3

/C-

/3' '3.3-'3O !!3*'&'M(#3'
';877%<'
% ;
( ! ;
$
( %K 
 $ 
E

!
((%!>

2!
:I ;
-HHF/C>
/3' 'M 3 ';877G<'
 
2((%!>
 
%H9 ;
D..D.9>
/3 /'3 &3 /'3
)3 ' '3 ## 3 +' +'3 / NN3 +' *'3 / 3 *'3 /!# 3 ''3(#3'
'3/3' 'M.3 -' ;877H<'
7
%%@ 
2
B& %
2((%!
E
 

( ! ;
 
$%"
$
6  "5>
2  
%=9 ;
+.9->
/!# 3 ''3#! 3' '3)! 3 '/'M#)> )3' ';9II7<'
` !
2 %"
2

/
% 


"
 " 
2

 (% 
MO !&ON
 
2

B& %

(%!a>
2 2 
%: ;
CH/CHC>
/!# 3 ''3 !!3*' 'M/3' ';877%<'
B& %
2((%!>

2!
=9 ;
F/HFH/>
/.3'+';8779<'
3!"
%& (%!
2 
E %$E$>

-8
 
I:9 ;
/F-C>
/.3 ' +'3 1 3 /' M &>$$3' ;877H<'
((%!>
' 2  / :!) ! ;
//DC
/-D->
/.3 ' +'3  >3 '3 &!)3
'+'3  ;9IG8<'
%%  $
( %
 " $"  ;

$
&  
 $% >
                   
3!" ;
FCFFH+>
/3+'3*!#31''M 3/'
';8777<'
$! 
2

!"
% 
DI
&5
%2% ;
$ !&%$$
 ;
%
(%!
2 >
2  
8=H ;
-.9-/9>
/3+' '3("#)3+'M3
';877:<'
G

(%!
&

 
%$! 
E5
2

%2% >
32 
HI ;
/FCF/FD.>
(>31'3?)!3'M 3 '&';877I<'
B$
5

$ !
(%!
% E

"""
$

>
3( 2 
I7 ;
-.9H-/.I>
(3 '3 !!N3 ' M 1)#3 /' ;877H<'

6
7 
2"5
2
%2% $!$
(%
% >
5  ) 
I;
/IFI/ICF>
(#!3'';877H<'
 !>
 
%H7 ;
/ICC/ID->
(#!3''M+.# 3+';877%<'
0
>

2!
=9 ;
/.F/F+>
(# 3'
';877G<'
%
""&%
2! >
-   5   
9: ;
I9.I9+>
("#3''3C 3+'+'3O>3' '3 !!3 ''33 ''M
.3/'' ;877:<'
%  5;
  5;
$

"2 
2
G

(%!
$>
&)  %
99 ;
//HC//H9>
( N3'-'M # 3'';8777<'
%!!%
$
""

2((%!
2! >

2 !
:: ;
-F/-I9>
( N3'-'M # 3'';877%<'
((%!
%!!%
$
""&%
2! >

2!
:I ;
IF9H>
(3'+'3( 3''3? 3
'@'3?3?''3
)3*'3 3@''3
) 3'&'3 3 ' @'M3+'(';877:<'
 !
(%!
5
-
 @

&!
 
 ""
(%
22
(
$ E% !
5B-
 


!"
$$%
>
3 ) 
9HB ;
+/IF+/H->
(  3 '33?'31)#3/'31)">)3 ' 'M&> N3+'&';877H<'
G

(%!$!$
5 "
""&%
%!!%
% ($
%
%  
 

%2% $!$
(%
6
% >
3( 2 
GG ;
F./FF./H>
(#3'
'3 3*'('3*3'3&)3+'M
#3+';877I<'
" % 
$
  %! 
2
"!
% 5
2
$ !
(%!

%EB;
5>
2!
9B% ;
/ D>
( 31'3@ 3('3 >>3 'M  )3 ';877:<'
 ! 
2

5


$! 

!%%
(E>
) ) 
9H ;
/FIH/FH+>

44 7 3

/CF

 3 '3..)3 '3&! 3'3 !!)3'('3 3'M )3 ';877=<'
%
% 
2 %
($
(%
!%
""!
% 
2

 >
3 7   
HI ;
/IFD>
##3 '3   3+'M ! 3 '
';9I:H<'
%!
%2%>
>
 "
% %
2
%2% >
 !      
9BH ;
-I+-HF>
#)>L3 (' M .3 /' ' ;877G<'
 7


$ "
%!!"
$ %

2

""!
  >
- 
B:: ;
IHCIH+>
#)>L3('3&3O'M.3/' ';877I<'
 7
% !
%!%
 "$$;
5

%2% $$

""!5>
- 
B=9 ;
H++H9->
L#>3 'M&N)! 3 ';8797<'
 ! 
2
$(
""!5
&5


""!
5">
  
%8H ;
-9/-9D>
+.#3 &' /'3 . # 3 *' +'3 !)"3
'3 &> N3 +' &' M @ 3 *' ' ;8777<'
 !
(%!
 %!!%
% 
/

6%$


5 5 $5   B$
2 %"


&
2
 
%$! >
'
& 3
9B ;
/I.F/I/.>
+ L"3
' '3 +' ;9IGI<'
% 

5" b
( ! 
$
%( ! 

""!  5>
   . 9   
:B*!9 ;
//F>
+$$"3 ' ?'3 1!3 '3 &#3 ' '3 &!N3 '3 ) 3 &'3 )L3 &'3 (  ## 3 '/'M
 #3&'+';9II:<'
!%
 @ 
2

%2%  $!&
% 
B
L

!"

$
%2$
" !
>
&* !
89G ;
/H-H>
+) ## 3&'3>#3 '+'3+ #3' 'M#  3'/';8779<'

"
%  
2

$ !
(%! $$
 $$

 5"%;
D;
 2%
% 
E
& 

!%
% %
% %
" %&
$

(%
;
F>
3( 2 
G8 ;
HFDHCD>
+) # 3 ' ?'3
).#3 ' '3
.!3 &' '3 #3 ' &'3  )#3 *' '3 # 3 &'
'3 &# &3 ' M &3 ' ' ;8778<'
 %
 "
2
4%@
$
 5
$
>
& !
2 %

4%@
5 E
2(%
(%!
 >
+ ! + 5.
I= ;
I//I/F>
+ #3&'3 # 3 '3(..!3'3/ L3*'3)>3+'33'3#!3/' 'M +.#3&';877:<'
 !
(%!
&

%2% 
 
&5
%$!
 -
6% >
32 
HI ;
DC/CDC-.>
13/'M( 3 '';9IIH<'
%! $$

>
32 
H9 ;
-D+F-D9.>
1C#!,  !#>3 '3&#)3 '3 >3&'*'3/ !3+''3#3&'M##3 *';877=<'

-_;D_  $5
5
/&


 
& %
2
G

(%!
% 
$
2$
>
3  
8G9 ;
CI-CCIFH>
1!3('3 >)3 '3!3'3@ "3&'3@!3&'3&!#31'3!#3'3 + 3 '31L3 '3#)31'+'3@)3 '3 !#31'3 #C3 '31)3
''3 ##3
'3&!#3@'3C!3 'M >3 ' ;877=<'
22%
% 
2

D
$
7



%  
2

(%!>
- 
BB9 ;
/.//.D>
1!N3&'/'3(3@'M/3&'3+';8778<'
%!
$
%2% 

2 
2 %
!%"5>
-  !) 
8;
IHDI+H>
1L3 'M >3';877H<'

  >
 ) 
9I ;
-CF->
1L3 '3 >)#)31'3!3'3
. 3
'313('31!3('3#)31'+'3 >)3 'M >3';877:<'
/;

$ %
% %
7
$
$D"$$
5

%2% 
$! >
- ) 
=;
9+/9++>
13'
'3+) # 3
''3># 3 '1'M/3&'3+';877H<'

""!
( 
&5


(%!
$
G

(%!>
 7 ( : ' !
9G ;
DFDDCC>
1)">)3 ' '3 &>3 ('3 /"!!3 1' +' M ?#!L"3 ' /' ;8779<'

% 
2
5@ 

32 
H: ;
IH/9IH-+>

44 7 3

/CC

1)">)3 ' '3>3*''M?#!L"3'/';8777<'

$
%
"

2((%!

% >
32 
HB ;
F-DFF-IF>
13@'&'3 > 3&'&'3*A!3&''M*)3('';877G<'
9F4/
$(%
! $
 B
% %

$ 5  ">
-  >
1 "3 '&'3!!3'3
) 3/'+'3 #)"31' '3 )3+' '3) !3 'M/.3'+';9IIH<'
 %! 
2
2 !
 
 
2 %
$ !
-
(%!
%
/II+/
$

!$
(
$%((;
%
 LDF>
2  
8%7 ;
F..F.+>
1 3 ''M 3&'';877G<'
""!  
$ %
(%
""!
% 
% 
 
!% ((
G

(%!>
+ 5 5
9B ;
-+I-9C>
1C3 '3L!31'3"3 '3&!#!3&'3 3('3 !> # 3+'*'M #L3' ;9II%<'
"&%
 2 %
% 
M CIN
% 

%$ "5
2% "
%(
 ""
E5"$$
F2% "
$  
$
5 5>
3 ) 
9:9 ;
/D/9/D-H>
1  3'';9II9<'

5 5
2
 $$

 5"%
2
 (%$

(%!>
3( 2 
H8;*!I< ;
-/9H--.I>
13 ' &' M 3
' &' ;8778<'
! 


5 E
2(%
(%!
 %!!%
% 
-
(5
& B
% $! 
2
2 !
%>
32 
H= ;
CHHFCH+C>
3+'&'3
>#3 '+'M!) # 3+' ';9IGI<'

5
$
"!% 
2

  %!!%
6%!%
 
2% "
B& %

(%!
$

% 


%!%
/
% >
3( 2 
H7;*!8< ;
FFDFCF>
 3+' '3/) 3 '3 #> #3+'33+'1'3&#3' ('3 #3' 'M 3 ' &' ;877G<'

F
 
 "6
2 %"
&5
 %(
% %
$"%@ >
 - 
 4

97: ;
-D+-IF>
 3+'@'3*C 3/'*'31 ! 3 '3("3+'3&> N3+'&'M1)">)3 ' ';877G<'
 
2
 %!!%
% 
-

2((%!
"&5>
32 
G8 ;
CHF/ CHC/>
3('3/3*'3-3?'M1 > 3'';877H<'

%2% 
 
E %B
$
%% 
2 %
K4&>
5 ) 
B;
C.HC/+>
31'3"3'3 3+'
'3 >3&'3 3 '
'3>3&'3 "3'
'3/3 &'3+'M 3'&';877:<'
""!
( 
&5


(%!
FKC
%  "$$
( 
2

B
% %
F
$ %
% 
>
 - 
 4 

978 ;
-99--99H>
3'3>3'&'3@3'&'3O) 3@'31) 3 '+'3
) 3+'M ## 3&'/';877G<'

2((%!
%!% %
""&%( 
% 
 "6
%!!%
$
"!% >
  
%9I ;
/+F./+FC>
 3
''3
)3'
'3(#3@''3? 3'?'33('@'3)3 ''33(''3@)3 ' &'3
) 3'('33
'
'M 3@'';877:<'
6% 
2
5 B;
" B;
$
$ 
" !
$!%
$ 

( 
$!$
&5
& $
 
$ !
(%!
 %!!%
% 
/>
3) 
9HB ;
F9DC.F>
 3
'?'3
) 3
'?'3@ 3 '
'33'?'3
) 3&'('3? 3'3 3@'+'33
' ('3  3 ?' @' M 13 &'
' ;877G<'
%2% 
 
2! 
2
0

(%!
C
$

% 
E
 & 6


C->
2!
9%H ;
C9DD>
 3 '+'3
) 3''3@3('*'33
''M 3@'';877=<'
4 B
2
%2%  $!$
0B
 
&5
0

(%!
D
% ! 

% 
5% 
 "$$
"">
32 
G7 ;
D9.+D9/+>
 3 '+'33
''3 3'M 3@'';877B<'
4 B
2


%2% $!$
0B
 
E5
&5
0

(%!
2 >
3 2 
HG ;
9-+D 9-9C>

44 7 3

/CD

  .)3''M 3
' &';9IIH<'
% "" 
2
5 E
2(%
(%!
/
%(

% 

%5

% >
32 
H9 ;
9I.+9I/H>
  .)3 ' ' M 3
' &' ;9III<'
7
% 
2
2((%!
 %!!%
% 
/
$
C


$%"
2
%
2! >
32 
H% ;
CI//CI-/>
  .)3''M 3
'&';877%<'
!%
&  5
2
2((%!>

2!
:I ;
-FI/>
3 '3 !#3 '3 ? 3 @'3  3 +' '3 ))3 +'33 ' &' M  #3 ' ' ;877G<'
%!!%
&
2
 B
% %
F
 
E
$ !&%$$
 >
  
%87 ;
FH9F+/>
3?'+'3
) 3(''M1)">)3 ' ';877%<'
!%
$
2! 
5
2
L!,
(%!
2 !
 
 
$" %


% 
2 %
-

(%!
"&5
$
2 %

  %((
%$!

F


% >
32 
HH ;
H+.C H+/F>
3?'+'3
) 3(''3? 3-'+'3( 3('M1)">)3 ' ';877B<'
5
2
$(
"! 

L!,
(%!
% 

%(

 (
% 
2 %

2((%!
 %!!%
% 
-

& 
2
&
%2% 
% " %$%(
%% >
32 
HG ;
/---D/--FD>
3?'+'3? 3-'+'3
>3'
'3.#3&'3(3 ' 'M1)">)3 ' ';877=<'

 
"
$
!&! 


G

(%!
 %!!%
% 
-
$&

&5

&
K&
%2% 
$! 
$
!
(%!
(%!

">
32 
G7 ;
-F9I-C.C>
3?'+'3? 3-'+'3&>)  3''3)3*'@'3  3 ' M 1)">)3 ' ' ;877:<'
& 
2
%2% 
 
&5

E
1 %B
99
%
$
L!,
!&5
2
G

(%!
( (
& B 
2
 /
$
 -
( 
&5
 %!!%
% >
32 
HI ;
/9FC/9C->
.#3&'3&.)3 'M  3&';877%<'
3


!% ! 
&5
2((%!
""!
% 
E
!B E
$( 

 
%
 >
)   
G9 ;
-/H--F>
3 @' &'3  >3 +'3
)!3 '3 CL3 /'3 *L!#3 '3 &! N,.3 '3 >3 '3 /3 &' '3 /,#!3 '3 1!N3 &' /' M /3 &'3 +' ;877G<'

7
$
D
 
&5

(%!


""!5>
32 
G8 ;
FFD FCD>
3@'&'M/3&'3+';877H<'
%
% ! 
$
( 
2

 
% >
+  5   ) 
%9= ;
-9DF/F>
 N3'
'3 # 3''3( N3'-'M( #3 ';8778<'
 $
$
$"%@ 
2
B& %

(%!
( 
% 
%
$



$ "
%!!">
32 
H= ;
DC+.DC9/>
& 3+'3  "3+'3(3 ' '3?#3'3*#3/'3 #)>3&''3+.3 '3
>3'3 #)3 ''3#3 'M> 3?'';877:<'
/
% 
% 
$!%

!

2
G

(%!
2 >
32 
HI ;
/F9-C/F9FF>
&> N3+'&'31)">)3 ' '3+ #3&'1'M?#!L"3'/';9IIG<'
!&!%
 @ 
$
 "
& "
% %
2

2((%!
L!,
 %!!%
% 
-
$
C >
2  
8B: ;
-.F-/D>
&A#3+' 'M
!)L3 '?';877H<'



%"
 ( ! 
2
"
(%!
$
 

""!
% >
+ ( 
8% ;
FD9FIC>
&! 3 ' '3 1>3 *' M #!)#3 ' ' ;877I<'
 !
(%!
 

 %$
(E>
 5   !
88 ;
DICD+/>
&# 3 *' ?' ;9IGI<'
!% 
2
0

(%!
5 % 
% $!$
&5
2$
"""
$
" 
2  
9=I ;
FDCFIC>

44 7 3

/CI

&#! 3'3& 3&'3!3&'3#>3'3*" 3'3*  3'3 ! 3 /'3
 3 '3 1  3 *' '3   3 ' '3  .3 -'3
!3 '3 1)">)3 ' 'M #3&';877H<'
%5
%!!%
$
(5
2
L!,
(%!
F
T
% 
$

$ "
"&5


2!
 
F
% >
3 5   
%H8 ;
CCCCDD>
&!#!3&'31>>L3'31)#3&'3&">31'M"3 ';8778<'
&"
2

"   
& $5
 
!"
B
% %
F

& B
$ !&%$$
 "$$
  >
   ! 
8I% ;
/FIC/FI9>
&!N 3*';9IIG<'



2
$ %>
 ) 
97 ;
F99C/D>
&NN 3&'3+ #3&'3 # 3 '3
) 3'M+.#3 &' ;877I<'
 !
(%!
D
&
%2% 
 
&5
& B
 
%$!%
$
( %
2
%% 
-
  %5 >
3)  %
877 ;
/-I//-H.>
&N)! 3 ';8779<'
B
% %
$

""!5>
- !) 
9;
/FD/CD>
&N)! 3 ' M + L"3
' '3 +' ;9IIH<'

""!5

(%!
2

  
5"
2
%  >

I9 ;
-9D-9+>
&))3'3?)!."31'3 ) !3 '3&3 '3 3 &' M  3 &' ' ;877:<'
 ""
( 

%
32 
HI ;
HCIIHCHH>
&)C# 3+'3+ # 3''3&3'3 ### 3''3?.3'3L3 '/'3 &!>  3 ' M * 3 ' ' ;877:<'
( 
2

$2
 

%"56 (%!

"$$
&5
7
$
H
$
+


52
"%>
32 
HI ;
/-9CC/-9D/>
& 3 ' '3 ( N 3 '3 #>3 '3 !)3 ' '3 ?#3 ' &'3 L #3 ' '3 ! )3'+'3 3+'3 >3 '3 !> #3+''3(3 'M1)">)3 ' ';8797<'
/_
2
2((%!


0

(%!
% % !


% $!
2
%&  "
2%"2
$
5

% 

(%
!% ((>
3 2 
GB ;
/IC/ /ICH>
&! #3 '3 1C#!,  !#>3 '3 #3 '3  >3 '3  >3 &' *'3 ! 3 '3 1)">)3 ' 'M##3*';8797<'
%
$%"


F

$
-L
$

% " 
G

(%!
%

(%
 
2
-_;D_  $5
5
/&>
2  
%II ;
/HI/+D>
&" 3'M #)3+';877=<'
B
% %
$


E
%
E5

% %
(%
% >
5 
88 ;
DI/DI9>
&3'31#! 3'31C #,>3+'3)3'M! #)3 ';877H<'

  %!!%
% 
C
2
$ !
(%!


 %
""&%
% 
$!
""&%
% 


-L% !$
"%>
3  
8G8 ;
++HF+++->
&3'33'M! #)3 ';877=<'
!&!%
 @ 
$
""&%
   5
2

 !
(%!
5
-
 %!!%
% 
C4>
3  
8G9 ;
++DC ++IF>
&#3@'3 !3'3
 !#3'M(# 3'
';877%<'

 $&$
 B


$ !
(%!
( 
5 % >
 - 
 4

977 ;
I9+II99/>
& # 3 1' '3 L N3 &'3 .3 +'3 !$3 /' M N3 /' ;9III<'

5

%2% 
% %
%!!%;
2! ;
$
( ! 
2

2"5
&!>
3)    7 !
9I ;
/.I9/.9+>
& !)3 ' *' ;877H<'
 !
$
5 E
2(% 
2 %

$( "
$
!
2
(>
-& 35
%:H ;
-------D>
&" )3*' ';877:<'

 
$
( 
2

%(
$
2%$
2% "

B4
E5>
+ ) 
8= ;
CI9CHI>
&>))""3'31) 3 '+'M ## 3&'/';877:<'

%!!%
%(
2

2((%!
2
5>
- !5   
%;
/F-->

44 7 3

/CH

& N,+ 3 +' '3  !, 3 &'3 #)3 +'3 &! N,.3 '3 #)>3 &'3 > 3?''M/,#!3 ';877:<'
& 
2
K&
%2% 
 
&5

C4
% 
2
2((%!>
32 
HI ;
+..C+./F>
& N,+ 3 +' '3  )N,#3 /' /'3  !, 3 &' M /,#!3 ' ;877%<'
& 
2
%2% 
 
&5
$ !
(%!>
 - 
 4

977 ;
/CFFF/CFF+>
&"3+'&'3 #> 3+'/'M?)!3*'+';9II%<'
% 
2

$ !
(%!
5
-
% 
%
$
% >
3( 2 
HB;*!8< ;
/HD/+->
&!)"3 (' &'3
3 ' M * .) 3 ' ;9III<'
 !
(%!
F
%
% >
%5
%!!%
$
 

% 
2

(

E
!&%
&5
" !%
" $
$
%!!%
5
2
"! 
22>
3  
8HB ;
DDHF DD+.>
&!.3+'*'3/ 3 '3  ### #3 '3 #)3 ''3!!3 ''M(#3' ;877B<'
1 E
2(%
(%!
2(5
2 %
4 (
$
 5
" 
& )  %
97 ;
/IDH/II.>
&!.3+'*'3? 3('33'3" !3+''M!!3 ' ' ;8779<'
5 
$
( ! %5
% 
" 
5 E
2(%
(%!
 

2%>
32 
H: ;
I999 H..+>
 , )N3'3##3*'M
, 3';877:<'

""!
% 

$ !
(%!>

 5!
%= ;
C-DCFD>
# 3 ' /' ;877I<'

% 
2
%
""!  
 " 

$ !
 >
2 3
=;
-//>
#)"3 '31C!3'3&L3'M$ 3 '';877:<'
F
$
H
%
% $


"
%!%
 "%"
&5
$
% ! %5
">
3   
8G7 ;
FH/.HFH//H>
L>3 '3.3*'&'M? 3/';9IGI<'
5
2

%"

2  
9=I ;
FIDFHI>
* 3 '3
#3 &' +' M /N 3 &' /' ;877H<'
2
$
$
%2
 %"

""!  
2
$ !
" %% 
2(%$ !
 B
5$% "
M 3K N>
)   
G: ;
CFCD>
*!>3
'/'3+ #3
' '3
) 3@'('3?3 'M1) 3 '+';877H<'
! 
%
32 
G9 ;
ICH/IC+/>
*!>3
'/'M1) 3 '+';877G<'
1 E
(%
(%!
F
5


% 

(%!
"&5
$$
2

B E
@5"
2! >
32 
G8 ;
FFC-FFD->
*3 '31)A3&'M1) 3 '+';877G<'
 

$ %

2((%!
%5


% 
2 %
(%
$%!
$ >

!
G7 ;
//-->
*"3' '3*#!L3 ' '33'&'3 !3
' 'M)#!3';877I<'
%$2 "$$
 
 % 



% 

$ !
(%!

(( >
32 
G% ;
+-HI+-+/>
*NN3 '3 * 3 '3 (>"3 *'3
#3 /'3 L#3 ' M  43 ' ;877I<'
!%"
!%(5
$
 

+  5 )  . 
9B ;
FDDFI/>
*)3 'M ##3
';877H<'

%  
2
(%!>
)  
8H ;
FH.F+F>
*# 3 '
'3 3&''3 )3 ' '3 ! 3''3 #3
'?'33&' '3( 3''3*$$3' 'M#3 '?';877:<'

2 !
G

(%!

6%

7
% %%
>
2  
%%B ;
-+C.>

44 7 3

/C+

*)3 '3  !3 '3 3 &' M 3 ' ;9IGI<'
$2 
2
2 !%
 %($
" 2
" 

 $$
 5"%
 $
">
&5  3
G;
F+IH F+HC>
*#3'M
 31' ';9IIG<'
   


" $
2

!&! >
     
9B ;
+/H+/+>
*#!3 *' '3
 )3 'M /3 ';9II9<'
75 5 
$
% 
2
5 E
2(%
(%!
 %!!%
% 
/>
2!
9G ;
-9/F.->
*"3 &' +'3 L # 3 ' &'3 !)3 ' '3 ! 3
' '3 ?!)#3 ' '3 #  3'/'3 3*'/'3?)!3*'+'3+ #3' 'M # 3 '';877H<'
!%
 @ 
2
$ !
(%!
 %!!%
% 
D
% ! 

" %
K&% @$
!%
 @ 

+
G;
H9D+.H>
*"3 &' +' M ?)!3 *' +' ;9II%<'

22
2
$%$
"! 


$"%@ 
$
% 
2

/
% 
2$
&5
$ !
(%!>
2  
9IB ;
HI9H+.>
) 3'3)" 3' '3 #) 3 ''3 ! > 3''3!)3+'?'3 ! 3 '
' M !  3 ' @' ;877G<'
%!!%&$
"! 
$2
" %
 (
$%"
2

-4
 2 %
2

G

(%!
E  " 
-4F
% >
3( 2 
GI ;
IFIIC/>
.)3 /'3 ?N 3 ' M !)3
' ;877G<'
 ""

22
E %$
2

""!5>
 5   
9: ;
H+/..>
 )3'';877G<'
B& %

(%!;
B
$
!"
 %%"
$
   %"
$5">
  )  %
89 ;
CI-CIH>
"3'3))3 '3  3&'3/3@'3O)3@'3 3('3#3 ''3O)3@'33('M )3*'@';877=<'
G

(%!
D_
%!!%

2 %"$
&5

32 
G7 ;
+FI-+FH.>
3
'&'3 )#3'&'3"3' '3) 3'+'3)!#3 ''M!##3+'(';9IG:<'
! $

  
88I ;
H-IHFF>
3@'3 !3
'3  3 '+'3
>3'3"N3 '3   3'3  N3 ' '3 #3+''3( 3+'*'M.3 -';877I<'
!%
$"  
5
2
$ !
2(%

4 (
2% "
/99+

-..+>
2    ;   %
I;
FFHFCC>
>3''3( 3/'3  #3+'
'31#! 3 ' 'MN 3+'';9IIG<'

2"5
2
!"
% %
%!!%5
%$

%  
>
 - 
 4

I: ;
D++D9F>
# 3 +'3 ?#!L"3 ' /'3 1)">)3 ' M &> N3 +' &' ;877=<'
 !$
( 


G

(%!
C -LC4
,! 
5

", %
% 

%%% 
5 "
""&%
$
7  
%22B
2

C
% >
32 
G7 ;
CI-FCIF->
##3''3O)30'3 P 3'1'M&# 3*'?';877:<'
$ 
2
G

(%!
% 



!!%
$
!
2
% &%


% &
(%
 >
2  
%%9 ;
CDHCH.>
" 3&''3& ) 3'M !3&';9IIG<'
%! $$
% 
2

((%$>
3( 2 
HI;*!:< ;
9CH9D9>
!3 ' M /3 &'3 +' ;877H<'
%
2
%!%
(%
%  >
    ) 
9I ;
/H-F>
!)3 ' M * .) 3 ' ;877I<'
!%
% 
2 %
2((%!
$%!
$ (%5>

!
G9 ;
I/D>

44 7 3

/C9

 C 3&' '3 3
'*'3 !3'?'3&#))3'3#"3'3
 3'31!#3 &'3  >31'3
  3+''3?!)$$3'M/ 3' ';877H<'
B
% %
 

"%  
B

!  5
E5

 5 >
- 
B:7 ;
/-DF/-DH>
>3 ' ' M  3 /'
' ;877B<'
 (
2! 
2
% 
 $$
&5
(%
% $!&
>
 +
97% ;
-CD-D9>
)3'M&# 3*'?';877:<'
G

(%!
% 
%2%
E
& 
 5MN $!$
%2% 

%% 
$
% 

%2% 
%">
2  
%B8 ;
HH+H>
)N3 '3.3''3
) 3&'3 # 3 ''3 !3&' '3 43'3 N3 @' '3 3 ' '3C#!3*'M ##3
' ;877:<'
B
% %
F
% " 
% %"

(%!2$
>
- 
B%% ;
++H+9->
 3/'
'M*!#3/' ';877H<'
%
%$!&
>

2!
H7 ;
-FF-IF>
!)3 ''3 3'M
) 3O'+';877=<'
(%

""!5
E5>
!
9= ;
/C//CH>
 3 &'3
3 ' &' M !N3 1' ' ;877=<'
B
% %$$
$
 $$
(%

6% 
$
 !%% ! 
&5
 &$
2 % /K4  />
3  
8G9 ;
-I/++-I/9D>
"3 ' M !> # 3 +' *' ;9IGI<'
! 
% %
2
""&%
 2 %
% 
2
 "">
 3
8=B ;
D+/D++>
)3 '';877%<'
$"  5
2
%
%%  $& %
2((%!
2
!">

 2!
=9 ;
FHFF9/>
 )3 '3 1## 3 ' M  #3 ' ;877H<'
 !
$
$ !
" %% 
2(%
" "
!


(
%
!>
3 
!>3/' '313'&'3?#3' '3  3 '('M).3 '';9IIG<'
3 E

% $

%2% >

!  
=H ;
--H-IC>
!!# 3''M&N)! 3 ';877=<'
(%
$2
%2% 
$
&5 $>
3&* 5
87% ;
/+FH/+C/>
!!# 3''M&N)! 3 ';877=.<'
5

%2% 

 
$2>
)  
8: ;
FHF F+/>
!# "31'M( N3'-';877=<'
((%!
""&%
2! >
3( 2 
GH ;
-HDD-HII>
!3 '3+'33@'&'3"3'331'3@ "3&'3C!3 '3 3'&'M /3 &'3 +' ;877:<'
 !
%!%
(%
$2
$
%"(



(%!

% 
% ! 

!%

;
7>
3 2 
HI ;
-I+9-I99>
L$$3'';8777<'
 c>
5 
5
!
%" 5
Mc$
%
" $N>
=   
&   ##!# >
>31'M >3';877:<'
B
% %


""!5>
) ) 
9H ;
//C>
>31'31#)3 'M >3';877%<'
B
% %>

!) 
89 ;
FFD FHI>
>)3 'M >3';877G<'
DK7
$
(%!
%  >
  ) 
87 ;
/H-->
>)3 'M >3';877I<'

""!5

(%!
2 >
) !
88H ;
HD+I>
3 1'3 "3 +'3 3 &' M 13 ' ;877H<'
7 C
!%
( ! %5
7
5
M 7 N
 2E%
(% 
C>.>
5   & 
8B ;
/D9I/D99>
3 /'
' M ( 3 ?' &' ;9IHB<'
% %  
&E
% !
4
%& (%!
%

"

0
4

$
>
!

(%!
$!$
&5
 !
(%!
""!@ >
)  )
I;
9.99/D>
)# 3 '+'M  3' ';877H<'
B
% %;
7B


$

(%

""!
% >
)   
G: ;
CFDCCD>

44 7 3

/D.

)# 3+''3/.# 3 '+'3*L >3'3+  3'M( #3'/';9IIH<'

  d
E$ E
%2
26&
% 
2 %
"!

-  
!
8: ;
C+HIC++->
3&' ';877I<'

  
  . 
%I ;
9H /C.>
3('3
## ,*#3*'3&3'3*# 3&' '3.3-'3&3 *'M 3+'*';8777<'
 "
 "

   ! 
8HH ;
+99->
3 ' ;877B<'

@  
2!;
>
//H/
 5 
$
 
$
"$
  !
$
 
(  %%
2% ;
%
M 5 N

#3@' '3
) 3'@'33
' 'M13
'';877I<'
3!"
F
% @
$ !
(%!
$
" $!
(%
% 

(% >
5   
99 ;
I.CI/D>
!#3'M >3';877=<'
B
% %
$

""!5>
35 5
GB ;
H/- H-D>
!3 '3 )N3 ('3 &>L3 '3 (#.3 '3 &!3 1'3 #)3 '3 (!#3 &'3 >NL3 1' M &"3 ' ;8777<'
$2 
$
%%@ 
2



(5
2
0

(%!
F
% >
'&  
B=: ;
HCH+>
 4,N3 '('3  3&' 'M!>3/' ' ;877=<'
 "6
" $! 
2

52
 

% 

5

%2% >
)  
8: ;
FI/FH->
 !"$!3*'3/ 3 '3*3&'3
##3+'33+'3*3
'3* 3+' '3 3'M!3&';9III<'
%& 
2
5 E
2(%


%!@;
4 (>
 
%:% ;
/DD+/DI->
   !"$!3 *'3 / 3 '3 *3 &'3 (N 3 '3 *3
'3 !3 &' M  3 ' ;9IIG<'
 %
 ""! 
$ !
% 5
-
!&5

M_0"_N
$"


%!@;
4 (>
+ 5) . 
%;
+DH+D+>
) 3'?'3/ 3'31" C3'3 #3''3  !"3'3 !"> 3 '3 "3 '*'3  #>)3'3 !) 3 ''3  #3 ' ' M #>3 ' ;8777<'
 !
(%"
%;
& $5
% 
%;
$
(%!
% 5
 %%
E
$
(%5>
3)  %
9G9 ;
-9>
 3 '3 !3+' M "!3 ' ;877G<'

2
(%
2 
$
( 
2

""!5
&5
 B
% %
F>
 5   !
89 ;
/F-D>
#!3'+'M))3'';877H<'
%!
5 5 
% 

(%!
$
""!
% >
+ 5   
9: ;
-//-/+>
3'&'M/ >3 '';877I<'
7 "
5@ 

% 5
2 %
2((%!

% >
2!
9%I ;
-F.-F9>
? 3'3? 3'
'3)3 ''3 #)3 ''3?!!#3'&'M!!3 '';9II=<'
7
(% 

5 E
2(%
(%!
$! 


F_
  $
%  
2
%
2% "
2%>
2  
88: ;
-HC-+/>
? 3 '3 L 3 '3 43'3 # 3+''3>3'M 3 ' ';877B<'
B
% %
F
"$
G

(%!
%5


&%
!

>
- 5
97 ;
/FII/FHF>
? 3 *'3 3 +' 1' M
!!3 &' ' ;9II%<'
 "!$
 
(5
 $
E
5 E
2(%
(%!
F
% 
6%$

&%>
3 2 
=H ;
9+9 99I>
? 3'
'M # 3?'1';8797<'
%
$
2!!%
%& (%
%>

!
G: ;
F-+FCD>
?#)3
'3 .)!3&'3&"!3+'3( 3'3?>3&'?'3N 3
'3)3 '3 3 'MON3';877H<'
%!!%
$
2! 
 "% 
2

  %!!%
% 
C4

2((%$>
35 ( 5 
8= ;
DCIDDH>

44 7 3

/D/

? 3 /' ;9IG9<'
%"

2  
99% ;
DCCDDD>
? 3 /' ;9II%<'


F
 %!!%
% 
2
2((%!
 


% 
(5>
2  
9IH ;
-ID-HF>
? 3 /' M /##3 (' +' ;9IHG<'
!$

(%!2
!
$
5@$

(%&%
$
" 
2  
GI ;
C-FCFH>
? 3/'3 L>3 'M
#!3';9II7<'
% 
2

% $!%
E
 E
  
2
(%
 %!!%
% 
2% "
43L
(%&%

2$
E

G

2((%!



E
 E
%
$%
"
%%@ >
2  
9HH ;
H9D+./>
?#!L"3 ' /'3 &> N3 +' &' M 1)">)3 ' ' ;8778<'
 
$

2! 

L!,
(%!>
+ 5   ) 
8=H ;
F-FFD/>
?# 3 +' '3  ## #3 *' '3  3 &' ' M )3 ' ;877G<'
G

(%!
 %!!%
% 
/
&
F
 
%$! >
32 
G8 ;
+-I-+-H/>
? >3 /'3 ( N3 ' -' M 1 N3
' ;9IGH<'
!$


5 5 
2
2((%!

% 
$

% 
2
%& 5$%

 
%!!%>
2  
9:I ;
-FH-CF>
? >3/'3&4L3''3 3
'M!3';9IGI<'
E5
5@$
$ !-
(%!
 %!!%
% 
/


 !&
% 
&!
& "
%5
5$%  &
$
""&% $
2%
$"%@ >
2  
9H9 ;
F.-F.D>
? >3/'3  )3''3
 #3/' '3 " 3 ''M!3';9IGG<'
($


"!%
2 %"
2

2((%!
 %!!%
% 
/


$"%>
2  
9=8 ;
/+H/9I>
?$$3 'ML3';877I<'
2!@
(%!
 % 

(%&%
5

%2% 
5"
2 %;
"";
$
 
3)   7 !
8I ;
DC9DDH>
?3+'33 '1'31) 3 '+'M!)3+'';877:<'
%!!%
2

((%!

" 
2 %
5
(5;
% 
% ;
$
%  5
%  >
32 
HI ;
/.-I+/.-HH>
-3 '3!)3 '3
)3 '3? 3'3  3&'3
) 3*'3#  3'/'M#3 +' ;877:<'
%!!%
2

 !
(%!
K! $
% 
5
$ "


% ! 
2
->C
>
32 
HI ;
/.-H+/.-++>
@#)!3 '3  3 ('3 &3 @'3  3 ('3 &#)3 1'3 >NL3 '3 )3 &'3 #>)3 ' M &!#3 @' ;877G<'
%5
%!!%
2

5
$ "
2
0

(%!
F
K! $
% 


% ! 
2
/>+
>
2  
%H% ;
C-ICFI>
@ 3
'3 !3 '3 &3 '3 *) 3 +'3 / #)3 ' 1'3 &!)"3 1' (' M * .) 3 ' ;877:<'
$! 
2

! $
% K

$
D_
% 
(
2
 !
(%!
5
-
 %!!%
% 
F
MFN
&5
% 
E
D;

 $$

 5"%>
3  
8G7 ;
-HC/--HC/9>
@ "3 &' M C!3 ' ;877H<'
! 
2
7B
% %

(%

""!5>
3  
8G8 ;
/DF/D/DF/+>
@ "3 &' M C!3 ' ;877I<'

%  
$
 
%$! 
&5
7B
% %>
) !
88H ;
DCID>
@ "3 &'3 1>)3 &'3 !#>L3 '3 ) .3 '3 N3 '3 &"#)3 &'3 3 1'3 >3 ' M C!3 ' ;877B<'



7



2! 

$ !&%$$
 $!$

(%
% >
-  ) 
:;
HF.HFH>

44 7 3

/D-

@ 3 *' '3 (!)3 *' '3 !)"3
' M ( 3 ?' ;8777<'

 
!%
@5"B$
""!  %&
5
%(
 
(
2

$ !
(%!
% 
/


%
2
2$
>
3 5   
%G ;
/.DF/.DH>
@3'&'3O) 3?'3(L"3(' '33'31#!") >3' '3
) 3*' '31) 3 '+'3 ## 3&'/'M
) 3+';877G<'
%!!%
2

""!%
$ !
(%!

 E
3
%"
%  5
"!% >
  
%9I ;
/+FC/+FH>
@#$3 '3
3 '3 ?!N3 &'3 &!)"3 (' &' M * .) 3 ' ;8777<'
!%2$
-4KF
%
% 
2
$ !
(%!
5
-
6&
 2 %
-4
$$
2 %
( 
2
!&%
E
$&
"
$

(% >
3  
8H: ;
99IF99I9>
O) 3@'3
 3+'3
) 3*' '3O) 3?'3*!  3''3>3'3>3 ''3 !##3 +' ('3 1) 3 ' +' M ## 3 &' /' ;877%<'
%!!%
2
""!%
2((%!
%>
&5 3
88 ;
-I.C-I/F>
O) 3 @'3 1$ 3 '3
) 3 *' '3 1) 3 ' +' M ## 3 &' /' ;877H<'
%!!%
2
""!%
G

(%!>
32 
G9 ;
I/C/I/CD>
O) 3'3@ 3@'33'3? 3@'@'33@'33'33
'3(3-'3O) 3'3  3*' M)3('';877G<'

$ %
% 

B
(%!
% %

F
%% 
2 %
( >
)  
8I ;
DF+DD.>
OL>3 ' +' ;877=<'
7
 %"
% 
2 %

5 
5
2
% 
&  

%  
#!  )  #!"$ 4#! #! >


     -

!#.! #    / #   #!!6$ !  #!# $ ) # !  ! # $ !)   !$$$!  !  # 

7 !$ c;
4
 !%$;
3
;
4
%;
;

G(%;

7 %&5;
7
!%%!;

4%  ;

 !;

 %%%;

L (;

$
"&%;
4;
%$>


 


    #!"% $!  ! !  $L#! $  (
;
% 
;
4% 
 ;

"&%
;
7!%
;
!$%
4;
%%
>
&     

  !!  ! !"   # #,*! #*#! #$# ' 4 
;
(%@
L;
&%
;
4% 
;
%$
4;
 B
;
 !%$
4;
% 5
;

"&%
;
7 !$
 ;
7%%$
7;
7%"
0 ;
3 2$
;
0 
;

3;

0;
% 
;
(
;

;
!%!
7;
5
0;
E
0;

0;
B
4;
% 
;
!&%
3;
!%
;
 
;
4  
>


  

VECTOR-BORNE AND ZOONOTIC DISEASES Volume 9, Number 3, 2009 ª Mary Ann Liebert, Inc. DOI: 10.1089 =vbz.2008.0187

Short Communication

Molecular Epidemiological Analysis of Dengue Fever in Bolivia from 1998 to 2008

Yelin Roca, 1, * Ce´cile Baronti, 2, * Roberto Jimmy Revollo, 1 Shelley Cook, 3 Roxana Loayza, 1 Laetitia Ninove, 2 Roberto Torrez Fernandez, 4 Jorge Vargas Flores, 1 Jean-Pierre Herve, 5 and Xavier de Lamballerie 2

Abstract

Dengue fever was first recognized in Bolivia in 1931. However, very limited information was available to date regarding the genetic characterization and epidemiology of Bolivian dengue virus strains. Here, we performed genetic characterization of the full-length envelope gene of 64 Bolivian isolates from 1998 to 2008 and investi- gated their origin and evolution to determine whether strains circulated simultaneously or alternatively, and whether or not multiple introductions of distinct viral variants had occurred during the period studied. We determined that, during the last decade, closely related viruses circulated during several consecutive years (5, 6, and 6 years for DENV-1, DENV-2, and DENV-3, respectively) and the co-circulation of two or even three serotypes was observed. Emergence of new variants (distinct from those identified during the previous episodes) was identified in the case of DENV-1 (2007 outbreak) and DENV-2 (2001 outbreak). In all cases, it is likely that the viruses originated from neighboring countries.

Key Words: Dengue—Epidemiology—Genetics.

ver the past 50 years a variety of arboviral human was first detected in 2002 (Kochel et al. 2008) and has subse- Odiseases have been reported in Bolivia, including yellow quently been identified every year. It is now recognized that fever (Bevier et al. 1953), Mayaro fever (Taylor et al. 2005), and cases of dengue fever arise predominantly in Bolivia between dengue fever (Gianella et al. 1998, Van der Stuyft et al. 1998, November and June and are referred to as the ‘‘annual epi- Kochel et al. 2008), although many other arboviruses poten- demic’’ (in this paper, they are reported as E1998–E2007; see tially circulate and cause disease in this country. Dengue fever Fig. 1). Most dengue fever cases are reported in the eastern was first recognized in Bolivia in 1931 (Garret 1993). How- region of Bolivia—in particular, in the Santa-Cruz region. The ever, the first documented Bolivian case of dengue fever was western region is dominated by the Andes Cordillera, where notified in 1987. The virus was isolated at the Instituto Fio- the altitude is too high for sustained arbovirus circulation. Co- cruz, Rio de Janeiro, Brazil (Anonymous 1995) and was be- circulation of DENV-2 and DENV-3 was reported during lieved to belong to dengue virus serotype 1 (DENV-1). This E2003, E2004, E2005, and E2006. Co-circulation of DENV-1, serotype is believed to have circulated until 2003. It then ap- DENV-2, and DENV-3 was reported only during E2002. parently disappeared and was not identified again until 2008. However, before the current study, very limited information Dengue virus serotype 2 (DENV-2) was first detected in 1997 was available regarding the genetic characterization of DENV (Gianella et al. 1998, Van der Stuyft et al. 1998, Foster et al. strains. Here, we carried out the genetic characterization 2004), and evidence of its circulation in Bolivia has been re- of isolates from 1998 to 2008 stored in the Centro National ported every year to 2007. Dengue virus serotype 3 (DENV-3) de Enfermedades Tropicales (CENETROP) Microbiology

1Centro National de Enfermedades Tropicales, Santa-Cruz, Bolivia. 2Unite´ des Virus Emergents =UMR190, Universite´ de la Me´diterrane´e & Institut de Recherche pour le De´veloppement, Marseille, France. 3The Natural History Museum, London, United Kingdom. 4Servicio Departamental de Salud, Santa-Cruz, Bolivia. 5UR014, Institut de Recherche pour le De´veloppement, Montpellier, France. *These authors contributed equally to this study.

337 338 ROCA ET AL.

FIG. 1. Dengue IgM-positive sera and dengue virus isolation, CENETROP, Santa-Cruz, 1998–2008. ( A) Dengue IgM- positive sera. Detection of specific IgM to dengue virus was performed using an immuno-capture assay. The antigen was produced from a pool of supernatants of DENV-1 (strain Hawaii), DENV-2 (strain new Guinea C), DENV-3 (strain H87), and DENV-4 (strain H241) strains propagated on C6 =36-HT cells. ( B) Isolation and characterization of dengue strains. Isolates were obtained after inoculation of human sera on C6 =36-HT cells in EMEM (Eagle’s Minimum Essential Medium) at 34 8C. Identification of serotype was performed using specific monoclonal antibodies (DENV-1: D2-IFI-3; DENV-2: 3H5-1-21; DENV-3: D6-8A1-12; DENV-4: 1H10-6-7; all from CDC, San Juan, Puerto Rico) and an immunofluorescence assay. ( C) Isolates included in the current study. X-axis: The period studied (1998 outbreak to 2007 outbreak). Y-axis: (A) the number of sera with IgM antibody to DENV identified monthly by the CENETROP microbiology department (Santa Cruz, Bolivia). ( B) the number of DENV isolates made by the CENETROP laboratory and their serotype. ( C) the DENV isolates analysed in the current study. Table 1. Main Characteristics of DENV Isolates Studied

Serotypes Isolate name Date Department Province

DENV-1 Bolivia DEN1 JR-16 =99 E1998 03 =1999 Santa Cruz de la Sierra Ciudad Bolivia DEN1 JR-22 =99 E1998 03 =1999 Santa Cruz Tornos Bolivia DEN1 JR38 =99 E1998 03 =1999 Santa Cruz Tornos Bolivia DEN1 119 =02 E2001 02 =2002 Santa Cruz Ichilos Bolivia DEN1 163 =02 E2001 03 =2002 Santa Cruz de la Sierra Ciudad Bolivia DEN1 221 =02 E2001 03 =2002 Santa Cruz Obispo Bolivia DEN1 273 =02 E2001 03 =2002 Santa Cruz de la Sierra Ciudad Bolivia DEN1 289 =02 E2001 03 =2002 Santa Cruz Ichilos Bolivia DEN1 25 =99 E1998 a 03 =1999 Santa Cruz Torno Bolivia DEN1 71 =01 E2000 03 =2001 Santa Cruz de la Sierra Ciudad Bolivia DEN1 1868 =08 E2007 03 =2008 Santa Cruz de la Sierra Ciudad Bolivia DEN1 1877 =08 E2007 03 =2008 Santa Cruz Florida Bolivia DEN1 2369 =08 E2007 04 =2008 Santa Cruz de la Sierra Ciudad Bolivia DEN1 2370 =08 E2007 04 =2008 Santa Cruz de la Sierra Ciudad Bolivia DEN1 2575 =08 E2007 05 =2008 Santa Cruz de la Sierra Tornos Bolivia DEN1 391 =02 E2001 04 =2002 Tarija DENV-2 Bolivia DEN2 293 =02 E2001 03 =2002 Santa Cruz Ichilo Bolivia DEN2 297 =02 E2001 03 =2002 Santa Cruz Ichilo Bolivia DEN2 376 =02 E2001 03 =2002 Santa Cruz Ichilo Bolivia DEN2 4 =03 E2002 01 =2003 Santa Cruz de la Sierra Ciudad Bolivia DEN2 36 =02 E2002 12 =2002 Santa Cruz Ichilo Bolivia DEN2 133 =03 E2002 01 =2003 Santa Cruz Sara Bolivia DEN2 147 =03 E2002 a 01 =2003 Santa Cruz Ichilo Bolivia DEN2 159 =03 E2002 01 =2003 Santa Cruz Ichilo Bolivia DEN2 166 =03 E2002 01 =2003 Santa Cruz Obispo Santistevan Bolivia DEN2 354 =03 E2002 01 =2003 Santa Cruz Andres Ibanez Bolivia DEN2 404 =03 E2002 01 =2003 Santa Cruz Andres Ibanez Bolivia DEN2 641 =03 E2002 a 01 =2003 Santa Cruz Andres Ibanez Bolivia DEN2 4093 =03 E2002 04 =2003 Santa Cruz Andres Ibanez Bolivia DEN2 4749 =03 E2002 06 =2003 Santa Cruz Obispo Santistevan Bolivia DEN2 126 =05 E2004 01 =2005 La Paz Palos Blancos Bolivia DEN2 270 =05 E2004 01 =2005 La Paz Sud-Yungas Bolivia DEN2 760 =05 E2004 03 =2005 La Paz Caranavi Bolivia DEN2 269 =06 E2006 01 =2007 Cochabamba Carrasco Bolivia DEN2 318 =07 E2006 01 =2007 Santa Cruz Andres Ibanez Bolivia DEN2 663 =07 E2006 01 =2007 Santa Cruz Andres Ibanez Bolivia DEN2 2007 =07 E2006 02 =2007 Santa Cruz Obispo Santistevan Bolivia DEN2 2106 =07 E2006 02 =2007 Santa Cruz Andres Ibanez Bolivia DEN2 3118 =07 E2006 03 =2007 Santa Cruz Obispo Santistevan DENV-3 Bolivia DEN3 751 =03 E2002 01 =2003 Santa Cruz Andres Ibanez Bolivia DEN3 1465 =03 E2002 02 =2003 Santa Cruz Andres Ibanez Bolivia DEN3 1593 =03 E2002 02 =2003 Santa Cruz Andres Ibanez Bolivia DEN3 1726 =03 E2002 02 =2003 Santa Cruz Andres Ibanez Bolivia DEN3 4445 =03 E2002 04 =2003 Tarija Padcaya Bolivia DEN3 608 =04 E2003 02 =2004 Santa Cruz Andres Ibanez Bolivia DEN3 704 =04 E2003 03 =2004 Santa Cruz Andres Ibanez Bolivia DEN3 786 =04 E2003 03 =2004 Santa Cruz Andres Ibanez Bolivia DEN3 805 =04 E2003 03 =2004 Santa Cruz Andres Ibanez Bolivia DEN3 867 =04 E2003 03 =2004 Santa Cruz Andres Ibanez Bolivia DEN3 869 =04 E2003 03 =2004 Santa Cruz Andres Ibanez Bolivia DEN3 892 =04 E2003 02 =2004 Tarija Bermejo Bolivia DEN3 991 =04 E2003 03 =2004 Santa Cruz Andres Ibanez Bolivia DEN3 1023 =04 E2003 03 =2004 Santa Cruz Andres Ibanez Bolivia DEN3 1035 =04 E2003 03 =2004 Santa Cruz Andres Ibanez Bolivia DEN3 1951 =04 E2003 05 =2004 Santa Cruz Andres Ibanez Bolivia DEN3 67 =05 E2004 01 =2005 Beni Riberalta Bolivia DEN3 84 =05 E2006 01 =2005 Beni Riberalta Bolivia DEN3 334 =07 E2006 01 =2007 Santa Cruz Andres Ibanez Bolivia DEN3 377 =07 E2006 01 =2007 Santa Cruz Andres Ibanez Bolivia DEN3 2019 =07 E2006 02 =2007 Santa Cruz Obispo Santistevan Bolivia DEN3 2184 =07 E2006 02 =2007 Santa Cruz Andres Ibanez Bolivia DEN3 2201 =07 E2006 02 =2007 Santa Cruz Andres Ibanez Bolivia DEN3 2302 =07 E2006 02 =2007 Santa Cruz Andres Ibanez Bolivia DEN3 2322 =07 E2006 02 =2007 Santa Cruz Andres Ibanez

aPersons deceased after DENV infection.

339 FIG. 2. Phylogenetic analysis in the Envelope gene (nucleotide sequences). All trees presented were obtained using the Kimura 2-parameter algorithm for distance calculation, NJ, and 500 bootstrap replicates (values greater than 50% are indicated on major nodes branches). Horizontal bars are proportional to genetic distances. Identical topologies were obtained using the p-distance algorithm and ML methodologies, with the exception of DENV-1 2007 in the ML analyses, results of which are shown in addition to the NJ tree. ( A) DENV-1 sequences. Phylogenetic tree reconstructed using DENV-1 E gene sequences (nt 772–2617 referring to the numbering of strain Indonesia_A88_1988; GenBank accession number AB074761). The topology of 2007 Bolivian isolates in ML reconstructions is slightly different and shown on the right of the main figure. ( B) DENV-2 sequences. Phylo- genetic tree reconstructed using DENV-2 E gene sequences (nt 863–2411 referring to the numbering of strain Puerto- Rico_BID =V678_1998; GenBank accession number EU482735). To improve legibility, the reconstruction for the ‘‘Asia =America’’ genotype is presented after magnification. ( C) DENV-3 sequences. Phylogenetic tree reconstructed using DENV-3 E gene sequences (nt 817–2478 referring to the numbering of strain Thailand_C0331 =94_1994; GenBank accession number AY876494). To improve legibility, the reconstruction for genotype III is presented after magnification.

340 FIG. 2. (Continued )

341 FIG. 2. (Continued )

342 DENGUE FEVER IN BOLIVIA, 1998–2008 343

Diagnostics Department (Santa Cruz, Bolivia). Our objective distance in the E gene <0.010) and shared a common phylo- was to investigate the origin of viral strains and, more spe- genetic ancestor. These observations demonstrate that the cifically, to determine whether or not multiple introductions E1998–E2001 viruses circulated persistently during this period of distinct viral variants had occurred between 1998 and 2008. with no introductions of new viral variants. Moreover, on the Sixty-four selected DENV isolates (16 DENV-1, 23 DENV-2, basis of sequence homology and phylogenetic data, the most and 25 DENV-3) isolated at CENETROP between 1998 and probable origin of this Bolivian DENV-1 is South America, be- 2008 were studied (Table 1). Viruses were propagated using cause the E gene sequence is highly similar to viruses that cir- C6 =36-HT cells (Roche et al. 2000) in EMEM (Eagle’s Mini- culated previously in neighboring countries such as Brazil and mum Essential Medium) at 34 8C; 200 mL of supernatant me- Argentina. However, the virus that has circulated more recently dium was collected at day 5 postinfection, and viral RNA was in Bolivia (E2007) is more distantly related to viruses from the extracted using the QIAamp viral RNA minikit (Qiagen, E1998–E2001 period than it is to the 2006–2007 isolates from Dusseldorf, Germany) according to the manufacturer’s rec- Puerto Rico or Venezuela (uncorrected p-distance in the E gene ommendations. Reverse transcription was performed using 0.016–0.025 vs. 0.005–0.013) (Fig. 2A), suggesting that it may random hexamers and the TaqMan reverse transcription re- have been introduced more recently from one of these countries. agents kit (Applied Biosystems, Foster City, CA). The com- Isolate 1868 =08 is located apart from other Bolivian isolates plete viral envelope gene was then amplified using the based on NJ analysis, but clusters with other E2007 Bolivian Triplemaster PCR system kit (Eppendorf, Hamburg, Ger- isolates based on ML and Bayesian analyses. The possibility that many), standard PCR amplification conditions, and a series of emergence of this new variant might be explained by immune serotype-specific primers (DENV-1: E1F [AACAAGARCYGA selection seems unlikely because the E1998–E2001 DENV-1 RACRTGGATGTC] and E4R [YARTTCATTTGATATTTGYT strains would be expected to provide complete immune cross TCCACAT]; DENV-2: DEN2-env-S3 [ACACCATAGGRAC protection against the DENV-1 E2007 variant. GACRYATTT] and DEN2-env-R3 [CCRCTGCCACAYTTY In the case of DENV-2, isolates from E2001, E2002, E2004, AGTTCT]; DENV-3: DEN3-E2-S [GARAARGTAGARACA and E2006 were studied. When compared with two E gene TGGGC] and DEN3-E2-R [TCNGCYTGRAATTTGTATTG sequences available in GenBank and previously produced CTC]). Amplicons were directly sequenced using the ampli- from Bolivian strains isolated in 1997 (Foster et al. 2004), all fication primers. Viral nucleotide sequences of each dengue Bolivian viruses belonged to the ‘‘Asia =America’’ genotype serotype were aligned using ClustalX (Thompson et al. 1997) (see Fig. 2B). Isolates from E2001–E2006 are very similar together with relevant sequences retrieved from GenBank. (maximum uncorrected p-distance in the E gene <0.008) and Phylogenetic analyses were performed using three different share a recent common phylogenetic ancestor. This suggests methods. First, analyses were conducted within MEGA ver- that viruses belonging to a unique and recent DENV-2 lineage sion 3.1 (Kumar et al. 2004) using either the uncorrected p- circulated during the E2001–E2006 period. However, the distance or the Kimura 2-parameter algorithm for distance virus that circulated in Bolivia in 1997 is more distantly re- calculation, and Neighbor-Joining (NJ) and 500 bootstrap lated (uncorrected p-distance *0.025), and more similar to replicates for construction of unrooted trees (see Fig. 2). Brazilian strains (uncorrected p-distance in the E gene 0.003– Maximum likelihood (ML) analyses were also carried out. 0.009), indicating that viruses from 1997 and from the E2001– The model of nucleotide substitution and parameter values E2006 period originate from two separate introductions. The were selected via Modeltest (Posada and Crandall 1998). ML precise origin of the viruses is difficult to establish but prob- phylogenetic trees were then estimated in GARLI (Zwickl ably in neighboring South =Central American countries or the 2006) using the GTR þ G(gamma) þ I model of nucleotide Caribbean (Fig. 2B), as suggested by all phylogenetic analyses substitution. The substitution matrix, base composition, performed. In addition, viruses from the E2001–E2006 period gamma distribution of among-site rate variation (G), and the share considerable identity with isolates from Paraguay in proportion of invariant sites (I) are available from the corre- 2001 and 2005, suggesting sustained circulation of DENV-2 sponding author on request. A bootstrap resampling analysis between the two bordering countries. was conducted using 1000 replicate NJ trees based on the ML In the case of DENV-3, isolates from E2002, E2003, E2004, substitution matrix described above, in PAUP (Swofford and E2006 were studied. When compared with two E gene 2000). Phylogenetic analyses were also performed via Bayes- sequences available in GenBank and previously produced ian analysis in MrBayes v3.1.2 (Huelsenbeck and Ronquist from Bolivian strains isolated in 2003 (Kochel et al. 2008), all 2001) with model parameters again specified according to these Bolivian viruses belonged to genotype III (Fig. 2C) with Modeltes, one million generations and a burnin of 10%. Sta- uncorrected p-distances in the E gene <0.010 and a common tionary was assessed using Tracer v1.4.1 (part of the BEAST recent phylogenetic ancestor, regardless of the method used package; Drummond and Rambaut 2007). All methods pro- for phylogenetic reconstruction. Viruses belonging to this duced similar topologies at the genotype level with one lineage, presumably originating from South America (Brazil, exception—namely, DENV-1 2007 isolates. In this case, the Venezuela) or the Caribbean, circulated in Bolivia during the ML reconstruction is shown in addition to the NJ trees pre- E2002–E2006 period. sented in Figure 2. Other detailed phylogenetic reconstruc- In conclusion, our study of the molecular epidemiology of tions are available upon request to the corresponding author. dengue fever in Bolivia during the last decade reveals that clo- In the case of DENV-1, isolates from E1998, E2000, E2001, sely related viruses (i.e., viruses with a common recent ancestor) and E2007 were studied. No previously characterized se- circulated during several consecutive years (5, 6, and 6 years for quences from Bolivian DENV-1 were available in public data- DENV-1, DENV-2, and DENV-3, respectively). Emergence of bases. Based on E gene analysis, all Bolivian viruses belonged to new variants (distinct from those identified during the pre- the ‘‘America =Africa’’ genotype V (see Fig. 2A). Isolates from vious episodes) was identified in the case of DENV-1 (E2007) E1998, E2000, and E2001 were very similar (uncorrected p- and DENV-2 (E2001). In all cases, it is likely that the viruses 344 ROCA ET AL. originated from neighboring countries. The determinants of the Kumar, S, Tamura, K, Nei, M. Integrated software for molecular emergence and disappearance of variants remain poorly un- evolutionary genetics analysis and sequence alignment. Brief derstood in Bolivia in the absence of comprehensive epidemio- Bioinform 2004; 5:150–163. logical and entomological studies. The co-circulation of two or Posada, D, Crandall, KA. Modeltest: testing the model of DNA even three serotypes was observed. Future studies may reveal substitution. Bioinformatics 1998; 14:817–818. whether these distinct viral populations can be associated with Roche, RR, Alvarez, M, Guzma´n, MG, Morier, L, et al. Com- different epidemiological (temporal or geographical distribu- parison of rapid centrifugation assay with conventional tissue tion, clinical presentation, and previous immunity) or ecological culture method for isolation of dengue 2 virus in C6 =36-HT characteristics such as vector subpopulations. cells. J Clin Microbiol 2000; 38:3508–3510. Swofford, DL. PAUP*. Phylogenetic Analysis Using Parsimony Disclosure Statement (*and other methods) , 4th ed. Sunderland, MA: Sinauer Asso- ciates; 2000. No competing financial interests exist. Taylor, SF, Patel, PR, Herold, TJ. Recurrent arthralgias in a pa- tient with previous Mayaro fever infection. South Med J 2005; References 98:484–48. Thompson, JD, Gibson, TJ, Plewniak, F, Jeanmougin, F, et al. The Anonymous. Dengue y dengue hemorragico en las Americas: Guia ClustalW windows interface: flexible strategies for multiple para su prevencion y control. Organizacion Panamericana de la sequence alignment aided by quality tools. Nucleic Acids Res Salud. Publicacion Cientifica. Washington, DC. 1995; 548. 1997; 25:4876–4882. Bevier, G, Torres-Munoz, N, Doria-Medina, J. Yellow fever in Van der Stuyft, P, Gianella, A, Pirard, M, Holzman, A, et al. Bolivia, its history and epidemiology. Am J Trop Med Hyg Dengue serotype 2 subtype III (‘Jamaica’) epidemic in Santa 1953; 2:464–482. Cruz, Bolivia. Trop Med Int Health 1998; 3:857–858. Drummond, AJ, Rambaut, A. BEAST: bayesian evolutionary Zwickl, DJ. Genetic algorithm approaches for the phylogenetic analysis by sampling trees. BMC Evol Biol 2007; 7:214. analysis of large biological sequence datasets under the max- Foster, JE, Bennett, SN, Carrington, CV, Vaughan, H, et al. imum likelihood criterion. Ph.D. dissertation. Austin: The Phylogeography and molecular evolution of dengue 2 in the University of Texas at Austin, 2006. Caribbean basin, 1981–2000. Virology 2004; 324:48–59. Garret, J, ed. Historia de la medicina en Santa Cruz . Sirena: Bolivia, 1993. Address correspondence to: Gianella, A, Pirard, M, Holzman, A, Boelaert, M, et al. Epidemic Xavier de Lamballerie outbreak of dengue virus 2 =Jamaica genotype in Bolivia. Sa- Unite´ des Virus Emergents =UMR190 lud Publica Mex 1998; 40:469–473. Faculte´ de me´decine de Marseille Huelsenbeck, JP, Ronquist, F. MRBAYES: bayesian inference of 27 Bd Jean Moulin phylogeny. Bioinformatics 2001; 17:754–755. 13005 Marseille Kochel, T, Aguilar, P, Felices, V, Comach, G, et al. Molecular France epidemiology of dengue virus type 3 in Northern South America: 2000–2005. Infect Genet Evol 2008; 8:682–688. E-mail: [email protected] LETTERS

Dengue Virus a DENV-1–4 real-time RT-PCR ( 4), a pore, Taiwan, Sri Lanka, India, and positive result in a DENV-3–speci [ c Saudi Arabia. Its closest relatives were Type 3 Infection in RT-PCR, and a negative result in a the strain from Saudi Arabia isolated Traveler Returning speci [ c RT-PCR for DENV-1, -2, and in 2004 and another strain claimed from West Africa -4 ( 4). The concomitant [ nding of in ProMED by Japanese researchers DENV RNA and IgG against DENV to have been isolated in 2008 from a To the Editor: GeoSentinel, the suggests the patient had dengue infec- Japanese traveler returning from Côte global surveillance program of the tion before this episode. d’Ivoire ( 6). Therefore, this strain is International Society of Travel Medi- DENV-3 viremia was con [ rmed likely to have originated in the Mid- cine, recently reported that returning by sequencing a 547-nt region of the dle East, the Indian subcontinent, or travelers may serve as sentinels for envelope gene (GenBank accession Southeast Asia rather than in Central local outbreaks of dengue fever in no. FJ587232, nt 852–1398 referring or South America. tropical areas to which it is endemic to the H87 DENV-3 prototype strain). In Africa, most data on epidemic (1). We investigated a case of dengue Our sequence aligned with homolo- or endemic dengue activity originate virus (DENV) type 3 (DENV-3) infec- gous DENV-3 sequences retrieved in East Africa. Dengue fever was sel- tion in a traveler returning to France from GenBank and used for phyloge- dom reported in West Africa. In 2000, from West Africa, which provided evi- netic analysis (Figure). Our patient, in DENV-1 was isolated from a French dence for DENV-3 circulation in Côte whom classic dengue fever was diag- soldier in Côte d’Ivoire ( 7). More re- d’Ivoire. nosed, was infected with a strain that cently, an outbreak caused by DENV-2 A 53-year-old French expatriate belonged to genotype III most closely occurred in Gabon in West Africa living in Abidjan, the economic capi- related to strains isolated in Singa- (8). In this context, it is notable that tal of Côte d’Ivoire, arrived in France Indonesia den3 98 1998 Indonesia FW01 2004 Singapore D3/SG/05K4477DK1 2005 on July 17, 2008, and was hospitalized Indonesia TB55i 2004 86 Indonesia FW06 2004 79 Indonesia PI64 2004 4 days later with fever of 40°C, head- 91 Indonesia KJ71 2004 100 Indonesia PH86 2004 94 Indonesia KJ46 2004 ache, asthenia, anorexia, chills, diffuse Indonesia BA51 2004 96 Indonesia den3 88 1988 73 Thailand D88.303 1988 arthralgia, and myalgia. Results of a Philippines 168.AP-2 1983 Indonesia Sleman/78 1978 Indonesia 228761 1973 physical examination were normal ex- I 100 97 Fr Polynesia PF90/3050 1990 Fr Polynesia PF94/136116 1994 99 Fr Polynesia PF89/27643 1989 cept for a diffuse nonpetechial macular Fr Polynesia PF90/3056 1990 Fr Polynesia PF92/2986 1992 97 Indonesia InJ-16-82 1982 Philippines PhMH-J1-97 1997 rash and moderate hepatosplenomega- 72 Taiwan 95TW466 1995 97 China 80-2 1980 IV 100 Puerto Rico PR6 1963 ly. A tourniquet test was not performed. Philippines H87 1956 Malaysia LN2632 1993 II Thailand D88.086 1988 At admission, platelet count was 103 Bangladesh BDH02-3 2002 100 100 Bangladesh BDH02-7 2002 3 Bangladesh BDH02-4 2002 cells/mm and leukocyte count was 82 Bangladesh BDH02-1 2002 Thailand ThD3 0007 87 1987 3 Thailand D92.431 1992 2,410 cells/mm . Thin and thick blood Indonesia 98TW182 1998 Thailand ThD3 0055 93 1993 83 Thailand D94.122 1994 smears and results of the immunochro- Thailand D97.0106 1997 Thailand C0331/94 1994 Thailand C0360/94 1994 matographic test (Binax NOW malaria 94 China 07CHLS001 unknown Thailand ThD3 1687 98 1998 86 Thailand D94.283 1994 tests; Binax, Portland, ME, USA) were Thailand ThD3 1283 98 1998 100 Singapore D3/SG/05K4648DK1 2005 81 Singapore D3/SG/SS710/2004 negative. The patient recovered with- 100 Singapore unknown 77 Sri Lanka SRI/1266 2000 Taiwan 99TW628 1999 out sequelae and was discharged 6 Sri Lanka 1594 1985 India 1416 1984 Sri Lanka 2783 1991 days after admission. Saudi Arabia 6805 2004 97 Cote d'Ivoire 8417234 2008 (case imported into France) III 100 Cote d'Ivoire NIID48 20 2008 (case imported into Japan) At admission, chikungunya vi- 100 95 Puerto Rico BID V1450 1998 Puerto Rico BID V1620 2005 [ Peru FSL706/Loreto 2002 rus–speci c immunoglobulin (Ig) G 95 Brazil BR74886/02 2002 Martinique 1567 2000 98 Martinique 1706 2000 and IgM were not detected by indirect Martinique 2012 2001 Martinique 1243 1999 \ 0.01 Venezuela BID/V911 2001 immuno uorescence tests ( 2). IgG, Brazil 68784 2000 Brazil BR DEN3 290-02 2002 [ 82 Brazil BR DEN3 95 04 2004 but not IgM, speci c for DENV was Brazil BR DEN3 98 04 2004 Brazil BR DEN3 97 04 2004 99 Bolivia FSB 413 2003 detected by an immunochromatic test Bolivia FSB 439 2003 (Panbio Dengue Duo Cassette; Bi- Figure. Phylogenetic analysis of selected dengue virus type 3 (DENV-3) sequences. The otrin, Lyon, France) and con [ rmed main genotypes are indicated using roman numerals at the node of the lineage. Sequence by ELISA (PanBio dengue duo test). identi [ cation is as follows: country of origin, strain name, year of isolation/detection. The However, DENV RNA was demon- sequence determined in our study is underlined and designated by an arrow. Phylogenetic strated in serum by using 4 reverse- studies were conducted by using MEGA version 2.1 ( 5). Genetic distances were calculated with the Kimura 2-parameter method at the nucleotide level. Phylogenetic trees were transcription–PCR (RT-PCR)–based constructed using the neighbor-joining method. The robustness of the nodes was tested assays: a positive result in a \ avivirus by 500 bootstrap replications. The tree was rooted with DENV-1, DENV-2, and DENV-4 universal assay ( 3), a positive result in sequences. Scale bar indicates nucleotide substitutions per site.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 11, November 2009 1871 LETTERS

DENV-3 strains recently caused un- References Low Immunity to expected outbreaks of dengue hemor- 1. Schwartz E, Weld LH, Wilder-Smith A, Measles and Rubella rhagic fever in Sri Lanka, East Africa, von Sonnenburg F, Keystone JS, Kain and Latin America ( 9). KC, et al. GeoSentinel Surveillance Net- among Female The case presented here demon- work. Seasonality, annual trends, and Guest Workers, strates that epidemics may be unde- characteristics of dengue among ill re- [ turned travelers, 1997–2006. Emerg In- Northern Mariana tected or unidenti ed until diagnosis fect Dis. 2008;14:1081–8. DOI: 10.3201/ is assessed in another country from a eid1407.071412 Islands returning infected visitor, thus draw- 2. Fulhorst CF, Monroe MC, Salas RA, To the Editor: ing attention to an unidenti [ ed poten- Duno G, Utrera A, Ksiazek TG, et al. Iso- The Common- lation, characterization and geographic wealth of the Northern Mariana Is- tial epidemic situation. This situation distribution of Cano Delgadito virus, a can be unraveled by a clinician who lands (CNMI), a group of northern Pa- newly discovered South American hanta- [ considers geographic factors in the virus (family Bunyaviridae ). Virus Res. ci c islands in political union with the diagnostic workup and has access to 1997;51:159–71. DOI: 10.1016/S0168- United States, was exempt from US and uses appropriate laboratory capac- 1702(97)00091-9 labor laws until 2007. This exemption 3. Moureau G, Temmam S, Gonzalez JP, attracted business opportunities, which ity to diagnose imported infections. Charrel RN, Grard G, de Lamballerie X. A At the time dengue was diagnosed real-time RT-PCR method for the universal led to a high demand for guest work- in this patient, cases of yellow fever detection and identi [ cation of \ aviviruses. ers. The Centers for Disease Control were reported in the same location of Vector Borne Zoonotic Dis. 2007;7:467– and Prevention advises the US Citi- 77. DOI: 10.1089/vbz.2007.0206 zenship and Immigration Services of Côte d’Ivoire (Abidjan) ( 5), illustrat- 4. Leparc-Goffart I, Baragatti M, Temman S, ing concomitant circulation of 2 vi- Tuiskunen A, Moureau G, Charrel R, et al. vaccination requirements for those ap- ruses in which dengue may have re- Development and validation of real time plying for immigration and work visas mained undetected in the absence of a one-step reverse transcription-PCR for the before the applications are approved detection and typing of dengue viruses. J (1). Since 1996, all applicants born af- laboratory-con [ rmed case in the trav- Clin Virol. 2009;45:61–6. eler’s home country. Therefore, this 5. Kumar S, Tamura K, Jakobsen IB, Nei M. ter 1957 and >12 months of age have case reinforces the utility of travelers MEGA2: Molecular Evolutionary Genet- been required to provide evidence of as sentinels for infectious diseases as ics Analysis software. Tempe (AZ): Ari- completed vaccination against, or of zona State University; 2001. immunity to, measles, mumps, and ru- previously reported ( 10 ). Our [ nd- 6. Dengue in Africa: emergence of DENV-3, ings reiterate the need for technologic Côte d'Ivoire, 2008. Wkly Epidemiol Rec. bella viruses. Those unable to provide transfer of PCR-based direct diagnos- 2009;84:85–8. such evidence must receive at least 1 tics to reference centers in areas where 7. Durand JP, Vallée L, de Pina JJ, Tolou H. dose of the vaccines recommended by Isolation of a dengue type 1 virus from the US Advisory Committee on Im- emergence is likely. These efforts also a soldier in West Africa (Côte d'Ivoire). should embrace serology and encour- Emerg Infect Dis. 2000;6:83–4. DOI: munization Practices before visa ap- age close collaboration with world 10.3201/eid0602.000211 proval. The Committee also advises reference centers for con [ rmation and 8. Leroy EM, Nkogue D, Ollomo B, Nze- applicants to receive additional doses Nkogue C, Becquart P, Grard G, et al. of the required vaccines after arrival in characterization ( 10 ). Concurrent chikungunya and dengue virus infections during simultaneous the Mariana Islands. We aimed to de- termine the proportion of CNMI guest Acknowledgments outbreaks, Gabon, 2007. Emerg Infect Dis. 2009;15:591–3. DOI: 10.3201/ workers who were immune to measles We thank David Freedman, Eu- eid1504.080664 and rubella by testing a convenience roTravNet members, and the reviewers for 9. Messer WB, Gubler DJ, Harris E, Si- sample of serum collected during Sep- helpful comments and discussion. vananthan K, de Silva AM. Emergence and global spread of a dengue serotype tember and October 2006. However, 3, subtype III virus. Emerg Infect Dis. procedures for validating the vaccina- Laetitia Ninove, Philippe Parola, 2003;9:800–9. tion status for our sample population Cécile Baronti, 10. Freedman DO, Weld LH, Kozarsky PE, Fisk T, Robins R, von Sonnenburg F, et al. are unknown. Given our results, it Xavier De Lamballerie, Spectrum of disease and relation to place appears that validation procedures of Philippe Gautret, of exposure among ill returned travelers. immunity status in guest workers or Barbara Doudier, N Engl J Med. 2006;354:119–30. DOI: immigrants to the United States were 10.1056/NEJMoa051331 and Rémi N. Charrel suboptimal at the time of this study. Author af [ liation: Fédération de Microbiolo- Address for correspondence: Rémi N. Charrel, Serum samples from 210 female gie Clinique Assistance Publique Hôpitaux Université de la Mediterranee–Unite des Virus workers from 17 through 51 years of de Marseille, Marseille, France Emergents, 27 bd Jean Moulin, Marseille 13005, age were collected opportunistically DOI: 10.3201/eid1511.081736 France; email: [email protected] when, as a requirement for annual con-

1872 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 15, No. 11, November 2009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

Antiviral Research xxx (2009) xxx–xxx

Contents lists available at ScienceDirect

Antiviral Research

journal homepage: www.elsevier.com/locate/antiviral

Review Structure and functionality in flavivirus NS•proteins: Perspectives for drug design

Michela Bollati a, Karin Alvarez b, René Assenberg c, Cécile Baronti d, Bruno Canard b, Shelley Cook e, Bruno Coutard b, Etienne Decroly b, Xavier de Lamballerie d, Ernest A. Gould d,f, Gilda Grard d, Jonathan M. Grimes c, Rolf Hilgenfeld g, Anna M. Jansson h, Hélène Malet b, Erika J. Mancini c, Eloise Mastrangelo a,i, Andrea Mattevi j, Mario Milani a,i, Grégory Moureau d, Johan Neyts k, Raymond J. Owens c, Jingshan Ren c, Barbara Selisko b, Silvia Speroni j, Holger Steuber g, David I. Stuart c, Torsten Unge h, Martino Bolognesi a,∗ a Department of Biomolecular Sciences and Biotechnology, University of Milano, Via Celoria 26, 20133 Milano, Italy b Laboratoire Architecture et Fonction des Macromolécules Biologiques, CNRS UMR•6098, Universités Aix•Marseille I et II, ESIL Case 925, 163 Avenue de Luminy, 13288 Marseille, France c Oxford Protein Production Facility, Wellcome Trust Centre for Human Genetics, Roosevelt Drive, Headington, Oxford OX3 7BN, UK d Unité des Virus Emergents, Faculté de Médecine, 27 Bd Jean Moulin, 13005 Marseille, France e The Natural History Museum, Cromwell Road, London, United Kingdom f Centre for Ecology and Hydrology, Mansfield Road, Oxford OX1 3SR, United Kingdom g Institute of Biochemistry, Center for Structural and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany h Department of Cell and Molecular Biology, Uppsala University, Biomedical Center, Box 596, SE•751 24 Uppsala, Sweden i CNR•INFM S3, National Research Center on Nanostructure and BioSystems at Surfaces, Via Campi 213/A, 41100 Modena, Italy j Department of Genetics and Microbiology, University of Pavia, Via Ferrata 7, 27100 Pavia, Italy k Rega Institute for Medical Research, KULeuven • University of Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium article info abstract

Article history: Flaviviridae are small enveloped viruses hosting a positive•sense single•stranded RNA genome. Besides Received 2 July 2009 yellow fever virus, a landmark case in the history of virology, members of the Flavivirus genus, such as Received in revised form 8 September 2009 West Nile virus and dengue virus, are increasingly gaining attention due to their re•emergence and inci• Accepted 21 November 2009 dence in different areas of the world. Additional environmental and demographic considerations suggest that novel or known flaviviruses will continue to emerge in the future. Nevertheless, up to few years ago Keywords: flaviviruses were considered low interest candidates for drug design. At the start of the European Union Flavivirus VIZIER Project, in 2004, just two crystal structures of protein domains from the flaviviral replication Flaviviral NS3 protein Flaviviral NS5 protein machinery were known. Such pioneering studies, however, indicated the flaviviral replication complex Protease as a promising target for the development of antiviral compounds. Here we review structural and func• Helicase tional aspects emerging from the characterization of two main components (NS3 and NS5 proteins) of the Polymerase flavivirus replication complex. Most of the reviewed results were achieved within the European Union Methyltransferase VIZIER Project, and cover topics that span from viral genomics to structural biology and inhibition mech• Flavivirus protein structure anisms. The ultimate aim of the reported approaches is to shed light on the design and development of Antivirals antiviral drug leads. VIZIER Consortium © 2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction ...... 00 1.1. Emergence and re•emergence of pathogenic flaviviruses ...... 00

Abbreviations: BVDV, bovine viral diarrhea virus; C, capsid protein; CSFV, classical swine fever virus; CCHFV, Crimean•Congo hemorrhagic fever virus; CPE, cyto• pathogenic effect; dsRNA, double•stranded RNA; ER, endoplasmic reticulum; E, envelope protein; GMP, guanosine monophosphate; GTP, guanosine triphosphate; GTase, guanylyltransferase; NS3Hel, helicase; HIV, Human Immunodeficiency Virus I; HCV, hepatitis C virus; HBS, high affinity binding site; IMP, Inosine 5 ′•monophosphate; LBS, low• affinity binding site; M, membrane protein; NS5MTase, methyltransferase; N7MTase, (guanine•N7)•methyltransferase; 2 ′OMTase, (nucleoside•2 ′•O•)•methyltransferase; NS, non•structural; NLS, nuclear localization sequences; NS3Pro, protease; RC, replication•competent complex; RSV, respiratory syncytial virus; NS5RdRp, RNA•dependent RNA polymerase; NS3RTPase, RNA triphosphatase; AdoMet, S•adenosyl•L•methionine; ssRNA, single•stranded RNA; T•705 RMP, T•705•ribofuranosyl•5 ′•monophosphate; VIZIER, Viral Enzymes Involved in Replication. ∗ Corresponding author. Tel.: +39 02 5031 4893; fax: +39 02 5031 4895. E•mail address: [email protected] (M. Bolognesi).

0166•3542/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi: 10.1016/j.antiviral.2009.11.009

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

2 M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx

1.2. Development of flavivirus treatments ...... 00 1.3. Molecular biology of flavivirus polyprotein processing and replication: the roles of NS5 and NS3 ...... 00 1.4. The VIZIER context ...... 00 2. Flavivirus genomics ...... 00 2.1. The first steps in flavivirus genomics ...... 00 2.2. E gene and NS5 datasets...... 00 2.3. Recent advances in flavivirus genomics ...... 00 2.3.1. Sequencing methods ...... 00 2.3.2. Sequencing of previously discovered flaviviral species ...... 00 2.3.3. Newly discovered flaviviruses ...... 00 3. Structure and function of flaviviral enzymes ...... 00 3.1. The flaviviral NS3 protein ...... 00 3.1.1. NS3 protease domain ...... 00 3.1.2. NS3 helicase domain...... 00 3.1.3. The full•length NS3 protein ...... 00 3.2. The flaviviral NS5 protein ...... 00 3.2.1. NS5 methyltransferase domain ...... 00 3.2.2. NS5 RNA•dependent RNA polymerase domain...... 00 4. Antivirals ...... 00 4.1. A broad•spectrum antiviral molecule with weak activity ...... 00 4.2. Selective inhibitors of viral replication ...... 00 4.3. Identification of novel antivirals...... 00 4.3.1. High•throughput screening approach...... 00 4.3.2. Virtual docking of small molecules ...... 00 Acknowledgements ...... 00 Appendix A. Supplementary data ...... 00 Appendix A. Supplementary data ...... 00 References ...... 00

1. Introduction about 12,500–25,000 deaths annually, DENV is robustly emerging in a growing number of countries ( Vasilakis and Weaver, 2008 ). The The genus Flavivirus, together with Pestivirus and Hepacivirus, two remaining clinically significant flaviviruses are the Japanese belongs to the family of Flaviviradae. Flaviviridae are small encephalitis virus (JEV) and tick•borne encephalitis virus (TBEV), enveloped viruses hosting a positive•sense single•stranded RNA for which existing vaccines should help reduce the current mor• genome. The complete genome is 9500–12,500 nucleotides long. bidity burden, mostly in Asia and central Europe, respectively. Most It encodes a large polyprotein precursor, which is co• and post• flaviviruses are arthropod•borne viruses (arboviruses), transmitted translationally processed by viral and cellular proteases into three either by ticks (tick•borne viruses, TBV) or mosquitoes (mosquito• structural proteins, building the capsid, and seven non•structural borne viruses, MBV), but a number of flaviviruses have no known proteins involved in virus replication. vectors (NKV) and/or have been isolated from infected animals without a link to any specific disease ( Table 1 ). 1.1. Emergence and re•emergence of pathogenic flaviviruses 1.2. Development of flavivirus treatments In the Flaviviridae family, the genus Flavivirus occupies a special space within the RNA virus world. The family derives its name from There are a number of environmental, demographic and eco• the word flavus (Latin for yellow), with one prominent member logical reasons to believe that either novel or known flaviviruses being the yellow fever virus (YFV) a landmark reference system will continue to emerge. In this respect, the success of vaccina• in the history of virology. It was introduced in the Americas in the tion against YFV has been temperated by difficulties encountered 16th century as a consequence of the African slave trade, recognized when such programs were launched against DENV. In particu• by Carlos Finlay as a vector•borne disease as early as 1881, before lar, the presence of four DENV serotypes has complicated vaccine any virus was isolated. YFV was the first human pathogenic virus design because incomplete protection against one serotype may isolated in 1927 ( Staples and Monath, 2008 ). Although a safe and influence the disease outcome once infection is established by a dis• efficient vaccine designed in 1937 by Max Theiler shaped our view tinct serotype, through a process referred to as antibody•mediated on the control of viruses, there are still more than 200,000 annual disease enhancement ( Guzman and Kouri, 2008 ). Therefore, in cases in Africa alone, and about 15% of the cases enter a critical addition to vaccine design efforts, there has been a growing inter• phase that only 50% of the patients survive ( Ellis and Barrett, 2008 ). est in discovering drugs against DENV and WNV. For instance, a In more recent years, members of the Flavivirus genus gained public moderate, borderline effect, whose mechanism of action is contro• visibility due to re•emergence and steadily increasing incidence, versial, was reported for the activity of ribavirin against flaviviruses such as for West Nile virus (WNV) in the Americas and dengue (Huggins, 1989; Day et al., 2005; Leyssen et al., 2006; Takhampunya virus (DENV) in subtropical areas of the world. et al., 2006 ). Prior to 2004 there were very few coordinated efforts WNV, isolated in Uganda in 1937, is endemic in Africa and towards the design of antiflavivirus compounds, flaviviruses being southern Europe, but its appearance in the Americas in 1999 was hardly considered interesting candidates for drug design. A notable followed by a rapid geographic extension from Canada to Argentina exception has been the activity at the Novartis Institute for Tropi• by 2008, leaving behind thousands of deaths and disabled patients cal Disease in Singapore that focused its research efforts on dengue (Petersen and Hayes, 2008 ). Likewise, the four DENV serotypes disease since its first opening (in 2003) ( Gubler and Clark, 1995; have considerably expanded their geographic distribution in recent Kroeger et al., 2004 ). Perhaps even before the launch of the Euro• years. With billions of people at risk, more than 50 million cases, and pean Union VIZIER Project (Viral Enzymes Involved in Replication)

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx 3

Table 1 in October 2004, the lack of viral genomics programs was recog• Flaviviral abbreviation. nized as a problem for any research activity aiming at the discovery Tick•borne viruses TBVs and design of antiviral drugs based on crystal structure information. Gadget Gully virus GGYV Indeed, since a single amino acid substitution can determine resis• Kadam virus KADV tance to a given drug, systematic benchmarking of starting genetic Kyasanur Forrest disease virus KFDV Langat virus LGTV material and resulting data was highly sought after. Few complete Omsk hemorrhagic fever virus OHFV flavivirus genome sequences were known at the launch of VIZIER Powassan virus POWV (less than 30 out of >70), and the commitment to sequence the Royal farm virus RFV entire Flavivirus genus in VIZIER (see below) was of key impor• Karshi virus KSIV tance in the standardization of cDNA targets and their referencing tick•borne encephalitis virus TBEV Louping ill virus LIV during the project. Meaban virus MEAV Saumarez Reef virus SREV 1.3. Molecular biology of flavivirus polyprotein processing and Tyuleniy virus TYUV replication: the roles of NS5 and NS3 Ngoye virus NGOV

Mosquito•borne viruses MBVs The ∼11 kb flavivirus RNA genome is a positive•sense, single• Aroa virus AROAV stranded, 5 ′•capped RNA ((+)ssRNA) that is released into the Bussuquara virus BSQV Iguape virus IGUV cytoplasm immediately following cell entry. It encodes a single, Naranjal virus NJLV large polyprotein, which is proteolytically processed to yield three Dengue virus DENV structural proteins (envelope, E; membrane precursor, PrM; and Kedougou virus KEDV capsid C) and seven non•structural (NS) proteins (NS1, NS2a, NS2b, Cacipacore virus CPCV NS3, NS4a, NS4b, and NS5). The polyprotein is cleaved co• and Koutango virus KOUV Japanese encephalitis virus JEV post•translationally by a combination of cellular proteases of the Murray Valley encephalitis virus MVEV furin•type or other Golgi•localized proteases and the viral serine Alfuy virus ALFV protease embedded in the N•terminal domain of non•structural St Louis encephalitis virus SLEV protein 3 (NS3Pro), which requires NS2B for its activity. NS proteins Usutu virus USUV West Nile virus WNV are thought to co•translationally assemble on the endoplasmic Kunjin virus KUNV reticulum (ER) membranes forming the replication competent Yaounde virus YAOV complex, which consists morphologically distinct, membrane• Kokobera virus KOKV bound compartments that also differ with respect to both function Stratford virus STRV and NS proteins composition (reviewed in: Mackenzie, 2005 ). The Bagaza virus BAGV Ilheus virus ILHV NS3 and NS5 proteins are central to the viral RC, as together they Rocio virus ROC harbour most, if not all, of the catalytic activities required to both Israel turkey meningoencephalomyelitis virus ITV cap and replicate the viral RNA. NS3 is a multidomain protein, with Ntaya virus NTAV an N•terminal NS3Pro as discussed above, and a C•terminal portion Tembuzu virus TMUV Zika virus ZIKV containing the RNA triphosphatase (NS3RTPase) and RNA helicase Spondweni virus SPOV (NS3Hel) activities involved in capping and viral RNA synthe• Banzi virus BANV sis, respectively. NS5 consists of an N•terminal methyltransferase Bouboui virus BOUV (NS5MTase) domain and the C•terminal RNA•dependent RNA poly• Edge Hill virus EHV merase (NS5RdRp) domain. During these processes, the (+)ssRNA Jugra virus JUGV Potiskum virus POTV viral genome acts as a template for: (1) the synthesis of the inter• Saboya virus SABV mediate ( −)ssRNA strand by the NS5 RdRp, which in turn acts as Sepik virus SEPV template solely for the synthesis of (+)ssRNA genomic RNAs (again Uganda S virus UGSV by the NS5), and (2) the synthesis of the viral polyprotein. Sitiawan virus SV ′ Kamiti River virus KRV The 5 •end of the (+)ssRNA genome is decorated by a RNA cap ′ Wesselsbron virus WESSV structure (N7meGpppA2 Ome•RNA). It plays an essential role, act• Yellow fever virus YFV ing, as for eukaryotic mRNAs, to initiate the process of translation Nounané virus NOUV and to protect the viral RNA from degradation by endogenous RNA Barkedji virus exonucleases. It is also a unique feature of the flavivirus genome in Viruses with no known arthropod vector NKVs the context of the Flaviviridae family as a whole, since pesti• and Entebbe bat virus ENTV hepaciviruses do not possess it. In flaviviruses, mRNA capping is Sokoluk virus SOKV ′ Yokose virus YOKV thought to start with the conversion of the 5 •triphosphate mRNA Apoi virus APOIV into a diphosphate by the RNA triphosphatase domain embedded in Cowbone Ridge virus CRV the C•terminal domain of the NS3 protein (NS3RTPase). The second Jutiapa virus JUTV reaction is the transfer of a guanosine monophosphate (GMP) moi• Modoc virus MODV ety from a guanosine triphosphate (GTP) to 5 ′•diphosphate RNA, Sal Vieja virus SVV ′ San Perlita virus SPV to yield G5 •ppp•N, by a guanylyltransferase (GTase). Afterwards, Bukalasa bat virus BBV the transferred guanosine moiety is methylated by the N•terminal Carey Island virus CIV methyltransferase domain of the NS5 protein (NS5MTase). To date, Dakar bat virus DAKV the molecular species responsible for the GTase activity remains Montana Myotis leukoencephalitis virus MMLV Phnom Penh bat virus PPBV unknown ( Ahola and Kaariainen, 1995; Furuichi and Shatkin, 2000; Batu Cave virus BCV Egloff et al., 2002; Ray et al., 2006 ), although recent evidences sug• Rio Bravo virus RBV gest that it might be associated to the NS5MTase domain ( Egloff et Cell fusing agent virus CFAV al., 2007; Bollati et al., 2009b ). Tamana bat virus TABV Although the details of flavivirus replication have seen major advances in the past years, many aspects remain not fully under•

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

4 M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx stood. For instance, an increasing number of studies have shown (3) YFV is the prototype species of the genus flavivirus. The 17D that specific RNA structures present in the 5 ′ and 3 ′ UTR regions vaccine is one of the most efficient vaccines ever developed play a critical role in replication and capping, with genome cycliza• and was derived from a strain of YFV isolated from a man who tion being one of several processes identified on which replication recovered from infection by the virus. depends. However, the precise details, such as how NS5 and/or (4) Flaviviruses are “complex” viruses, with various – and poorly NS3Hel activities might be controlled by such structures, remain understood – ecological cycles. Importantly, most of the to be established. Equally, the role of NS3Hel in these processes human pathogens are transmitted by arthropods (i.e. they remains to be established formally and hence, analyzed in more are “arboviruses”). However, viruses with no known vector, detail, although it is thought to be at least involved in the formation or viruses that infect only arthropods (tentatively referred to of the 5 ′ cap structure of viral RNA and in the unwinding of dsRNA as “insect•only” flaviviruses) have also been identified. This intermediates that arise during replication. Observations such as remarkable diversity is associated with broad genetic variabil• those showing that NS3Hel has an apparently unrelated function in ity and complex mechanisms of pathogenesis. the downstream assembly of the virion indicate that many aspects (5) The genus flavivirus underlines the history of mammalian virol• of NS3 as well as NS5 function remain to be established. ogy since the first human viral pathogen to be demonstrated as Following replication the protected, genomic RNA is packaged a filterable agent was YFV ( Reed, 1901a,b ) and subsequently by the C protein in a host•derived lipid bilayer in which the E protein the first human viral pathogens isolated experimentally were is embedded. The mature particles subsequently exit from the host YFV ( Theiler, 1930 ) and Louping ill virus (LIV) ( Greig et al., cell by exocytosis. 1931 ), followed soon after by DENV and African horse sickness virus. (6) Flaviviruses have historically been associated with changes in 1.4. The VIZIER context taxonomy that reflected advances in virology. Firstly, the term arborviruses (subsequently changed to arboviruses to avoid At the start of the VIZIER Project, crystal structures of only two confusion with the Latin word “arbor”, meaning tree) was flavivirus replication protein domains (DENV NS3Pro, Murthy et derived as a taxonomic criterion following the discovery of al., 1999 ; and the DENV NS5MTase, Egloff et al., 2002 ) were known. several arthropod•borne viruses. Subsequently, morphological In addition to their biological relevance (discussed below) these information obtained using electron microscopy supported the studies had a pioneering value since they showed that individual hypothesis of the existence of at least two virus groups: domains of NS3 and NS5 could be produced in isolation and their crystal structure solved. • One group includes non•enveloped viruses, which currently As a result, flavivirus NS3 and NS5 proteins were held as targets are classified within the family Reoviridae (genera Orbivirus, for the VIZIER Project. On one hand, NS3 and NS5 constitute impor• Coltivirus and Seadornavirus), i.e. viruses with an overall diam• tant drug targets, and on the other they were held s targets within eter of 60–80 nm, icosahedral symmetry and several concentric reach for large scale production and crystallization, thereby facili• capsid layers that surround a segmented double•stranded RNA tating the cementing of the VIZIER community, the beta•testing of (dsRNA) genome. the communication protocols and project pipelines, and the estab• • A second group includes enveloped viruses (inactivated by ether lishment of bridges between the structural biology and virology and deoxycholate), 50–60 nm in diameter, with an infectious laboratories expertises. ssRNA genome of positive polarity. The development of serologi• In the following sections of this paper, we will present the collec• cal methods led to the identification of two antigenically distinct tive efforts developed for the characterization of several flavivirus sub•groups. This division was subsequently confirmed by anal• molecular aspects within the VIZIER Project ( http://www.vizier• ysis of the genome sequences and the viruses were divided as europe.org/ ), from viral genomics to structural biology approaches follows: focused on flavivirus NS3 and NS5, emphasizing the implications (a) The “Group A arboviruses”, comprising viruses currently that the data produced bear for antiviral drug development. classified within the genus Alphavirus, family Togaviridae (together with the non•arboviral genus Rubivirus). (b) The “Group B arboviruses”, comprising viruses currently 2. Flavivirus genomics classified within the genus Flavivirus, family Flaviviridae (together with non•arboviral genera Hepacivirus and Pes• The flaviviruses comprise a fascinating group of viruses, occu• tivirus). pying a very special position in the history of virology due to their taxonomic, epidemiological and pathogenetic characteristics, 2.1. The first steps in flavivirus genomics which include the following:

The history of flavivirus genomics did not start with the progres• (1) The genus Flavivirus, contains an unusually large number of sive accumulation of partial genome sequences but, surprisingly, viruses (more than 70), that are distributed globally. The genus with the publication in 1985 of a seminal study by Rice et al. also includes a large, and increasing, number of unclassified or (1985) who determined the complete genome sequence of YFV. “tentative” species that have very different characteristics from The work of Rice and his collaborators was remarkable because it those currently recognized as members of the genus. unexpectedly established that the flavivirus genome strategy was (2) Among the flaviviruses there are more than 40 human very distinct from that of the alphaviruses that had been grouped pathogens, responsible for a variety of diseases ranging taxonomically in the same virus family. Indeed, results demon• from poorly specific pseudo•flu•like syndromes, to severe strated the existence of 5 ′ and 3 ′ non•coding regions and, a unique encephalitic or hemorrhagic disease. One flaviviral disease of single open reading frame that encoded a polyprotein containing particular note is dengue fever, which is estimated to cause all the structural proteins in the N•terminal region of the genome in excess of 50 million cases per year (WHO, Fact sheet No. and all the non•structural proteins in the C terminal region of the 117, March 2009). Many other flaviviral diseases, such as West genome. Nile fever, Japanese encephalitis, and Zika fever are classified This founding discovery was followed by the rapid characteriza• as emerging diseases. tion of a large number of complete sequences for other flaviviruses:

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx 5

WNV ( Castle et al., 1986 ), JEV ( Sumiyoshi et al., 1987 ), Kunjin virus 2.3.2. Sequencing of previously discovered flaviviral species (KUNV) ( Coia et al., 1988 ), DENV4 ( Zhao et al., 1986; Mackow et al., Since the year 2000, significant progress has been made in 1987 ), DENV2 ( Hahn et al., 1988; Irie et al., 1989 ), TBEV ( Mandl et the field of flavivirus genomics. Billoir et al. (2000) produced the al., 1989; Pletnev et al., 1990 ), DENV3 ( Osatomi and Sumiyoshi, first complete sequences of NKVs (i.e. APOIV and RBV). This was 1990 ), DENV1 ( Fu et al., 1992 ), Powassan virus (POWV) ( Mandl followed by the characterization of other NKVs: the MODV and et al., 1993 ), LIV ( Gritsun et al., 1997 ), Murray Valley encephali• Montana Myotis leukoencephalitis viruses (MMLV) ( Charlier et tis virus (MVEV) ( Hurrelbrink et al., 1999 ), and Langat virus (LGTV) al., 2002; Leyssen et al., 2002 ), YOKV ( Tajima et al., 2005 ) and (Campbell and Pletnev, 2000 ). EBV ( Kuno and Chang, 2006 ). The highly atypical Tamana bat This first series of full•length genome sequences included the virus (TABV) was also characterized. TABV was isolated in 1973 first “atypical” flavivirus. In 1992, Cammisa•Parks et al. (1992) in Trinidad from a Pteronotus parnelii bat ( Price, 1978 ) and its tax• reported the discovery and complete characterization of Cell• onomic position remained unresolved for nearly 30 years. Genome Fusing Agent virus (CFAV). For the first time, a very distantly related sequencing finally revealed that the virus was clearly, but very dis• virus was studied and, importantly, results implied that the fla• tantly, related to other known flaviviruses ( de Lamballerie et al., vivirus lineage included viruses which infect only mosquitoes, in 2002 ). The evolutionary relationship of this virus (which chroni• other words they are insect viruses which do not appear to infect cally infects bats and has no known vector) with other flaviviruses mammals. Together with the previous isolation and antigenic char• remains unclear. Complete sequences were also established for acterization of a number of viruses with no identified vector (i.e. a number of “classical” arboviruses within the genus: St. Louis infecting only vertebrates) such as Rio Bravo virus (RBV) ( Burns encephalitis virus (SLEV) ( Billoir et al., 2000 ), Usutu virus (USUV) et al., 1957; Johnson, 1957 ), this provided robust evidence that (Bakonyi et al., 2004 ), Iguape (IGUV), Bussuquara (BSQV), Kokobera the ecological and genetic complexity of the flaviviruses had been (KOKV) and Ilheus (ILHV) viruses ( Kuno and Chang, 2005 ), Alfuy under•estimated. virus (ALFV) ( May et al., 2006 ), Sepik virus (SEPV) ( Kuno and Chang, 2006 ), Kedougou (KEDV), Zika (ZIKV) and Bagaza (BAGV) viruses (Kuno and Chang, 2007 ), and Rocio virus (ROCV) ( Medeiros et al., 2.2. E gene and NS5 datasets 2007 ). The VIZIER Project has enabled full•length genome characteri• In parallel, studies of partial sequences commenced, focusing zation of all previously identified flavivirus species. The analysis of mainly on flavivirus E genes. Increased availability of E gene data all tick•borne flavivirus species ( Grard et al., 2007 ) led to significant enabled the construction of the first robust phylogenies for the development of the previously recognized taxonomic classification, genus. Importantly, these studies globally confirmed the previous e.g. the creation of the Kadam TBV group, and of the Karshi virus classification of flaviviruses ( Porterfield, 1980; Calisher et al., 1989 ) species, and the assignment of TBEV and LIV to a unique species based on antigenic relationships ( Blok et al., 1992; Lewis et al., (TBEV) which included the four viral types: Western TBEV, Eastern 1993; Mandl et al., 1993; Zanotto et al., 1996 ), but also established TBEV, Turkish sheep TBEV and LIV. milestone observations regarding flaviviral evolution. In particu• Within VIZIER, similar studies devoted to other flavivirus groups lar, they suggested that TBVs and MBVs evolved independently have been conducted. In the Aedes•borne virus group, the com• from a common ancestor, that viruses belonging to the tick•borne plete coding sequences of Potiskum (POTV), Saboya (SABV), Jugra encephalitis complex evolved as an arboviral cline across the north• (JUGV), Banzi (BANV), Uganda S (UGSV), Bouboui (BOUV), Edge ern hemisphere, and that, within the group of MBVs, the lineage of Hill (EHV), Sepik (SEPV), Wesselsbron (WESSV), Kedougou (KEDV), Culex spp.•associated flaviviruses emerged from that of Aedes spp. Zika (ZIKV) and Spondweni (SPOV) viruses have now been estab• associated viruses. lished or verified ( Grard et al., in press ). In the group of Culex•borne In 1998, Kuno et al. (1998) published a genetic study based on viruses (Moureau et al., unpublished data), the complete coding partial NS5 RdRp sequences. For the first time, phylogenies included sequences of Aroa (AROAV), Stratford (STRV), Naranjal (NJLV), Israel a very large number of flaviviruses from different genetic or eco• Turkey (ITV), Ntaya (NTAV), Sitiawan (SV), Tembuzu (TMUV), Caci• logical groups, i.e. MBVs and TBVs, also in addition to NKVs, plus pacore (CPCV), Koutango (KOUV) and Yaounde (YAOV) viruses CFAV. This study confirmed the major findings of previous E gene have been characterized. In the case of the NKV flaviviruses phylogenies, but also led to clarification of the two different groups (Moureau et al., unpublished data), the sequences of Sokuluk of NKV: one that constitutes a large independent lineage (e.g. RBV, (SOKV), Bukalasa bat (BBV), Dakar bat (DAKV), Batu cave (BCV), Apoi virus (APOIV), Bukalasa bat virus (BBV), Modoc virus (MODV), Phnom Penh bat (PPBV), Carey Island (CIV), Cowbone Ridge (CRV) etc.) and one that is related to YFV, within the group of Aedes•borne and Sal Vieja virus (SVV) were obtained. In all cases, the addi• viruses (Entebbe bat (ENTV), Yokose (YOKV) and Sokuluk (SOKV) tional information has enabled new, further analyses of a large viruses). panel of flaviviral species to be performed and provided relevant information regarding taxonomic classification and evolutionary 2.3. Recent advances in flavivirus genomics relationships.

2.3.1. Sequencing methods 2.3.3. Newly discovered flaviviruses Most complete flaviviral sequences characterized to date have In recent years, a number of interesting atypical viruses related been produced using complementary DNA clone(s) of the viral to known flaviviruses have been identified: genome, or, more recently, following overlapping PCR amplifica• tions along the viral genome. The latter method was optimized THE CFAV GROUP —A second virus related to CFAV, Kamiti River within the framework of the VIZIER Project: the LoPPS method, a virus (KRV), was isolated in 2003 from African Aedes Macintoshi shotgun•based approach applied to long PCR amplification prod• mosquito ( Crabtree et al., 2003; Sang et al., 2003 ). Subsequently, ucts, was proven to be cost•effective and enabled the complete field isolates of CFAV were identified from New World Aedes and sequencing of large PCR products in a high•throughput for• Culex mosquitoes ( Cook et al., 2006 ). Recently, a new flavivirus mat ( Emonet et al., 2006, 2007 ). More recently, high•throughput associated with phlebotomines has been detected by molecular pyrosequencing methods ( Margulies et al., 2005 ) have shown biology in Algeria ( Moureau et al., 2009 ), and another new insect potential for the rapid characterization of viruses produced in cell flavivirus associated with Ochlerotatus caspius , Ae. vexans , Cx. thei• cultures. leri , Anopheles atroparvus and Culiseta annulata has been detected

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

6 M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx

in Spain ( Aranda et al., 2008 ). An additional insect flavivirus asso• ciated with Culex spp. has been also described from Japan ( Hoshino et al., 2007 ), in Guatemala ( Morales•Betoulle et al., 2008 ), in Mex• ico ( Farfan•Ale et al., 2009 ), and in both the USA and Trinidad (Kim et al., 2009 ). Taken together, these studies have revealed that the genetic and ecological diversity of CFAV•related viruses is much higher than previously thought. Indeed, apparently such viruses commonly infect a large range of mosquito species all over the world and are hypothesized to be more accurately described as “insectiviruses” (as opposed to arboviruses). The discovery of long CFAV•related sequences inserted into the cellular genomes of Aedes albopictus and Ae. aegypti mosquitoes ( Crochu et al., 2004 ) provided an unexpected and intriguing suggestion of an intimate and complex relationship between Aedes spp. mosquitoes and CFAV•related viruses. NGOYE VIRUS —Another unique virus, “Ngoye virus” (NGOV), was Fig. 1. Model representation of NS3 (full•length) anchoring via NS2B to the ER mem• identified by molecular methods from Rhipicephalus ticks sam• brane. The N•terminal NS3Pro domain is shown in blue, the NS3Hel domain in green. pled from Bovidae in Senegal. This virus has not yet been The crystal structure of DENV4 NS2B/NS3 (PDB entry 2VBC, Luo et al., 2008a ) was successfully propagated in cell cultures or newborn mice ( Grard et used for model preparation. The NS2B protein is shown in yellow, modeled regions al., 2006 ). It is more closely related to “classical” flaviviruses than it are shown as dashed lines and helices anchoring the complex to the membrane. is to TABV, but it also constitutes a new independent evolutionary lineage within the genus Flavivirus. NS3Pro resulting in the formation of a heterodimeric complex. NEW AEDES ASSOCIATED VIRUSES —Recently, Nounané virus NS2B is a small protein ( ∼14 kDa) with a central hydrophilic part (NOUV) was isolated from Uranotaenia in Côte d’Ivoire ( Junglen (residues 49–89) involved in binding to NS3, thereby fulfilling a et al., 2009 ) and Barkedji virus in Senegal (Dupressoir et al., chaperone•like role in stabilizing the latter protein, and two ter• unpublished data, GB EU078325, 2008). These viruses seem to minal hydrophobic regions responsible for membrane association represent a new and distinct group inside the MBV group ( more of the NS2B/NS3 complex ( Fig. 1 ) ( Chambers et al., 1991, 1993; information on Flavivirus phylogeny is reported as Supplementary Falgout et al., 1991, 1993; Lindenbach and Rice, 2003; Lescar et al., Information ). 2008 ). The co•localization of NS2B and NS3 in convoluted mem• branes suggests these as the location for polyprotein processing by NS2B/NS3Pro, whereas Golgi•derived vesicle packets (the com• 3. Structure and function of flaviviral enzymes partment presumably involved in RNA replication by NS3 and NS5) lack the presence of NS2B. Accordingly, the relevance of NS2B for 3.1. The flaviviral NS3 protein non•proteolytic NS3 activities, such as helicase, nucleoside triphos• phatase and 5 ′RNA phosphatase activities located in the C•terminal The bipartite NS3Pro•NS3Hel is an enzyme central to flavivirus two•third of NS3, is yet unclear. replication and polyprotein processing. Dissecting the structural Even though the minimum requirements for proteolytic activ• and functional properties of this protein in its full•length state ity comprise the NS3 residues 1–160 (in WNV) or 1–167 in DENV2 is therefore key to improving our understanding of the flavivirus (Li et al., 1999; Leung et al., 2001 ), the maximum activity con• life cycle and informing the design of effective antiviral drugs. cerning WNV NS3Pro, for an optimized fusion construct containing It remains unclear why NS3 harbours several catalytic activities 44 NS2B residues covalently connected via a G4SG3 linker to within one polypeptide chain, however the conservation of this the NS3Pro domain, has been observed for the N•terminal 1–184 arrangement across the Flaviviridae genus suggests some func• residues ( Chappell et al., 2007 ). Interestingly, a comparative analy• tional relevance. Crucially, it is a matter of debate whether there sis of the proteolytic activity of the full•length NS3 protein (1–618) is an interplay between the catalytic activities of the individual fused to the optimized NS2B•G4SG3•linker region showed only domains and whether there is a functional role for the linker region, marginal influence of the larger C•terminal domain on the NS3Pro a poorly conserved, acidic stretch of residues connecting the two kinetic parameters ( Chappell et al., 2007 ). In contrast, studies of domains (see below). WNV full•length NS2B/NS3 and full•length NS3 exhibited differ• ent catalytic activities with respect to the unwinding of DNA and 3.1.1. NS3 protease domain RNA: whereas full•length NS3 is capable to unwind both DNA 3.1.1.1. Functional aspects of the NS3 protease. The 375•kDa fla• and RNA templates, full•length NS2B/NS3 unwinds only RNA tem• viviral polyprotein precursor is processed by host proteases and a plates, and DNA unwinding is severely repressed ( Chernov et al., virus•encoded protease activity localized at the N•terminal domain 2008 ). Accordingly, the NS2B/NS3Pro part restricts substrate speci• of NS3. Whereas the cleavage at the junctions C•prM, prM•E, E•NS1, ficity of the C•terminal NS3Hel domain, however, in the absence of NS4A•NS4B ( Speight et al., 1988; Nowak et al., 1989 ), and likely also NS2B, the NS3 protein might dissociate from membranes and inter• NS1•NS2A ( Falgout and Markoff, 1995 ), is performed by the host fere with host DNA after translocation into the host•cell nucleus signal peptidase located within the lumen of the ER, the remain• (Chernov et al., 2008 ). ing peptide bonds between NS2A•NS2B, NS2B•NS3, NS3•NS4A and NS4B•NS5 are cleaved by the virus encoded NS3Pro. Cleavage at the 3.1.1.2. Three•dimensional structures determined for NS3 protease. NS2B/NS3 site is performed in cis, but is not necessary for protease In 1989, sequence and structural comparison studies of the flavi• activity ( Leung et al., 2001; Bera et al., 2007 ). Thus, the protease and pestiviral genomes suggested the presence of a serine protease activity of NS3 is essential for viral replication and its inhibition related to the trypsin family, comprising a His, Asp, Ser catalytic has to be considered as a valuable intervention strategy for the triad ( Bazan and Fletterick, 1989; Gorbalenya et al., 1989a ). One treatment of flaviviral infections. year later, this prediction was verified for YFV NS3 by mutagen• The activity of NS3Pro is strongly dependent on the associa• esis and characterization of segments of the NS3 gene ( Chambers tion of a 40•amino acid region of NS2B acting as a cofactor for et al., 1990 ). The first crystal structure of a flavivirus NS3Pro was

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx 7

Table 2 Overview about the currently PDB•deposited crystal structures of Flavivirus NS3 proteases (April 2009).

Viral Protease Resolution (Å) Ligand/inhibitor PDB entry Reference, year of publication

DV2 NS3pro 2.1 Uncomplexed 1BEF Murthy et al. (1999) DV2 NS3pro 2.1 Mung Bean Bowman•Birk inhibitor 1DF9 Murthy et al. (2000) DV2 NS3pro 2.1 Mung Bean Bowman•Birk inhibitor 2QID Murthy et al. (to be published) DV4 NS2b/NS3 protease•helicase 3.15 Uncomplexed 2VBC Luo et al. (2008a,b) DV2 NS2B/NS3pro 1.5 Uncomplexed 2FOM Erbel et al. (2006) WNV NS2B/NS3pro 1.68 Covalently bound peptide•type inhibitor 2FP7 Erbel et al. (2006) WNV NS2B/NS3pro, His51Ala mutant 1.8 Uncomplexed 2GGV Aleshin et al. (2007) WNV NS2B/NS3pro 2.3 Aprotinin 2IJO Aleshin et al. (2007) WNV NS2B/NS3pro 2.45 Covalently bound peptide•type inhibitor 3E90 Robin et al. (2009)

described in 1999 ( Murthy et al., 1999 ) for DENV2. This crystal crystal structures presently available suggest conformational plas• structure served as a template for homology modeling studies and ticity of the NS2B peptide: whereas in those protease structures interpretation of biochemical data ( Nall et al., 2004; Zhou et al., hosting a small•molecule inhibitor in the active site, NS2B forms a 2006 ). Table 2 provides an overview of the crystal structures from belt around NS3Pro by contributing one ␤•strand to the N•terminal flavivirus NS3Pro currently available. The binding mode of a pep• and two ␤•strands as ␤•hairpin motif to the C•terminal b•barrels, in tidic mung•bean Bowman•Birk inhibitor in complex with DENV2 the unbound state, the latter ␤•hairpin does not contribute to the NS3Pro has been reported subsequently (see below). Even though C•terminal ␤•barrel ( Fig. 2 c). Instead, while the N•terminal NS2B these structures helped to explain various biochemical observa• fragment (residues 52•58) remains associated with the N•terminal tions, such as the redundant nature of interactions formed by Arg b•barrel, the C•terminal residues form a short helical segment and and Lys residues in the S1 substrate•recognition sub•site, they were a short ␤•strand interacting with strand B2a of NS3Pro, but the substantially different from the more relevant picture represented following hairpin motif points into the solvent and interacts with by the recently described crystal structures of flaviviral NS3Pro in symmetry•related NS3Pro molecules. A similar fold for NS2B is complex with its cofactor NS2B ( Fig. 2 a and b). Accordingly, recent observed in the inhibitor•free DENV2 NS2B/NS3Pro, with a disor• structure determination attempts were focused on the crystalliza• dered region corresponding to the b•hairpin in WNV NS2B/NS3Pro. tion of fusion proteins containing the hydrophilic part of NS2B and The reasons for this unexpected NS2B plasticity are not completely the NS3Pro domain, linked via a glycine•rich linker ( Erbel et al., understood ( Erbel et al., 2006; Aleshin et al., 2007; Chappell et al., 2006; Aleshin et al., 2007; Robin et al., 2009 ). So far, only crys• 2008 ). Nevertheless, the fold adopted by NS2B appears relevant for tal structures of flavivirus NS3Pro from DENV and WNV have been structure•based ligand design of inhibitors of WNV NS3Pro, as the described ( Table 2 ). NS2B b•hairpin tip in complexed NS2B/NS3Pro partly contributes In its activated state, the flavivirus NS3Pro consists of the to the formation of the S2 as well as the S3 pockets and may thereby N•terminal domain of the full•length NS3 protein and its NS2B directly interact with the bound ligand (see below). cofactor. The hydrophilic region of NS2B strongly interacts with NS3Pro, whereas both N• and C•terminal moieties of NS2B form 3.1.1.3. Flavivirus NS3 protease complexes with inhibitors. In order two hydrophobic helices putatively acting as membrane anchors to analyze the substrate preference of the proteases and to establish (Fig. 1 ). NS2B/NS3Pro adopts a chymotrypsin•like fold comprising the basis for structure•based drug lead design, various contribu• two b•barrels, each formed by six ␤•strands, embedding the pro• tions analyzed the interaction of peptide•like ligands with the tease catalytic triad (His51, Asp75, Ser135) in the cleft between protein active site (see Table 2 ). The first complex structure of the two ␤•barrels ( Fig. 2 a). The presence or absence of the NS2B NS3Pro lacking the NS2B cofactor described by Murthy et al. (2000) cofactor substantially influences the NS3Pro structure with respect revealed the interaction of the DENV2 NS3Pro binding pocket with to the extension and location of secondary structure elements a polypeptide•type Bowman•Birk inhibitor isolated from mung (Erbel et al., 2006 ). Notably, in the cofactor•free DENV2 NS3Pro beans, this being the only structure of a complex of DENV2 NS3 structure, the secondary structure elements are either shorter or available to date. Despite the absence of NS2B, the structure allows even absent relative to DENV NS2B/NS3Pro. In the latter protein, general conclusions about the properties and ligand preference of the hydrophilic region of NS2B forms a link between the two b• the NS3Pro substrate•recognition pockets. The bivalent inhibitor barrels and contributes an anti•parallel ␤•strand to each of the possesses a lysine•head and an arginine•head, both occupying the b•barrels. The arrangement of the catalytic triad of the NS2B•bound substrate binding pockets of two different NS3Pro molecules simul• NS3Pro suggests an exhaustive H•bonded network between the taneously ( Murthy et al., 2000 ). Both basic residues occupy the catalytic residues, in particular, a single•donor–double•acceptor S1 pocket while establishing different interactions. The NS3Pro (three•center) hydrogen bond between His51 and Asp75, whereas molecule hosting the inhibitor lysine head adopts virtual identical the structures lacking NS2B in the free or inhibitor•bound state side•chain conformations as observed in the inhibitor•free NS3Pro. exhibit an interaction geometry where only one weak H•bond However, the second NS3Pro molecule exhibits strong conforma• between His51 and Asp75 is observed ( Murthy et al., 1999, 2000 ). tional changes, particularly in the region Val126•Gly136, to adopt These structural differences and the less constrained framework in a binding•competent conformation ( Fig. 3 ). The complex structure absence of NS2B will presumably be related to the low proteolytic shows that Asp129 points either to the solvent (in the P1•Lys• activity described for the non•cofactor•bound NS3Pro ( Falgout et bound molecule), or interacts with the basic residue (with P1•Arg al., 1991, 1993 ). While cleavage of small substrates by DENV2 bound) of the ligand, but the latter is involved in further charge• NS3Pro is virtually not affected by the presence or absence of NS2B, assisted hydrogen•bonds in a fashion obviously compensating the degradation of larger substrates is strongly stimulated by presence mutational loss of the interaction to Asp129 ( Murthy et al., 2000 ). of NS2B ( Yusof et al., 2000 ). Additionally, a comparison of both NS3Pro molecules of the DENV2 Sharing a sequence identity of 50%, the overall fold observed NS3Pro•inhibitor complex reveals remarkable plasticity of active for the NS2B/NS3Pro from WNV and DENV2 is very similar, with site residues ( Fig. 3 ). only subtle deviations in length and location of secondary structure The flexible behavior of DENV2 NS3Pro is not observed in the elements ( Aleshin et al., 2007 ). Interestingly, WNV NS2B/NS3Pro three available WNV NS3Pro•ligand complexes, all containing the

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

8 M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx

Fig. 3. DENV2 NS3Pro complexed with a Bowman•Birk inhibitor from Mung Bean (PDB entry 1DF9, Murthy et al., 2000 ). The representation shows a superimposition of the two protein molecules present in the asymmetric unit and the relevant pep• tide region of the inhibitor (lysine head, depicted in orange). The crystal structure suggests a pronounced mobility for the region 126–136 lining the specificity pock• ets of the NS3Pro. Particularly Asp129 (equivalent to Asp189 in trypsin) is capable of pointing either towards the solvent or contributing to the S1 pocket.

NS2B cofactor. One of them hosts aprotinin as the inhibitory lig• and, the other two are complexed with a peptide•type substrate analogue covalently bound to the catalytic triad residue Ser135. Whereas the overall structure of the three protein/inhibitor com• plexes is very similar, in contrast to the peptidic inhibitor described by Erbel et al. (2006) , aprotinin occupies all the major specificity pockets of the NS3Pro (S2•S2 ′). Additionally, it induces a catalyt• ically competent conformation with a fully structured oxyanion hole established by the main•chain nitrogens of Gly133 and Ser135 (Aleshin et al., 2007 ). In the ligand•free state, the peptide bond between Thr132 and Gly133 is flipped, thereby forming a helical 310 conformation for residues 131–135. A superposition of the two conformations observed for the aprotinin•bound and ligand•free states is shown in Fig. 4 . The main chain of aprotinin residues 13–19 forms antiparallel ␤•sheet interactions with the strands E2B and B1 of WNV NS2B•NS3Pro. In contrast, the substrate analogue inhibitor described by Erbel et al. adopts a loop conformation supported by favourable cation–p interactions between the P1•Arg residue and the inhibitor benzoyl cap ( Fig. 2 c). The protein–ligand complexes provide structural evidence for the strong preference for ligands Fig. 2. Crystal structures of NS3Pro from DENV2 and WNV viruses. (a) Overall fold of (substrate or inhibitor) comprising basic residues at the P1 and P2 NS2B/NS3Pro from DENV2 (PDB entry 2FOM, Erbel et al., 2006 ). NS3Pro is shown in sites. Next to other interactions, the properties of the S1 pocket are blue, the NS2B region, ordered in the crystal structure, is shown in yellow. (b) Super• position of DENV2 NS2B/NS3Pro as depicted in (a) and the crystal structure of DENV2 governed by the salt•bridge between Asp129 and P1•Arg, as well as NS3Pro without the stabilizing cofactor shown in orange (PDB entry 1BEF, Murthy et by Tyr161 contributing a face•to•face ␲•cation stacking with the P1 al., 1999 ). Remarkably, substantial differences with respect to secondary structure residue. Interactions with the P2 moiety are mainly contributed by elements are observed. (c) Superposition of the WNV NS2B/NS3Pro in ligand•bound the tip of the b•hairpin formed by the NS2B cofactor whose back• and uncomplexed state. The NS3Pro covalently linked to the inhibitor (PDB entry bone oxygen atoms of Asp82 and Gly83 and the Od1 atom of Asn84 2FP7, Erbel et al., 2006 ) is shown in blue with the cofactor and ligand colored in orange and light blue, respectively. In the uncomplexed state (H51A mutant, PDB act as acceptors of charge•assisted H•bonds donated by the hosted entry 2GGV, Aleshin et al., 2007 ) shown in green, the NS2B colored in yellow exhibits P2•Arg residue. The latter forms an additional H•bond to Asn152 remarkable plasticity compared to the ligand•bound conformer. O␦1. A P2•Lys moiety is capable of mimicking two of these interac• tions (H•bonds accepted by the backbone oxygen of Gly83 and by Asn152 O ␦1), and additionally establishes a hydrogen•bond to one of the carboxylate oxygens of Asp75. However, replacement of the P2•basic residue by alanine leads to a total loss of binding ( Erbel et

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx 9

Fig. 4. Induction of the oxyanion hole in WNV NS3B/NS3Pro by the polypeptide• type inhibitor aprotinin (for clarity, only Pro13•Lys15 are shown in yellow). Residues of the uncomplexed NS3Pro (H51A mutant, PDB entry 2GGV, Aleshin et al., 2007 ) are shown as green sticks, residues of the aprotinin•bound enzyme are depicted in blue (PDB entry 2IJO, Aleshin et al., 2007 ). The peptide bond Thr132•Gly133 flips and contributes via its backbone nitrogen atom to the formation of the oxyanion Fig. 5. The structure of DENV NS3Hel with its three domains (I red, II blue and III hole. H•bonding interactions between the ligand carbonyl oxygen and the backbone green) bound to AMPPNP (left, molecule in green) and RNA (7 bases are visible: nitrogens of Gly133, Thr134 and Ser135 are shown as orange dashes. AGACUAA in yellow), adapted from PDB entry 2JLV ( Luo et al., 2008a ). al., 2006 ). The S1 ′ pocket is lined on one side by the catalytic His51 cleavage of host cell proteins (see above), inhibition of the prote• as well as by Gly37, providing only sufficient space to accommodate olytic activity is a promising antiviral strategy. small P1 ′ side chains such as Gly, Ser, or Thr ( Aleshin et al., 2007 ). The different properties and plasticity behavior of the DENV2 and 3.1.2. NS3 helicase domain WNV specificity pockets could be exploited to design substrates 3.1.2.1. Functional aspects of the NS3 helicase domain. It is well with selectivity for only one of the flavivirus NS3Pro. Whereas prob• understood that RNA synthesis by the viral replication machinery ing the cleavage activity revealed a strict substrate specificity of requires unwinding of the RNA secondary structure in the tem• WNV NS2B•NS3Pro, in agreement with the described mobility of plate RNAs. The NS3Hel domain is held to assist in initiation of the Val126•Gly136 segment ( Fig. 3 ), the DENV2 NS2B•NS3Pro was (−)ssRNA synthesis by unwinding the RNA secondary structure in less selective and tolerated well the presence of a number of amino the 3 ′ UTR ( Takegami et al., 1995 ). The key role of helicase activity acid types at either the P1 ′ or the P2 ′ site ( Shiryaev et al., 2007 ). in viral replication has been demonstrated through site•directed Very recently, Robin et al. (2009) described a crystal structure mutagenesis ( Grassmann et al., 1999; Matusan et al., 2001 ). Crystal of WNV NS2B•NS3Pro in complex with a substrate•based tripep• structures of the flavivirus C•terminal NS3RTPase/Hel domain have tide inhibitor capped at its N•terminus by a naphthoyl moiety and been solved for YFV ( Wu et al., 2005 ), DENV ( Xu et al., 2005; Luo at its C•terminal end by an aldehyde. The latter acts as an elec• et al., 2008b ) and JEV ( Yamashita et al., 2008 ). In the context of the trophilic warhead for covalent inhibition. Interestingly, in one of VIZIER Project, three new structures have been obtained for MVEV the two NS3Pro molecules present in the asymmetric unit, the cat• (Mancini et al., 2007 ); 1.9 Å resolution), KUNV, an Australian variant alytic His51 side chain adopts a split conformation. One conformer of WNV ( Mastrangelo et al., 2007b ; 3.1 Å) and KOKV ( Speroni et al., hydrogen•bonds to the aldehyde oxygen directing it for a nucle• 2008 ; 2.1 Å). In particular, the KOKV NS3Hel domain features high ophilic attack by the catalytic Ser from the re side, whereas the thermostability and good propensity to crystallize, making this an other His conformer, inconsistent with a catalytic triad, points away attractive model system for structural and biochemical analysis of from the reaction center enabling the oxyanion hole to direct the inhibitor binding. nucleophilic attack from the side. These observations suggested a role for the ligand to stabilize the His in its catalytically competent 3.1.2.2. Three•dimensional structures determined for the NS3 helicase conformation. domain. The flaviviral NS3Hel tertiary structure is characterized by Proteases related to the occurrence of pathobiochemical pro• three domains, each of about 130–150 amino acids ( Fig. 5 ). The first cesses have raised the interest of biochemists and drug designers two domains (domains I and II) are structurally similar, displaying for many years ( Mittl and Grutter, 2006 ). Benefiting from the an ␣/␤ open sheet topology (Rossman fold), composed of six ␤• knowledge thereby generated, current efforts to develop flavivirus strands (topology ␤1– ␤6– ␤5– ␤2– ␤4– ␤3), surrounded by four and NS3Pro inhibitors suitable for clinical use are indeed promising. three ␣•helices, respectively. Domain III is mainly composed of five Due to the increasing prevalence of infections caused by pathogenic approximately parallel ␣•helices and two antiparallel ␤•strands flaviviruses such as WNV, different types of DENV, and SLEV, devel• (Fig. 5 ). In domain II, a ␤•hairpin (residues 426–450) protrudes opment of anti•flaviviral drugs is of utmost importance ( Ghosh from the core domain projecting towards domain III and is held to and Basu, 2008 ). Even though lessons from the treatment of be a critical element in unwinding activity ( Luo et al., 2008b ). The Human Immunodeficiency Virus (HIV) and HCV infections show first two domains, likely originated by gene duplication ( Caruthers in a dramatic way the development of escape mutations confer• and McKay, 2002 ), host seven characteristic sequence motifs of the ring resistance to viral proteases upon single therapy with only one NS3Hel superfamily 2 ( Gorbalenya et al., 1989b; Cordin et al., 2006 ), inhibitor ( Manns et al., 2007 ), the protease inhibitors developed do associated with nucleic•acid binding and NTP hydrolysis ( Caruthers contribute to an efficient combination therapy. Since NS2B•NS3Pro and McKay, 2002 ). In particular, the conserved motifs I (GAGKTRR) is obviously not only responsible for processing of the viral polypro• and II (DEAH), also known as Walker A (ATP phosphate•binding tein, but also appears to contribute to further pathogenic processes loop, or ‘P•loop’) and Walker B motifs (Mg 2+ •binding; Walker et al., such as induction of membraneous structures, neurovirulence and 1982 ), are located in the amino•terminal region (domain I) where

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

10 M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx they interact with the NTP substrate and Mg 2+ , respectively ( Xu residues and of the conformational changes that underlie the cou• et al., 2005 ). In addition, two conserved Arg residues in domain II pling between ATP hydrolysis and RNA unwinding activity. (Arg458 and Arg461: “arginine fingers” in motif VI) are involved in ATPase and RTPase activities ( Sampath et al., 2006 ), and in the 3.1.3. The full•length NS3 protein structural rearrangement that results in RNA unwinding, follow• 3.1.3.1. Functional aspects of the NS3 protein. Understanding the ing ATP hydrolysis. A similar role in coupling the ATPase and RNA biologically relevant functional properties of NS3 is complicated unwinding activities is played by the residues of the Ia motif, as by the fact that in infected cells NS3 acts anchored to or in close gathered from mutagenesis studies on the KOKV NS3Hel ( Speroni proximity of membranes ( Lindenbach and Rice, 2003 ), whereas et al., 2008 ). The central region of the NS3Hel, where the three most in vitro characterization has been done in solution. Fur• domains contact each other, hosts a cleft held to be involved in thermore, as the virus progresses towards maturation, different ssRNA binding during the helicase activity ( Xu et al., 2005; Luo et protein–protein and protein–RNA interactions occur which demar• al., 2008b ). cate specific points in its life cycle, although the details remain Differences between the YFV, DENV, KUNV, KOKV, JEV and unclear. Polyprotein processing and replication occur in dis• MVEV structures were found to be confined primarily to the relative tinct, membrane•bound compartments (convoluted membranes orientation/distance of domain II to domains I and III, suggesting and vesicle packets, respectively), and in each compartment, NS3 that movement of domain II can affect nucleic•acid translocation engages with different proteins ( Mackenzie, 2005 ). An intrigu• in an ATP•dependent mode according to the ‘inchworm’ model ing finding has been the apparent absence of NS2B, the essential (Mancini et al., 2004; Mastrangelo et al., 2007b ). Such overall cofactor of the NS3Pro, in vesicle packets ( Westaway et al., 1997; structural rearrangement was recently confirmed by a detailed Mackenzie, 2005 ) suggesting that in the transition from polyprotein structural study on DENV4 NS3Hel, which describes the structures processing to replication, the NS3Pro becomes inactive. Finally, the of various complexes with ATP analogues and ssRNA of 12–13 structure and dynamics of the polyprotein as it emerges follow• nucleotides ( Luo et al., 2008b ) ( Fig. 5 ). In particular, upon ssRNA ing translation remain largely unexplored and little information binding domain III rotates about 11 ◦ away from domain I with exists on interactions between NS3 and other parts of the polypro• the simultaneous narrowing the of cleft between domains I and II tein, which might be important for priming NS3Pro for its first and (12 ◦ rotation). The overall movement can be described as an open• subsequent cleavage activities. ing of both the ssRNA access site, located between ␣•helices ␣2 (domain II) and ␣9 (domain III; as showed in normal mode analysis; 3.1.3.2. Three•dimensional structures determined for the NS3 protein. Mastrangelo et al., 2007b ), and the ssRNA exit site, by the reposi• Two full•length NS3 structures have been solved by X•ray crystal• tioning of a loop (disordered in many crystal structures) located lography: those of DENV4 NS3 ( Luo et al., 2008a ) and of MVEV NS3 between strands ␤3 and ␤4 in domain I. (Assenberg et al., submitted for publication) ( Fig. 6 ). Both struc• tures were solved in the presence of a fragment of protein NS2B, the essential co•factor and activator for NS3Pro, by producing a 3.1.2.3. Characterization of helicase activity. RNA unwinding activ• single polypeptide chain where this region was linked via a flexible ities are assessed using a partially dsRNA molecule consisting of tether to NS3. One difference between the two studies is that for a 14 base 3 ′ single•stranded tail followed by a 16 base•pair dsRNA DENV4 only part of the NS2B activating region was coupled to NS3 region ( Wu et al., 2005 ). Generally, NS3Hel containing longer linker (18 amino acids of NS2B, DENV4 NS2B 18 NS3), whereas for MVEV regions show higher activity than those with short linkers. The the full activating region was included (45 residues of NS2B, MVEV DENV4 NS3Hel (NS3 178•end) used by Luo et al. (2008b) was trun• NS2B 45 NS3). cated close to boundaries shown previously for DENV, WNV and In both the DENV4 NS3 ( Luo et al., 2008a ) and MVEV NS3 (Assen• YFV to yield inactive or significantly impaired domains with respect berg et al, submitted for publication) structures the NS2B•NS3 to ATPase and helicase activities when compared to constructs molecule consists of two separate globular folds linked by a short with N•terminally extended linker regions ( Li et al., 1999; Wu et linker, an arrangement consistent with SAXS data for full•length al., 2005; Xu et al., 2005 ). In contrast, the MVEV NS3Hel construct KUNV NS3 in solution ( Mastrangelo et al., 2007b ). The individual includes a significantly longer linker suggesting that the observed domains are very similar between the two molecules (r.m.s.d. of reduction in activity for the DENV4 NS3Hel domain ( Luo et al., 1.5 and 1.6 Å for the NS3Hel and NS3Pro, respectively) and to the 2008b ) was due to the short linker. The reason why truncation of structures of isolated domains. Yet the relative orientations of the the linker region can have a detrimental effect on the activity of the NS3Pro and NS3Hel domains are dramatically different between C•terminal domain of NS3 remains unclear; it may have a functional MVEV NS2B 45 NS3 and DENV4 NS2B 18 NS3. When superimposing role ( Matusan et al., 2001 ), or cause structural artefacts as observed the NS3Hel domains of the two structures, a rotation of ∼180 ◦ for KUNV NS3Hel (aa186•619) which forms a dimer ( Mastrangelo and translation of 17 Å are required to align the NS3Pro domains. et al., 2007b ). The 13•residue “linker” between the NS3Pro and NS3Hel domains The VIZIER Project has substantially enhanced our knowledge (residues 169–181) is ordered in DENV4 NS2B 18 NS3 but partially on flaviviral NS3Hel, providing the bases for the structure•based disordered in MVEV NS2B 45 NS3. Even though the buried surface design and development of specific antiviral molecules target• area between the NS3Pro and the NS3Hel in DENV2 NS2B 18 NS3 ing this essential class of enzymes. The remarkable similarities in is only 568 Å 2, the NS3Pro domain and the linker loop engage the Hel/ATPase catalytic regions indicate that it might be possi• in possibly significant interactions with subdomains 1 and 2 of ble to develop compounds with a broad spectrum of activities – NS3Hel. Specifically, the linker interacts with the catalytic P•loop i.e. which are able to act on different flaviviral enzymes – and/or of the NS3Hel, which assumes the distinctive apo conformation lead molecules that can be targeted to a specific viral enzyme seen in the NS3Hel domain crystal structure in the absence of through minimal ad hoc chemical modifications. Medicinal chem• bound nucleotides. These interactions are not seen in the MVEV 2 istry studies on protein kinases have shown that the most effective NS2B 45 NS3 structure where the buried surface area is only 30 Å ; inhibitors are conformationally based; they exert their inhibitory however, the P•loop is found in the same apo conformation. Thus, effect through an allosteric alteration of the equilibrium among a possible role for the linker loop could be to stabilize the apo con• different protein conformations ( Vajpai et al., 2008 ). Likewise, formation of the P•loop, in line with recent studies suggesting a future drug•design studies on flaviviral NS3Hel will benefit from functional role for the linker (truncating this acidic linker can have our improved understanding of the role of the various fingerprint a substantial effect on the activity of isolated NS3Hel domains; Li

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx 11

questions over the role of the NS3Pro domain in regulating helicase activity since in the absence of NS2B the NS3Pro domain partially unfolds.

3.1.3.3. General structural properties of the NS3 protein. The struc• tures of full•length NS3 raise two important questions: (1) do these structures represent two distinct and stable conformations of NS3 possibly adopted at different stages of the flavivirus life cycle or are they merely snap•shots of a highly dynamical interconversion process and (2) given the segregated nature of the two catalytic domains, what is the functional significance of this arrangement? Our analysis suggests that the relative domain organization is probably highly dynamic, given the linker flexibility (disordered in the MVEV structure) and the small buried surface areas between the two domains in both structures. Further, the configurations are in principle inter convertable via simple rotations around the linker loop, and linker flexibility is probably paramount to the NS3 activ• ities and its ability to associate with other proteins and RNA. On the other hand, specific configurations may be stabilized during a particular stage of the virus life cycle. To gain further insights into these issues, we modeled the MVEV and DENV NS2B•NS3 struc• tures in the presence of a membrane (Assenberg et al., submitted for publication). Previous studies have shown that when in complex with NS2B, and in particular when fully activated, NS3Pro sits in a rather tight membrane•anchored sling ( Clum et al., 1997; Robin et al., 2009 ). Fig. 7 shows a model of MVEV and DENV4 NS2B•NS3, with their NS3Pro domains superimposed and associated to the mem• brane as inferred from the anchoring of the published NS2B•NS3Pro structures ( Erbel et al., 2006; Robin et al., 2009 ). In this model, the DENV4 NS2B 18 NS3 NS3Hel is positioned near the membrane with the active site orientated towards the membrane and with lit• tle space to accommodate RNA, whilst in the MVEV structure the NS3Hel domain is positioned away from the membrane, with the Fig. 6. Comparison of the NS2B•NS3 structures of MVEV (upper panel) and DENV4 active site facing the cytoplasm. Thus, the DENV4 NS2B•NS3 struc• (lower panel); in both panels the NS3Hel domain is in the upper part of the figure, ture appears incompatible with RTPase/helicase activity. Although the NS3Pro domain is in blue–cyan colors, hosting the NS2B segment (red color). this could be taken as an argument that the DENV4 conformation may not be physiologically relevant, the strength of conforma• et al., 1999; Wu et al., 2005; Mastrangelo et al., 2007b ). tional constraints imposed by the cellular environment is difficult A striking observation relating to the influence of the NS3Pro to assess. Prior to cleavage of the NS3•NS4A junction, NS3 is also domain on the NS3Hel domain emerges from a comparison of the anchored to the membrane at its C•terminus via the membrane• two structures. In DENV4 NS2B 18 NS3, the interactions between bound NS4A. However, although there are 50 residues separating NS3Pro and NS3Hel are such that motions of domain 2 of the the NS3 C•terminus and the first trans•membrane ␣•helix of NS4A NS3Hel, known to be important for helicase activity, would be (Miller et al., 2007 ), the structure of the first 50 residues of NS4A constrained. In the MVEV NS2B 45 NS3 structure in contrast, such remains unknown. Thus, the presence of NS4A may limit the abil• interactions are weak and therefore there are no constraints on the ity of NS3 to change its conformation in vivo . This leads to the motility of the NS3Hel domain. This would suggest that the NS3Pro interesting possibility that NS3 may adopt this conformation dur• domain might repress helicase activity and that such activity might ing polyprotein processing where helicase activity is probably not be regulated by switching between various transient configura• wanted. Formation of the replication complex, where the helicase tions, such as those observed in the two structures. This conclusion activity is presumably needed, would release NS2B, inactivating the contrasts that of Luo et al. (2008a) , who saw an increased affin• NS3Pro domain. In this view, the MVEV NS2B•NS3 conformation is ity for ATP when the activity of the DENV4 NS2B 18 NS3 protein likely to be relevant later in the virus life cycle, during the assembly was compared to that of the isolated NS3Hel domain. Although and functioning of the replication complex. The regulation of the this enhancement was explained by a positive contribution of activities of NS3 by an environment•dependent re•configuration of the NS3Pro domain to the electrostatic potential of the NS3Hel the molecule offers a simple temporal and spatial control mech• nucleotide binding pocket raising helicase activity, an alternative anism, coupling activities appropriately with the virus life cycle. explanation is that the isolated NS3Hel domain chosen for compari• This model provides answers to both of the questions posed in the son was ‘hobbled’ by truncation of the linker. Indeed the truncation previous paragraph. used has been shown to significantly reduce helicase activity of In vivo , the situation is probably complicated by the modulation DENV2 and other flavivirus NS3Hel domains ( Li et al., 1999; Wu of the structure and function of NS3 by additional binding partners. et al., 2005 ). The latter interpretation is supported by biochemical Thus, the activity of NS3 may be affected by interactions within the analysis of the helicase activity of MVEV NS2B 45 NS3, which showed polyprotein ( Zhang and Padmanabhan, 1993 ), and NS3 binds to free no significant difference between the activity of full•length MVEV NS5 ( Johansson et al., 2001; Yon et al., 2005 ), NS4A ( Mackenzie et NS2B 45 NS3 and that of the isolated NS3Hel domain (using a more al., 1998 ) and NS4B ( Umareddy et al., 2006 ) as well as viral RNA. appropriate linker than in the DENV4 study). Finally, NS2B is not In particular, it has been suggested that the C•terminal domain of found in vesicle packets and therefore not part of the replication NS3 binds NS5 ( Liu et al., 2002; Wu et al., 2005 ) during the forma• complex ( Westaway et al., 1997; Mackenzie, 2005 ), posing further tion of the replication complex. Unfortunately, the details of these

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

12 M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx

Fig. 7. Models for membrane association of MVEV and DENV4 NS2B•NS3. (a) Schematic diagram of the flavivirus polyprotein organization and processing. The upper figure shows the linear organization of the structural and non•structural proteins within the polyprotein. The lower figure shows the putative membrane topology of the polyprotein, as predicted from biochemical and cellular analyses, which is then processed by cellular and viral proteases (denoted by arrows). (b) Predicted structural organization of

MVEV NS2B 45 NS3 and DENV4 NS2B 18 NS3 at the cellular membrane. A model for the membrane is shown as van der Waals balls, atomic structures are shown in a surface representation and color coded according to the following convention: NS3 protein (pale yellow) and NS2B stretches (blue). The NS4A (shown schematically in pink) was positioned at the NS3 C•terminus (domain3) and the RNA (shown schematically in grey) is positioned in the ssRNA binding groove. interactions remain poorly understood. Clearly, further studies are Guyatt et al., 2001; Nomaguchi et al., 2003, 2004; Kim et al., 2007 ). required to test the functional significance of the two conforma• It has been demonstrated that NS5 initiates RNA synthesis de novo tions in vivo , as well as the influence of the interactions between (i.e. primer•independent) ( Ackermann and Padmanabhan, 2001; NS3 and other viral proteins, RNA, and lipids on the conformation Nomaguchi et al., 2004; Selisko et al., 2006 ). of NS3. The N•terminal boundary of the RdRp domain of protein NS5, The structures of full•length NS3 reveal that the molecule which comprises around 900 amino acids, has long remained can assume two radically different configurations, defined by the unknown. Usually two nuclear localization sequences (NLS) local• relative positioning of the NS3Pro and NS3Hel via a flexible inter• ized between amino acid residues 320 and 405 were supposed domain linker. We suggest that these may be important in its to represent the inter•domain region between MTase and RdRp interactions with other proteins and RNA and, possibly, in mod• (Brooks et al., 2002 ). Within the VIZIER Project, structure•based ulating the switch to helicase and triphosphatase activities during sequence analysis of NS5 was conducted and allowed the defi• replication. nition and subsequent production of a recombinant soluble and enzymatically active RdRp domain of DV2 (NS5Pol DV ) and WNV 3.2. The flaviviral NS5 protein (NS5Pol WNV ) starting at DV2 NS5 residue 272 ( Selisko et al., 2006 ). More recently, we expressed full•length NS5 proteins of two strains With a molecular mass of about 100 kDa, NS5 is the largest (Vasilchencko and Oshimo) of TBEV in Escherichia coli (cloned in flaviviral protein; NS5 is also the most conserved one across the pDest14 and expressed as described, Selisko et al., 2006 ). The genus. Early on a motif of AdoMet•dependent MTases was identi• recombinant proteins were purified by IMAC followed by size fied within the N•terminal domain of NS5 ( Koonin, 1993 ) whereas exclusion chromatography. NS5 was obtained but also to a large RdRp motifs were identified in the C•terminal domain of protein extent a degradation product of around 30 kDa. Western blot anal• NS5 ( Rice et al., 1985; Poch et al., 1989; Koonin, 1991; Bruenn, ysis against the N•terminal hexahistidine•tag revealed that this 2003 ). The MTase functions were demonstrated in 2002 and 2007 pool was the N•terminal part of NS5. The mass of these proteins using the recombinant N•terminal MTase domains of DENV2 and were checked by matrix•assisted laser desorption ionization•time WNV ( Egloff et al., 2002; Ray et al., 2006 ). The RdRp activity was of flight mass spectrometry (MALDI•TOF) rendering a unique peak first demonstrated by the use of NS5•specific antisera that inhib• at 30,299 Da for NS5 of TBEV Vasilchenko and 30,335 Da for NS5 ited RdRp activity in assays using DENV2•infected cell lysates of TBEV Oshima (data not shown). These data suggest that the (Bartholomeusz et al., 1994 ), as well as by DENV1 recombinant main cleavage occurs after Arg•264 or Cys•265, depending on the NS5 ( Tan et al., 1996 ). The latter bound RNA template and showed presence or not of the start methionine that can be, in E. coli , RdRp activity as detected by the incorporation of radiolabel into a removed. In summary, we conclude that the linker region between neosynthesized RNA strand ( Tan et al., 1996 ). NS5 of flaviviruses the MTase and the RdRp domain of flavivirus NS5 can be assigned has subsequently been expressed in various in vitro systems, and to residues 266 to 272. Interestingly, all solved NS5MTase struc• shown to have RdRp activity ( Ackermann and Padmanabhan, 2001; tures (see Section 3.2.1 ), which were obtained either from protein

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx 13

Table 3 Flaviviral MTases crystal structures.

Virus PDB ID Ligand(s) Reference Vizier

3ELY AdoHcy Bollati et al. (2009a) Yes 3ELU AdoMet Bollati et al. (2009a) Yes 3ELW AdoMet + GpppG Bollati et al. (2009a) Yes WESSV 3EMB AdoMet + N7MeGpppG Bollati et al. (2009a) Yes 3ELD Sinefungin Bollati et al. (2009a) Yes 3EMD Sinefungin + N7MeGpppA Bollati et al. (2009a) Yes

2PX2 AdoHcy Assenberg et al. (2007) Yes 2PX4 AdoHcy Assenberg et al. (2007) Yes 2PX5 AdoHcy Assenberg et al. (2007) Yes MVEV 2PXC AdoMet + GpppA Assenberg et al. (2007) Yes 2PX8 AdoHcy + N7MeGTP Assenberg et al. (2007) Yes 2PXA AdoHcy + GpppG Assenberg et al. (2007) Yes

1L9K AdoHcy Egloff et al. (2002) No 3EVG AdoHcy Geiss et al. (2009) No 1L9K AdoHcy + Ribavirin triphosphate Egloff et al. (2002) No 2P41 AdoHcy + N7MeGpppG2 ′OMe Egloff et al. (2007) Yes DENV 2P40 AdoHcy + N7MeGpppG Egloff et al. (2007) Yes 2P3Q AdoHcy + GpppG Egloff et al. (2007) Yes 2P3O AdoHcy + N7MeGpppA Egloff et al. (2007) Yes 2P3L AdoHcy + GpppA Egloff et al. (2007) Yes

3EVA AdoHcy Geiss et al. (2009) No 3EVB AdoHcy Geiss et al. (2009) No 3EVC AdoHcy + GTP Geiss et al. (2009) No YFV 3EVD AdoHcy + GTP Geiss et al. (2009) No 3EVE AdoHcy + GpppA Geiss et al. (2009) No 3EVF AdoHcy + N7MeGpppA Geiss et al. (2009) No

WNV 2OY0 AdoHcy Zhou et al. (2007) No MEAV 2OXT AdoMet Mastrangelo et al. (2007a,b) Yes YOKV 3GCZ AdoMet Bollati et al. (2009b) Yes constructs consisting of about 265 or of 293 residues, comprise (Bollati et al., 2009a ) and MODV ( Jansson et al., 2009 ) ( Table 3 ). approximately 265 residues only, since the 266–293 stretch of Moreover, structures of DENV NS5MTase, MVEV NS5MTase and the long constructs are usually disordered. The only exception are WESSV NS5MTase in complex with GTP or several cap analogues, two WESSV NS5MTase domain structures (PDB entries 3ELD and GpppA/G and N7meGpppA/G ( Egloff et al., 2002, 2007; Assenberg et 3ELU, Bollati et al., 2009b ), which include the linker region and a al., 2007; Bollati et al., 2009b ) have been reported, shedding light on C•terminal helix from residues 274 to 285. Interestingly, residues the substrate•binding mode during methylation and on the enzyme 267–269 are disordered, what supports our proposal of the linker mechanism of action ( Table 3 ). region. The C•terminal region is nevertheless characterized by high The flaviviral NS5MTase domain consists of a 33•kDa protein mobility, which may be functional for the interaction between comprising residues 1–260/270 of the N•terminus of the NS5 the NS5MTase and the NS5RdRp domains of the full•length viral protein. It is characterized by an overall globular fold consist• protein. ing of a core domain (residues 59–224) flanked by an N•terminal region (residues 1–58), and a C•terminal region (residues 225–265) 3.2.1. NS5 methyltransferase domain (Fig. 8 ). The core domain comprises a seven•stranded ␤•sheet sur• 3.2.1.1. Functional background aspects of the NS5 methyltransferase rounded by four ␣•helices and two 310 helices, closely resembling domain. The flavivirus RNA is decorated with a conserved type• the topology observed in the catalytic domain of other AdoMet• 1 cap (N7meGpppA2 ′Ome•RNA) at its 5 ′•end, a unique structure dependent MTases ( Fauman et al., 1999; Bugl et al., 2000; Egloff consisting of an inverted guanosine linked to the first transcribed et al., 2002 ). The N•terminal segment comprises a helix•turn•helix RNA nucleotide by a 5 ′–5 ′ triphosphate bridge. Viral MTases are motif followed by a ␤•strand and an ␣•helix. The C•terminal region involved in the mRNA capping process, transferring a methyl group consists of an ␣•helix and two ␤•strands ( Fig. 8 ). The core subdo• from the cofactor S•adenosyl• l•methionine (AdoMet) onto the N7 main hosts the active site and the cofactor binding site ( Ingrosso atom of the cap guanine and onto the 2 ′OH group of the ribose et al., 1989; Egloff et al., 2002, 2007; Assenberg et al., 2007; moiety of the first RNA nucleotide. In the genus Flavivirus, both Mastrangelo et al., 2007a; Zhou et al., 2007; Bollati et al., 2009a,b ). In (guanine•N7)•methyltransferase (N7MTase) and (nucleoside•2 ′•O• all the structures a cofactor molecule, in some cases the co•product )•methyltransferase (2 ′OMTase) activities have been associated AdoHcy, originated from E. coli and co•purified with the enzyme, with the N•terminal domain of the viral NS5 protein ( Egloff et al., is bound in this binding site, where it is stabilized by a network 2002; Ray et al., 2006; Zhou et al., 2007 ). of hydrogen bonds and van der Waals contacts involving several residues – Ser56, Gly86, Trp87, Thr104, Leu105, His110, Asn131, 3.2.1.2. Three•dimensional structures determined for the NS5 methyl• Val132, Asp146, and Ile147 (residue numbering refers to WESSV transferase domain. Crystal structures of the NS5MTase domain NS5MTase) – and a series of interactions that are well conserved have been reported for different mosquito•borne flaviviruses, such within the flaviviruses NS5MTases ( Fauman et al., 1999; Egloff et as DENV ( Egloff et al., 2002 ), WNV ( Zhou et al., 2007 ) and YFV al., 2002, 2007; Assenberg et al., 2007; Mastrangelo et al., 2007a; (Geiss et al., 2009 ). In the context of the VIZIER Project new high• Zhou et al., 2007; Bollati et al., 2009a,b ). resolution structures of NS5MTases have been obtained for MEAV, The known flaviviral NS5MTases show a large degree of struc• a TBV ( Mastrangelo et al., 2007a ), for the MBV MVEV ( Assenberg et tural homology (r.m.s.d. < 1 Å), which reflects the high amino acid al., 2007 ) and WESSV ( Bollati et al., 2009b ), and for two NKVs: YOKV sequence conservation (between 30% and 90%). Comparison of

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

14 M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx

guanylyltransfer reaction would be the only occasion where the GTP•binding site of DENV NS5MTase needs to accommodate a non• methylated GTP. Since flaviviral NS5MTases display both N7MTase and 2 ′OMTase activities ( Ray et al., 2006; Egloff et al., 2007; Zhou et al., 2007; Dong et al., 2008 ), in order for capped RNA to be methylated at the two different sites, it must adopt two distinct binding modes relative to the enzyme active site. To date structural details on catalytically relevant states for both N7 or 2 ′O methyl transfer are missing. Nev• ertheless, it has been speculated that when the cap is bound in the HBS, the 2 ′OH group of the ribose moiety of the nucleotide follow• ing the cap is the closest atom to the AdoMet exchangeable methyl group ( Egloff et al., 2007; Mastrangelo et al., 2007a; Bollati et al., 2009b ). For this reason, the HBS is assumed to host the cap during 2′O methylation. Moreover, structural considerations suggest the existence of a secondary, putative low•affinity binding site (LBS) located in a positively charged region close to the AdoMet binding site which could be involved in binding the capped RNA substrate (Egloff et al., 2007; Mastrangelo et al., 2007a; Dong et al., 2008; Bollati et al., 2009a,b ). Fig. 8. Crystal structure of DENV NS5MTase in complex with AdoHcy. A ball•and• In this context, two different models of the mechanism of action stick representation is used for AdoHcy, whereas DENV NS5MTase is drawn as a of the flaviviral NS5MTase have been proposed. Model 1 ( Egloff et ribbon ( Egloff et al., 2002 ). The loops differing between NS5MTases representative of the three Flaviviral branches are highlighted with a star and an identification al., 2007; Mastrangelo et al., 2007a; Zhou et al., 2007; Dong et al., number referring to what has been described in the text. 2008; Bollati et al., 2009a,b; Milani et al., 2009 ) suggests that when the capped RNA substrate binds in the LBS, it undergoes methyl transfer in position N7 of the guanine with the cap guanine fixed in the NS5MTases representative of each of the three flaviviruses the active site. Afterwards, the NS5MTase slides on the RNA chain branches (the NKV, YOKV NS5MTase; the TBFV, MEAV NS5MTase, positioning the cap in the HBS and locating the first RNA adenine and the MBFV, DENV NS5MTase) shows that most differences in in the active site for 2 ′O methylation. In this binding mode, the the structures are caused by surface•loop flexibility and amino rest of the RNA chain is still stabilized by the interaction with the acid variability, displayed in four regions of the enzyme: (1) helix• residues of the LBS, but shifted by one nucleotide. The analysis of loop•helix motif in the N•terminal domain, involved in the binding conserved residues within flaviviral NS5MTases shows that there is of the substrate (residues Gly21•Lys22•Thr23 in YOKV NS5MTase, an almost continuous and conserved region extending away from substituted with Gly•Lys•Ser in DENV NS5MTase, and Thr•Lys•Glu the active site towards the back of the protein—residues Tyr89, in MEAV NS5MTase); (2) ␣3• ␣X loop (Asn47•Ile53; insertion of Pro113, Gly120, Trp121, Asn122, Leu123, Ile124, Phe126, Lys127, one amino acid in MEAV NS5MTase); (3) ␣D• ␤5 loop (Leu172• Asp131, Gly263, Thr264 and Arg265 on the side of the NS5MTase, Thr178; deletion of two amino acids in DENV NS5MTase); and (4) and residues Ala60, Trp64, Leu207, Val208, Arg209, Pro211, Met220 ␣4• ␤8 loop (Leu246•Thr252; deletion of two amino acids in MEAV and Arg244 on the back (numbering referring to YOKV NS5MTase NS5MTase) (highlighted in Fig. 8 ) ( Bollati et al., 2009a ). (Bollati et al., 2009a ). This region may play a role in stabilization of the rest of the RNA chain following the cap (no crystal struc• 3.2.1.3. Structures of protein/ligand complexes. In the context of ture available so far) ( Bollati et al., 2009a ). In order to predict the the VIZIER Project, structures of different NS5MTases in complex interactions between the protein and the RNA during N7 methyl with cofactor and several capped substrate analogues have been transfer, Milani et al. presented a model of a short capped RNA solved ( Egloff et al., 2002, 2007; Assenberg et al., 2007; Bollati et (GpppAGUp) bound to the LBS of WESSV NS5MTase ( Milani et al., al., 2009b ). All the complexes show that the cap guanine moiety 2009 ). The model produced after 9 ns of Molecular Dynamic simu• (methylated or not) binds to a site next to the N•terminal helix• lation shows that the cap guanine was located close to AdoMet and turn•helix motif, called the high affinity binding site (HBS) formed to residues Glu149 and Arg213; other residues found to interact by residues Lys13, Leu16, Asn17, Leu19, Phe24, Ser150, Arg197, with capped RNA were: Arg37, Arg41, Leu44, Ser56, Arg57, Arg84, Arg213, Ser215. In particular, Phe24 plays an essential role in driv• Glu149, Lys112, Ser150, Arg160, Ser215. In line with this model, ing the cap binding by stacking against the cap guanine base during it has been recently shown that point mutations of a good part of the interaction ( Egloff et al., 2002, 2007; Assenberg et al., 2007; these amino acids (Arg37, Arg57, Arg84, Trp87, Glu149, Arg213, and Mastrangelo et al., 2007a; Bollati et al., 2009b; Milani et al., 2009 ). Tyr220) dramatically inhibit N7 methyl transfer activity in WNV The first nucleotide after the cap is often disordered, reflecting a NS5MTase ( Dong et al., 2008 ). lack of strong interactions with the enzyme. This is in line with Based on the structure of MVEV NS5MTase in complex with other studies showing that a minimal number of nucleotides in GpppA, which reveals a crystallographic dimer with two GpppA capped RNA are required for substrate binding and methylation molecules bound per monomer, Assenberg et al. (2007) proposed (Ray et al., 2006; Egloff et al., 2007; Dong et al., 2008; Selisko et al., Model 2. In this structure, the first cap analogue binds in the HBS in press ). A unique conformation of the cap analogue is adopted by with the adenosine base approaching the AdoMet binding pocket. GpppA in complex with DENV NS5MTase. The guanine is bound The second analogue binds to the LBS and stacks against the first cap in the HBS and GpppA displays an overall hairpin•like shape in analogue, with the guanosine base stacking against the adenosine which the two bases stack against each other in a way that allows base of the first analogue ( Fig. 9 ) ( Assenberg et al., 2007 ). Although to accommodate a longer capped RNA because the 3 ′•position of this indicates that the LBS can bind cap analogues, supporting in this the A ribose is oriented towards a positively charged surface region way Model 1, the authors note that in the MVEV co•crystal struc• (Egloff et al., 2007 ). It was proposed that this conformation might ture the N7 and 2 ′O guanosine group of the two analogue bound mimic the reaction product of a putative flavivirus GTase activ• to the dimer are too far away (>10 and >8 Å, respectively) from ity: pppG + ppAGN leading to GpppAGN + pyrophosphate. Such a the AdoMet methyl leaving group to be catalytically relevant. This

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx 15

Fig. 9. Stereo view of the complex formed by MVEV NS5MTase with AdoHcy and two molecules of GpppA: the first cap analogue binds in the HBS, the second is adjacent and interacting with the positively charged residues near the AdoMet•binding cleft in the LBS ( Assenberg et al., 2007 ). casts some doubt over the catalytic relevance of the structure and no available data fully proving the transfer of GMP on a ppRNA indeed the significance of the LBS in controlling N7MTase activity. substrate catalyzed by a flavivirus NS5MTase domain. Further analysis of the crystallographic dimer show that two LBS regions form a nearly continuous positively charged groove, with 3.2.2. NS5 RNA•dependent RNA polymerase domain the four GpppA molecules interacting in a manner resembling that The first structure of a flavivirus NS5RdRp domain KUNV of a continuous strand of RNA. Based on this, Model 2 proposes that NS5RdRp was determined within the VIZIER Project ( Malet et al., the dimerization of two NS5MTase monomers generates a large 2007 ). The low sequence identity of flavivirus RdRps compared to positively charged RNA binding cleft that facilitates binding and other RdRps with existing structures ( Ferrer•Orta et al., 2005 ) pre• translocation of the capped RNA. As the RNA moves into the cat• cluded the use of the molecular replacement method. Starting from alytic site of the first monomer, N7 methylation occurs, followed by a number of constructs of the KUNV NS5RdRp domain comprising RNA translocation via the LBS to the HBS of the second monomer residues 273–905 with N• and C•terminal deletions, the structure ′ where 2 O methylation occurs. In this model the LBS acts merely as of a shorter form (317–905) could be solved using single anomalous an RNA•binding domain, in contrast to Model 1 discussed above. dispersion at 2.35 Å resolution. This form of KUNV NS5RdRp then allowed structure determination of the longer form (273–905) at 3.2.1.4. Functional characterization. The first evidence of enzymatic 3.0 Å resolution. The longer form was enzymatically active whereas activity from a flavivirus NS5MTase domain was demonstrated the shorter form was not ( Malet et al., 2007 ). The KUNV NS5RdRp with DENV NS5MTase using short capped RNA substrates structure then allowed the determination of the structure of the N7me ± GpppAC5 ( Egloff et al., 2002 ). Such short substrates, even NS5RdRp domain (starting at residue 273) of DENV3 NS5RdRp at starting with GpppAG (the first two nucleotides strictly conserved 1.85 Å resolution by molecular replacement ( Yap et al., 2007 ). in the 5 ′•end of the flavivirus genome), support specifically the 2′OMTase activity ( Kroschewski et al., 2008; Lim et al., 2008 ). In 3.2.2.1. General structural properties of the polymerase domain. the context of the VIZIER Project, a protocol was set•up to pro• The structure of the two flavivirus NS5RdRp domains have been duce pure N7me ± GpppACn substrates of varying chain lengths recently analyzed and reviewed in ( Malet et al., 2008 ). They adopt (n = 1–7) in high amounts ( Peyrane et al., 2007 ). The substrates were a typical RdRp right•hand structure comprising three subdomains: used to prove 2 ′OMTase activity in a variety of flavivirus NS5MTase fingers, palm and thumb ( Fig. 10 ). The fingers subdomain of the domains ( Mastrangelo et al., 2007a; Bollati et al., 2009a,b ) and short KUNV NS5RdRp construct was partially disordered, whereas set•up inhibition assays ( Luzhkov et al., 2007; Milani et al., 2009; the long constructs started with the ordered N•terminal helix Selisko et al., in press ). Interestingly, it was found that DENV (277–287 and 275–285 for KUNV NS5RdRp and DENV NS5RdRp, 2′OMTase activity and binding increases with the substrate chain respectively). Both flavivirus NS5RdRp domains display a closed length until they reach a plateau at n = 5. N7meGpppAC5 sub• conformation, where fingers and thumb subdomains are con• strates were used to determine kinetic parameters of the 2 ′OMTase nected. This is a characteristic of RdRps and in particular of activity of DENV NS5MTase ( Selisko et al., in press ). On the other primer•independent (de novo) RdRps (reviewed in Ferrer•Orta hand, in order to measure N7MTase activity of flavivirus NS5MTase et al., 2005 ). A structural element named the priming loop pro• domains, a substrate of 74 nucleotides is needed, which contains a vides the initiation platform. It belongs to the thumb subdomain conserved stem•loop structure ( Ray et al., 2006; Dong et al., 2007 ). and points towards the active site in the palm. The active site is In the context of VIZIER, capped subgenomic RNA of DENV was located at the intersection of two tunnels. Other de novo RdRps produced and used to demonstrate N7MTase activity of DENV and solved in complex with ssRNA template, NTPs and/or dsRNA prod• WESSV NS5MTases ( Milani et al., 2009 ). Moreover, in accordance uct ( Butcher et al., 2001; Tao et al., 2002; O’Farrell et al., 2003 ) with the hypothesis that the flavivirus NS5MTase domain contains suggest the following scenario shown in Fig. 10 . The first tun• the GTase activity, the WESSV NS5MTase domain was shown to nel, located between the fingers and the thumb, should allow the covalently bind GMP at Lys28 ( Bollati et al., 2009b ). Following the ssRNA template to access the active site. The second tunnel, roughly classic scheme of cap formation ( Furuichi and Shatkin, 2000 ) to perpendicular to the first, stretches across the entire protein. The complete the guanylyltransfer, GMP would be transferred to newly incoming NTP should arrive from the back of the tunnel and, after synthesized ppRNA (see introduction). To date, however, there are the polymerization has started, the nascent dsRNA should go out

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

16 M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx

Fig. 10. Overview of the flavivirus RdRp structure based on WNV NS5Pol ( Malet et al., 2007 ) as an example; a “Front” view is presented here in ribbon representation. Fingers, palm and thumb subdomains are colored in blue, green and red, respec• tively. The ssRNA template entry and the dsRNA exit are shown by black arrows. A dotted arrow points to the NTP entry tunnel at the back of the RdRp. Motifs A, C, E, F, the G•loop and the priming loop are colored in orange, yellow, grey, magenta, cyan and purple, respectively. The Asp residues of catalytic motifs A and C (Asp•533, Asp•663 and Asp•664) are represented as stick models. N•ter and C•ter indicate the termini of the RdRp domain. through the front of this tunnel. However, as for other de novo RdRps, a conformational change is necessary to avoid a steric clash Fig. 11. Structural formulae of (a) ribavirin and (b) EICAR. with the priming loop and allow neo•synthesized RNA to exit. As expected, the most closely related structures are those of de Fig. 11 ), run across the back of the NS5RdRp domain and end in an novo RdRps from members of the Flaviviridae family, namely HCV ␣•helix, which comes to the front and forms the interface between NS5RdRp ( Ago et al., 1999; Bressanelli et al., 1999; Lesburg et al., the bottom of the fingers subdomain and the palm. 1999 ) and bovine viral diarrhea virus (BVDV) NS5RdRp ( Choi et al., The NLS of DENV2 NS5 has been shown to be functional and the 2004 ). They show sequence identities as low as 11–21%, depending transport of NS5 to the nucleus vital for virus replication ( Pryor et on the subdomain considered ( Malet et al., 2007 ). Special features of al., 2007 ). Especially two lysine residues at the beginning of the ␣• flavivirus NS5RdRp structures are as follows ( Malet et al., 2008 ): (1) helix seem to be important. In contrast, KUNV NS5 does not localize The fingers subdomain was captured in a pre•initiation conforma• to the nucleus. Subtle differences in NLS geometry and charge dis• tion since motif F, normally comprising the NTP•binding sites does tribution may be responsible for distinct behavior towards nuclear not form the upper part of the NTP tunnel but is perpendicular to its import in closely related viruses, but this is not yet deducible from normal position and partially disordered ( Fig. 11 ). Additionally, the the structures. It has not been shown if NS5 of DENV3 localizes to fingers subdomain presents a loop, named G•loop, which protrudes the nucleus ( Malet et al., 2008 ). towards the active site (see Fig. 11 ). It was given the name G• loop because it harbours RdRp motif G in primer•dependent RdRps 3.2.2.2. Three•dimensional structures determined for the flaviviral (Gorbalenya et al., 2002; Ferrer•Orta et al., 2004 ). This loop may play polymerase domain. Two flavivirus NS5RdRp domain structures are a regulatory role similar to the C•terminal in other RdRps ( Adachi known so far, KUNV NS5RdRp and DENV3 NS5RdRp ( Malet et al., et al., 2002; Leveque et al., 2003; Ng and Parra, 2004 ). In summary, 2007; Yap et al., 2007 ). The structures are very similar with an a concerted conformational change of motif F and G•loop of the r.m.s.d. of 1.9, 0.8 and 1.0 Å (C ␣ atoms of matched residues) for trapped pre•initiation conformation is expected before flavivirus the fingers, palm and thumb subdomains, respectively. The overall NS5RdRp initiate RNA synthesis. (2) The priming loop is provided r.m.s.d. is expected to be high because of a domain rotation based by the thumb domain as observed for other de novo RdRps (bacte• on the hinge region between the thumb and the palm subdomains riophage phi6, HCV and BVDV RdRps ( Ago et al., 1999; Bressanelli near the Zn atom (see above). The rotation of the fingers subdomain et al., 1999; Lesburg et al., 1999; Butcher et al., 2001; Choi et al., by 8 ◦ leaves the DENV3 NS5RdRp structure more open in compari• 2004 ) but does not contain any secondary structure. Two aromatic son to KUNV NS5RdRp. Another consequence is that the active site residues Trp795 or His798 (DENV NS5RdRp), may act as initiation of KUNV NS5RdRp is more tightly closed by the priming loop than platform stacking with the priming nucleotide. (3) Two Zn ions the active site of DENV3 NS5RdRp. KUNV NS5RdRp may need a were found, one in the fingers and one in the thumb subdomain. The wider opening movement upon the transition to elongation mode, latter is localized at a supposed hinge position between the thumb which could explain why the transition from initiation to elon• and the palm subdomains. It might play a role in the regulation gation seems to be kinetically more limiting for KUNV NS5RdRp of the conformational change between initiation and elongation compared to DENV2 NS5RdRp ( Selisko et al., 2006 ). state of flavivirus NS5RdRps. (4) Two NLSs are present at the sur• Both flavivirus NS5RdRp structures were obtained with a Mg 2+ face of the fingers subdomain. They comprise the first fingertip ion in a non•catalytic position near the active center. The role of the forming the connection between finger and thumb domain (see non•catalytic ion is not known. It was proposed that it might play a

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx 17 role in the de novo initiation mechanism facilitating the movement of the nascent dsRNA after formation of the first dinucleotide out of the active site ( Butcher et al., 2001 ). DENV3 NS5RdRp crystals were soaked with the nucleotide analog 3 ′dGTP and the complex structure solved at 2.6 Å resolution. Only the triphosphate moiety of 3 ′dGTP is visible. Nevertheless, its position near the priming loop and in particular next to residue Trp795 lead to the proposal that it represents the priming nucleotide and Trp795 acts as the initia• tion platform ( Yap et al., 2007 ). This is consistent with a model of the de novo initiation complex of KUNV NS5RdRp, which assigned the same Trp, conserved in all flavivirus NS5 proteins, as initiation platform ( Malet et al., 2007 ).

3.2.2.3. Characterization of polymerase activity. Within the VIZIER Project, a comparison of steady•state enzymatic activity param• eters of both full•length NS5 and NS5RdRp domain of DENV2 and KUNV on a homopolymeric template poly(rC) suggested that the NS5MTase domain does not influence de novo RdRp activity (Selisko et al., 2006; Zhang et al., 2008 ), although others did not support fully this view ( Yap et al., 2007 ). The NS5RdRp domain alone was used for screening processes and characterization of inhibitors of the flavivirus NS5RdRp activity. Furthermore, it has been shown by atomic force microcopy that a NS5RdRp domain of DENV2 with the same boundaries (272–900) binds to the cir• cularized DENV2 RNA genome and that de novo RNA synthesis of the negative strand is enhanced by the presence of a promoter ele• ′ Fig. 12. Mechanism of ribavirin action. Target enzyme: IMP dehydrogenase. Rib• ment, a large stem•loop structure, named SLA, present at the 5 •end avirin 5 ′ monophosphate inhibits the conversion of IMP to XMP resulting in a of the genome ( Filomatori et al., 2006 ). The authors demonstrated reduced supply of GTP, and, indirectly, also a reduced supply of ATP. the physical interaction of the NS5RdRp domain with SLA. They proposed a novel mechanism for −ssRNA synthesis in which the flavivirus NS5RdRp is recruited by and specifically binds SLA at the temperature and level of humidity). Moreover, (vi) the production 5′•end of the genome. It then reaches the site of initiation at the 3 ′• should be “easy” and low•cost (limited number of chemical steps end recruited to the 5 ′•end via long•range RNA–RNA interactions. and common availability of the starting material). The VIZIER Project contributed decisively to a precise identi• fication of the flaviviral NS5RdRp domain and to its subsequent 4.1. A broad•spectrum antiviral molecule with weak activity structural characterization ( Selisko et al., 2006; Malet et al., 2007; Yap et al., 2007 ). Our contributions will greatly facilitate the explo• Ribavirin (1•beta• d•ribofuranosyl•1,2,4•triazole•3•carboxam• ration of the flavivirus NS5RdRp as a drug target ( Rawlinson et al., ide, Fig. 11 a) is a broad•spectrum inhibitor of RNA viruses replica• 2006; Malet et al., 2008 ), hopefully leading to the discovery and tion proved to treat HCV infections, in combination with pegylated design of drugs against flaviviruses. interferon and in aerosol form, for the treatment of pediatric res• piratory syncytial virus (RSV) infections. Ribavirin has also been 4. Antivirals used experimentally against a number of other conditions, includ• ing Lassa fever, Crimean•Congo hemorrhagic fever virus (CCHFV), A safe and efficient anti•flavivirus/anti•DENV drug could poten• and hantaviruses ( Ergonul, 2008; Jonsson et al., 2008; Khan et al., tially be used for the treatment of patients living in endemic regions 2008 ). Almost all RNA viruses and even some DNA viruses are sen• and presenting symptoms of DENV infection, as well as patients sitive to the in vitro antiviral activity of ribavirin. Some viruses are with laboratory•diagnosed DENV infection. Such drug may also be more susceptible to the action of ribavirin than others; flaviviruses, of prophylactic relevance in case of an epidemic, in particular in for example, are much less sensitive than the paramyxovirus RSV regions where more than one serotype is circulating. Another pos• (Leyssen et al., 2005 ). sible prophylactic use can be by travelers to and through endemic The antiviral activity of ribavirin was reported almost four regions and by personnel of NGOs working in endemic regions or decades ago, but the molecular mechanism by which the compound by military personnel carrying out humanitarian actions. Because exerts its antiviral activity still remains a matter of debate. Inosine severe DENV disease has been associated with higher virus titres 5′•monophosphate (IMP) dehydrogenase, a cellular enzyme which (Whitehead et al., 2007 ), reduction of viral replication may be converts IMP to xanthosine 5 ′•monophosphate in the de novo syn• instrumental to limit the risk of developing such symptoms. thesis pathway of GMP, is inhibited by ribavirin 5 ′•monophosphate The ideal flavivirus drug should preferably be active against all (Streeter et al., 1973 ). As a consequence, intracellular GTP pools are four DENV serotypes (and even other flavivirus infections, such depleted, resulting in inhibition of viral (but also cellular) RNA syn• as WNV and JEV). Such drug should be (i) administered via the thesis ( Fig. 12 ). Several other mechanisms have been proposed to oral route and should thus have high oral bioavailability. Ideally, it contribute to the antiviral activity of ribavirin, including inhibition should be (ii) administered only once or twice (maximum 3 times) of viral capping (via an effect on the viral GTase or MTase activities) daily and (iii) have a high genetic barrier to resistance. Obviously, (Benarroch et al., 2004; Bougie and Bisaillon, 2004 ), and inhibition (iv) a drug that is to be used in the prophylactic setting as well as of RdRp activity by the 5 ′•triphosphate group of the drug ( Maag et in pediatric patients should be very safe. Moreover, since flavivirus al., 2001; Bougie and Bisaillon, 2003 ). Moreover, inhibition of the drugs will be used in tropical regions, the drug should also have viral helicase activity by ribavirin has been proposed for reoviruses (v) good thermal stability and a good hygroscopic parameter (high (Rankin et al., 1989 ).

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

18 M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx

Fig. 13. Structural formulae of (a) 2 ′•c•methylcytidine, (b) 4 ′•azidocytidine and (c) T•705.

Poliovirus cultured in the presence of ribavirin (concentrations Recently, a substituted pyrazine (T705, Fig. 13 c) has been up to 1 mM) accumulated mutations in its genome, a process called reported to be a potent inhibitor against influenza A, B and error catastrophe ( Crotty et al., 2001 ). For flaviviruses we demon• C viruses in vitro (Furuta et al., 2002, 2005 ). It has been strated that GTP pool depletion is the predominant mechanism by proposed that T•705 is converted intracellularly to the ribonu• which ribavirin exerts its antiviral activity ( Leyssen et al., 2005 ) and cleotide T•705•ribofuranosyl•5 ′•monophosphate (T•705 RMP) by that an error catastrophe based mechanism does not contribute to a phosphoribosyl transferase, and, upon phosphorylation, to its the in vitro antiviral activity of the drug ( Leyssen et al., 2006 ). The 5′•triphosphate. This metabolite would inhibit the influenza virus effect of ribavirin was studied in rhesus monkeys infected with YFV RdRp in a GTP•competitive manner ( Furuta et al., 2002, 2005 ). or DENV1. Either therapeutic or prophylactic protocols were stud• Unlike ribavirin 5 ′•monophosphate, T•705 RMP does not signifi• ied. Overall, no effect on viremia and survival was noted ( Huggins cantly inhibit IMP dehydrogenase, indicating that it may owe its et al., 1984; Huggins, 1989; Malinoski et al., 1990 ). Since the mech• anti•influenza virus activity mainly, if not exclusively to inhibi• anism of anti•flavivirus activity of ribavirin is based on an aspecific tion of the influenza virus RNA polymerase. Surprisingly, T•705 mechanism, the design of safe and more potent analogues of rib• has also been accredited with both activity against other viruses, avirin will likely be very difficult to achieve. EICAR ( Fig. 11 b), the i.e. arenaviruses (Pichinde), and bunyaviruses (Punta Toro) and fla• 5•ethynyl analogue of ribavirin, was shown to be roughly 10–20• viviruses. In addition to inhibiting YFV and WNV replication in vitro , fold more potent in inhibiting flaviviruses replication in vitro . This improvements in survival and disease parameters were observed improved activity came, however, at the price of a concomitant also after addition of T•705 to YFV• or WNV•infected rodents increase in toxicity ( Leyssen et al., 2005 ), which is explained by the (Morrey et al., 2008; Julander et al., 2009 ). It may be assumed fact that EICAR 5 ′•monophosphate is also more potent in inhibiting that the mechanism by which T•705 inhibits viruses other than the IMP dehydrogenase ( Balzarini et al., 1993 ). Recently, a hetero• influenza is similar to the mechanism by which it is believed to cyclic molecule with in vitro anti•DENV activity was reported; the inhibit influenza virus replication. This remains subject of further mechanism of action was suggested to be related to the inhibition studies. Such studies may also provide insight on how broad• of cellular IMP dehydrogenase ( Nair et al., 2009 ). spectrum inhibitors of RNA viruses encompassing both ( −)ssRNA and (+)ssRNA viruses should be designed,. 4.2. Selective inhibitors of viral replication So far, non•nucleoside inhibitors of flavivirus replication which target the viral NS5RdRp domain have not yet been reported. The HCV RdRp has been shown to be an excellent target for inhi• Within the VIZIER Project, potential allosteric inhibitor binding bition of viral replication. In fact, numerous selective inhibitors of sites were predicted on the NS5RdRp of DENV and WNV, using HCV replication that target this enzyme have been identified so far. two different programs ( Malet et al., 2008 ). Since several classes of These compounds can largely been classified as nucleoside (that non•nucleoside inhibitors of pestiviruses and HCV RdRp (and four need to be phosphorylated to their 5 ′•triphosphate metabolite) allosteric binding sites) have been identified, it may be assumed and non•nucleoside inhibitors (that act as allosteric site inhibitors) that this class of inhibitors may also have potential against fla• (De Clercq and Neyts, 2009 ). The nucleoside HCV inhibitors 2 ′• viviruses. C•methylcytidine ( Fig. 13 a), and related 2 ′•C•methyl nucleosides, inhibit the replication of a broad spectrum of (+)ssRNA viruses 4.3. Identification of novel antivirals including flaviviruses ( Eldrup et al., 2004 ). Another nucleoside inhibitor, i.e. 4 ′•azidocytidine ( Fig. 13 b), is solely active against As outlined above, target•based design of inhibitors of flavivirus HCV and does not show activity against flaviviruses and other RNA replication may be a promising strategy towards the development viruses ( Klumpp et al., 2006 , and unpublished data). It remains to be of selective anti•flaviviral drugs. Another strategy, with a proven studied what is the structural basis for the broad•spectrum activity success in the development of inhibitors of for example HIV, her• versus RNA viruses of the 2 ′•C•methyl nucleoside analogues and pes and HCV, has been based on the screening of large libraries the lack of activity of the 4 ′•azido nucleoside analogues. of molecules. Infected•cell•based screening assays offer the advan•

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx 19 tage that (i) novel targets for antiviral therapy in the replication DENV subgenomic replicons. Preincubation of high•titre DENV or cycle of the virus that would not have been discovered in target• YFV•17D stocks with 5 or 10 ␮g/ml SA•17 for 1 h resulted in 100% based assays may be identified, and (ii) compounds that do not inhibition of viral infectivity. Inhibition of viral infectivity by SA•17 enter the host cell or that are toxic to the host cell will be excluded in such pre•incubation experiments correlates with the antiviral for further validation. Several examples of antiviral drugs that effect obtained in virus•yield assays. Molecular modeling studies would not have been identified in target•based screenings assays identified a putative binding site for SA•17 in the DENV glycopro• include (i) the BVDV non•nucleoside inhibitors (VP32947, BPIP, tein E ( Kaptein et al., submitted for publication ). AG110, LZ37) that target the viral RdRp, but that do not exhibit A second compound class, identified in a large screening effort, inhibitory activity on the purified RdRp, (ii) the imidazopyridines consists of a series of small drug•like molecules that inhibit the with anti•HCV activity, of which one analogue (GS•9190), target• replication of both YFV and DENV2 replication (representative ing the HCV RdRp but not inhibiting the activity of the purified molecule CHI•104). Unlike SA•17, it appeared from time of drug enzyme, is currently in phase II clinical studies ( Vliegen et al., 2009 ), addition studies that this class of compounds interferes with a step and (iii) the cyclophilin•binding agent Debio•025, a potent inhibitor in the replication cycle of flaviviruses that coincides with the onset of HCV replication (currently in phase II clinical studies) that pre• of viral RNA synthesis. This was corroborated by the observation vents HCV replication by interfering with cyclophilins (which are that the compounds are active (like the reference compound rib• essential in the replication cycle of HCV), but that has not been avirin, but unlike compound SA•17 that acts at an early stage of shown to directly inhibit a particular enzymatic function of the the replication cycle) in the DENV subgenomic replicon system. virus ( Chatterji et al., 2009; Coelmont et al., 2009 ). Currently, drug•resistant variants against CHI•104 (and analogues) are being generated. It is expected that drug•resistant variants will 4.3.1. High•throughput screening approach carry mutations in the NS genes encoded by the replicon. Reintro• Most flaviviruses that are pathogenic to humans, readily cause a duction of the mutations identified in drug•resistant viruses in the cyto•pathogenic effect (CPE) in cell culture, being thus amenable to wild•type genome should result in a resistant phenotype. Once the high•throughput screening programs. Compounds to be screened target has been identified using this approach, the gene of interest should not contain potential toxic functions, should not have unsta• will be expressed and, if an enzymatic activity is associated with ble chemical groups, or poor solubility, and should ideally comply this protein, a potential inhibitory activity of the compound (class) with Lipinski’s rule of five or the Veber rules. Furthermore, the on this protein will be studied. If a crystal structure of the target library can be enriched for drug•like compounds (Comprehensive protein is available, this should allow to define (based on soaking Medicinal Chemistry mapping). To identify molecules with poten• or co•crystallization experiments with subsequent complex struc• tial in vitro anti•DENV activity (hit compounds), a primary screen ture determination) the precise molecular interactions between the is run (using multiple dilutions/compound to exclude too many inhibitor and the target. In turn, this will allow a rational approach false positives) and the potential inhibitory effect on virus•induced to optimize the antiviral activity/selectivity. CPE is quantified [employing a luminescence•based metabolic assay (ATP•lite)]. Concomitantly with the evaluation of the antivi• 4.3.2. Virtual docking of small molecules ral effect, the potential anti•metabolic effect of the compounds In the context of the VIZIER Project, about one hundred crys• on uninfected cells is quantified [employing an absorbance•based tal structures of important enzymes relevant for viral replication metabolic assay]. Once hit compounds have been identified, the have been determined. The 3D structure of an enzyme, and, in par• antiviral activity and selectivity need to be confirmed using a ticular, of its active site, can be an useful tool to identify possible newly synthesized batch of the molecule. Next, the antiviral activ• inhibitors of the target protein. In virtual docking, a library of small ity, either based on quantifying the effect of the compound on the molecules is used to identify ligands with high binding affinity to infectious virus•yield and/or the effect on viral RNA production, is the protein active site. In VIZIER Project, in order to find new fla• confirmed in virus•yield assays. Once this has been accomplished, vivirus inhibitors, many efforts have been spent on the study of a hit•to•lead optimization process can be initiated, given the fact the NS5MTase enzyme. In particular, the binding site of its cofactor that the compound class is chemically tractable. Meanwhile studies AdoMet was used to screen a set of 7836 potential ligand structures to characterize the antiviral activity and to identify the molecular by virtual docking ( Luzhkov et al., 2007 ). The structures were gen• target are performed. erated by geometry optimization and ligand preparation of 2566 Within the VIZIER consortium, two compound classes with hits that had been selected from a data base of 2.1 million com• antiflavivirus activity were identified. A first compound, SA•17, mercially available compounds after conducting a pharmacophore identified as a potent inhibitor of flavivirus replication, is an and a 2D similarity search. One of the top binders was found to ′ analogue of doxorubicin (an antineoplastic antibiotic from Strep• inhibit the 2 OMTase activity of DENV NS5MTase with an IC 50 value tomyces peucetius) that carries a squaric acid amide•ester moiety of 60.5 ␮M. Another approach for NS5MTase inhibition was based at the carbohydrate ( ␣•l•daunosaminyl) of doxorubicin. It should on the mechanism of action of the enzyme. In this case, the virtual be mentioned that this molecule does not comply with the Lipin• search was directed to find molecules potentially able to bind to ski rule of five. SA•17 was found to have excellent activity against the protein active site in presence of the AdoMet cofactor. In the DENV (EC50 = 0.3 ␮g/ml) and is markedly less cytostatic than the latter approach, it was chosen not to interfere with the binding of parent compound (CC50 = 28 ␮g/ml). SA•17 also inhibited YFV•17D the cofactor to the protein but with the methyl transfer activity replication, although less efficiently than DENV replication, but (Milani et al., 2009 ). Therefore, virtual screening of a compounds proved inactive against other viruses (the picornavirus coxsack• library yielded a ligand capable to bind to the NS5MTase active ievirus B3, the retroviruses HIV•1 and HIV•2, and the herpesvirus site in presence of AdoMet. Such a ligand can interfere with the HSV•1). SA•17 inhibits flavivirus replication in Vero cells in a dose• methyl transfer activity of the enzyme because of steric hindrance dependent manner, as assessed by virus•yield reduction assays and in the active site. In both cases, inhibition of AdoMet binding site quantification of viral RNA by means of q•RT•PCR. The anti•DENV or hindrance of the enzyme active site, the two molecules identi• activity was confirmed using a Renilla luciferase expressing DENV fied showed activity against the enzyme (N7MTase and 2 ′OMTase reporter. Time•of•drug addition studies revealed that SA•17 acts activities). The potential use of the two new inhibitors against viral at the very early stages of the viral replication cycle. This obser• replication is still under investigation. vation was corroborated by the observation that SA•17, unlike the In addition to the components of the flaviviral replicase com• nucleoside analogue ribavirin, does not inhibit the replication of plex, the protease is also of high interest as a target for new

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

20 M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx antivirals (see Section 3.1.1 above). Here, some lessons can be 2 virus and its candidate vaccine derivative: sequence relationships with the learned from drug discovery efforts performed in order to inhibit flaviviruses and other viruses. Virology 187, 573–590. Bollati, M., Milani, M., Mastrangelo, E., de Lamballerie, X., Canard, B., Bolognesi, the proteases of HCV and HIV, both well established drug targets. M., 2009a. Crystal structure of a methyltransferase from a no•known•vector As various high•throughput screening attempts did not produce the Flavivirus. Biochem. Biophys. Res. Commun. 382, 200–204. originally sought results, researchers chose structure•based design Bollati, M., Milani, M., Mastrangelo, E., Ricagno, S., Tedeschi, G., Nonnis, S., Decroly, E., Selisko, B., de Lamballerie, X., Coutard, B., Canard, B., Bolognesi, M., 2009b. Recog• strategies to develop potent inhibitors. This resulted in impres• nition of RNA cap in the Wesselsbron virus NS5 methyltransferase domain: sive success stories ( Anderson et al., 2009; Lamarre et al., 2003; implications for RNA•capping mechanisms in Flavivirus. J. Mol. Biol. 385, Tsantrizos, 2004 ). Similar strategies are currently being applied to 140–152. the flavivirus NS2B/NS3 proteases, not only in academic laborato• Bougie, I., Bisaillon, M., 2003. Initial binding of the broad spectrum antiviral nucle• oside ribavirin to the hepatitis C virus RNA polymerase. J. Biol. Chem. 278, ries, but increasingly also in the pharmaceutical industry. 52471–52478. Bougie, I., Bisaillon, M., 2004. The broad spectrum antiviral nucleoside ribavirin as a substrate for a viral RNA capping enzyme. J. Biol. Chem. 279, 22124–22130. Acknowledgements Bressanelli, S., Tomei, L., Roussel, A., Incitti, I., Vitale, R.L., Mathieu, M., De Francesco, R., Rey, F.A., 1999. Crystal structure of the RNA•dependent RNA polymerase of The work reported in this review was supported by the FP6 hepatitis C virus. Proc. Natl. Acad. Sci. U.S.A. 96, 13034–13039. Brooks, A.J., Johansson, M., John, A.V., Xu, Y., Jans, D.A., Vasudevan, S.G., 2002. The European Project VIZIER (Comparative Structural Genomics of Viral interdomain region of dengue NS5 protein that binds to the viral helicase NS3 Enzymes Involved in Replication—Contract number 2004•511960), contains independently functional importin beta 1 and importin alpha/beta• which is gratefully acknowledged. recognized nuclear localization signals. J. Biol. Chem. 277, 36399–36407. Bruenn, J.A., 2003. A structural and primary sequence comparison of the viral RNA• dependent RNA polymerases. Nucleic Acids Res. 31, 1821–1829. Appendix A. Supplementary data Bugl, H., Fauman, E.B., Staker, B.L., Zheng, F., Kushner, S.R., Saper, M.A., Bardwell, J.C., Jakob, U., 2000. RNA methylation under heat shock control. Mol. Cell 6, 349–360. Burns, K.F., Farinacci, C.J., Shelton, D.F., 1957. Virus of bats antigenically related to Supplementary data associated with this article can be found, in group B arthropod•borne encephalitis viruses. Am. J. Clin. Pathol. 27, 257–264. the online version, at doi:10.1016/j.antiviral.2009.11.009 . Butcher, S.J., Grimes, J.M., Makeyev, E.V., Bamford, D.H., Stuart, D.I., 2001. A mecha• nism for initiating RNA•dependent RNA polymerization. Nature 410, 235–240. Calisher, C.H., Karabatsos, N., Dalrymple, J.M., Shope, R.E., Porterfield, J.S., Westaway, References E.G., Brandt, W.E., 1989. Antigenic relationships between flaviviruses as deter• mined by cross•neutralization tests with polyclonal antisera. J. Gen. Virol. 70, Ackermann, M., Padmanabhan, R., 2001. De novo synthesis of RNA by the dengue 37–43. virus RNA•dependent RNA polymerase exhibits temperature dependence at the Cammisa•Parks, H., Cisar, L.A., Kane, A., Stollar, V., 1992. The complete nucleotide initiation but not elongation phase. J. Biol. Chem. 276, 39926–39937. sequence of cell fusing agent (CFA): homology between the nonstructural Adachi, T., Ago, H., Habuka, N., Okuda, K., Komatsu, M., Ikeda, S., Yatsunami, K., 2002. proteins encoded by CFA and the nonstructural proteins encoded by arthropod• The essential role of C•terminal residues in regulating the activity of hepatitis C borne flaviviruses. Virology 189, 511–524. virus RNA•dependent RNA polymerase. Biochim. Biophys. Acta 1601, 38–48. Campbell, M.S., Pletnev, A.G., 2000. Infectious cDNA clones of Langat tick•borne fla• Ago, H., Adachi, T., Yoshida, A., Yamamoto, M., Habuka, N., Yatsunami, K., Miyano, vivirus that differ from their parent in peripheral neurovirulence. Virology 269, M., 1999. Crystal structure of the RNA•dependent RNA polymerase of hepatitis 225–237. C virus. Structure 7, 1417–1426. Caruthers, J.M., McKay, D.B., 2002. Helicase structure and mechanism. Curr. Opin. Ahola, T., Kaariainen, L., 1995. Reaction in alphavirus mRNA capping: formation of a Struct. Biol. 12, 123–133. covalent complex of nonstructural protein nsP1 with 7•methyl•GMP. Proc. Natl. Castle, E., Leidner, U., Nowak, T., Wengler, G., 1986. Primary structure of the West Acad. Sci. U.S.A. 92, 507–511. Nile flavivirus genome region coding for all nonstructural proteins. Virology 149, Aleshin, A.E., Shiryaev, S.A., Strongin, A.Y., Liddington, R.C., 2007. Structural evi• 10–26. dence for regulation and specificity of flaviviral proteases and evolution of the Chambers, T.J., Grakoui, A., Rice, C.M., 1991. Processing of the yellow fever virus non• flaviviridae fold. Protein Sci. 16, 795–806. structural polyprotein: a catalytically active NS3 proteinase domain and NS2B Anderson, J., Schiffer, C., Lee, S.•K., Swanstrom, R., 2009. Viral protease inhibitors. are required for cleavages at dibasic sites. J. Virol. 65, 6042–6050. In: Kräusslich, H.•G., Bartenschlager, R. (Eds.), Antiviral Strategies. Handbook of Chambers, T.J., Nestorowicz, A., Amberg, S.M., Rice, C.M., 1993. Mutagenesis of the experimental pharmacology, pp. 85–110. yellow fever virus NS2B protein: effects on proteolytic processing, NS2B•NS3 Aranda, C., Sanchez•Seco, M.P., Caceres, F., Escosa, R., Galvez, J.C., Masia, M., Mar• complex formation, and viral replication. J. Virol. 67, 6797–6807. ques, E., Ruiz, S., Alba, A., Busquets, N., Vazquez, A., Castella, J., Tenorio, A., 2008. Chambers, T.J., Weir, R.C., Grakoui, A., McCourt, D.W., Bazan, J.F., Fletterick, R.J., Rice, Detection and monitoring of mosquito flaviviruses in Spain between 2001 and C.M., 1990. Evidence that the N•terminal domain of nonstructural protein NS3 2005. Vector Borne Zoon. Dis. (Larchmont, NY). from yellow fever virus is a serine protease responsible for site•specific cleavages Assenberg, R., Ren, J., Verma, A., Walter, T.S., Alderton, D., Hurrelbrink, R.J., Fuller, in the viral polyprotein. Proc. Natl. Acad. Sci. U.S.A. 87, 8898–8902. S.D., Bressanelli, S., Owens, R.J., Stuart, D.I., Grimes, J.M., 2007. Crystal structure of Chappell, K.J., Stoermer, M.J., Fairlie, D.P., Young, P.R., 2007. Generation and charac• the Murray Valley encephalitis virus NS5 methyltransferase domain in complex terization of proteolytically active and highly stable truncated and full•length with cap analogues. J. Gen. Virol. 88, 2228–2236. recombinant West Nile virus NS3. Protein Expr. Purif. 53, 87–96. Bakonyi, T., Gould, E.A., Kolodziejek, J., Weissenbock, H., Nowotny, N., 2004. Com• Chappell, K.J., Stoermer, M.J., Fairlie, D.P., Young, P.R., 2008. West Nile Virus plete genome analysis and molecular characterization of Usutu virus that NS2B/NS3 protease as an antiviral target. Curr. Med. Chem. 15, 2771–2784. emerged in Austria in 2001: comparison with the South African strain SAAR• Charlier, N., Leyssen, P., Pleij, C.W., Lemey, P., Billoir, F., Van Laethem, K., Vandamme, 1776 and other flaviviruses. Virology 328, 301–310. A.M., De Clercq, E., de Lamballerie, X., Neyts, J., 2002. Complete genome sequence of Montana Myotis leukoencephalitis virus, phylogenetic analysis and compar• Balzarini, J., Karlsson, A., Wang, L., Bohman, C., Horska, K., Votruba, I., Fridland, ′ A., Van Aerschot, A., Herdewijn, P., De Clercq, E., 1993. Eicar (5•ethynyl• ative study of the 3 untranslated region of flaviviruses with no known vector. J. 1•beta• d•ribofuranosylimidazole•4•carboxamide). A novel potent inhibitor of Gen. Virol. 83, 1875–1885. inosinate dehydrogenase activity and guanylate biosynthesis. J. Biol. Chem. 268, Chatterji, U., Bobardt, M., Selvarajah, S., Yang, F., Tang, H., Sakamoto, N., Vuagniaux, 24591–24598. G., Parkinson, T., Gallay, P., 2009. The isomerase active site of cyclophilin a is Bartholomeusz, A., Tomlinson, E., Wright, P.J., Birch, C., Locarnini, S., Weigold, critical for HCV replication. J. Biol. Chem.. H., Marcuccio, S., Holan, G., 1994. Use of a flavivirus RNA•dependent RNA Chernov, A.V., Shiryaev, S.A., Aleshin, A.E., Ratnikov, B.I., Smith, J.W., Liddington, polymerase assay to investigate the antiviral activity of selected compounds. R.C., Strongin, A.Y., 2008. The two•component NS2B•NS3 proteinase represses Antiviral Res. 24, 341–350. DNA unwinding activity of the West Nile virus NS3 helicase. J. Biol. Chem. 283, Bazan, J.F., Fletterick, R.J., 1989. Detection of a trypsin•like serine protease domain 17270–17278. in flaviviruses and pestiviruses. Virology 171, 637–639. Choi, K.H., Groarke, J.M., Young, D.C., Kuhn, R.J., Smith, J.L., Pevear, D.C., Rossmann, Benarroch, D., Egloff, M.P., Mulard, L., Guerreiro, C., Romette, J.L., Canard, B., 2004. M.G., 2004. The structure of the RNA•dependent RNA polymerase from bovine A structural basis for the inhibition of the NS5 dengue virus mRNA 2 ′•O• viral diarrhea virus establishes the role of GTP in de novo initiation. Proc. Natl. methyltransferase domain by ribavirin 5 ′•triphosphate. J. Biol. Chem. 279, Acad. Sci. U.S.A. 101, 4425–4430. 35638–35643. Clum, S., Ebner, K.E., Padmanabhan, R., 1997. Cotranslational membrane insertion of Bera, A.K., Kuhn, R.J., Smith, J.L., 2007. Functional characterization of cis and trans the serine proteinase precursor NS2B•NS3(Pro) of dengue virus type 2 is required activity of the Flavivirus NS2B•NS3 protease. J. Biol. Chem. 282, 12883–12892. for efficient in vitro processing and is mediated through the hydrophobic regions Billoir, F., de Chesse, R., Tolou, H., de Micco, P., Gould, E.A., de Lamballerie, X., of NS2B. J. Biol. Chem. 272, 30715–30723. 2000. Phylogeny of the genus flavivirus using complete coding sequences of Coelmont, L., Kaptein, S., Paeshuyse, J., Vliegen, I., Dumont, J.M., Vuagniaux, G., Neyts, arthropod•borne viruses and viruses with no known vector. J. Gen. Virol. 81, J., 2009. Debio 025, a cyclophilin binding molecule, is highly efficient in clearing 781–790. hepatitis C virus (HCV) replicon•containing cells when used alone or in combi• Blok, J., McWilliam, S.M., Butler, H.C., Gibbs, A.J., Weiller, G., Herring, B.L., Hemsley, nation with specifically targeted antiviral therapy for HCV (STAT•C) inhibitors. A.C., Aaskov, J.G., Yoksan, S., Bhamarapravati, N., 1992. Comparison of a dengue• Antimicrob. Agents Chemother. 53, 967–976.

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx 21

Coia, G., Parker, M.D., Speight, G., Byrne, M.E., Westaway, E.G., 1988. Nucleotide Filomatori, C.V., Lodeiro, M.F., Alvarez, D.E., Samsa, M.M., Pietrasanta, L., Gamarnik, and complete amino acid sequences of Kunjin virus: definitive gene order A.V., 2006. A 5 ′ RNA element promotes dengue virus RNA synthesis on a circular and characteristics of the virus•specified proteins. J. Gen. Virol. 69 (Pt 1), 1– genome. Genes Dev. 20, 2238–2249. 21. Fu, J., Tan, B.•H., Yap, E.•H., Chan, Y.•C., Tan, Y.H., 1992. Full•length cDNA sequence Cook, S., Bennett, S.N., Holmes, E.C., De Chesse, R., Moureau, G., de Lamballerie, X., of dengue type 1 virus (Singapore Strain S275/90). Virology 188, 953–958. 2006. Isolation of a new strain of the flavivirus cell fusing agent virus in a natural Furuichi, Y., Shatkin, A.J., 2000. Viral and cellular mRNA capping: past and prospects. mosquito population from Puerto Rico. J. Gen. Virol. 87, 735–748. Adv. Virus Res. 55, 135–184. Cordin, O., Banroques, J., Tanner, N.K., Linder, P., 2006. The DEAD•box protein family Furuta, Y., Takahashi, K., Fukuda, Y., Kuno, M., Kamiyama, T., Kozaki, K., Nomura, N., of RNA helicases. Gene 367, 17–37. Egawa, H., Minami, S., Watanabe, Y., Narita, H., Shiraki, K., 2002. In vitro and Crabtree, M.B., Sang, R.C., Stollar, V., Dunster, L.M., Miller, B.R., 2003. Genetic and phe• in vivo activities of anti•influenza virus compound T•705. Antimicrob. Agents notypic characterization of the newly described insect flavivirus, Kamiti River Chemother. 46, 977–981. virus. Arch. Virol. 148, 1095–1118. Furuta, Y., Takahashi, K., Kuno•Maekawa, M., Sangawa, H., Uehara, S., Kozaki, K., Crochu, S., Cook, S., Attoui, H., Charrel, R.N., De Chesse, R., Belhouchet, M., Lemasson, Nomura, N., Egawa, H., Shiraki, K., 2005. Mechanism of action of T•705 against J.J., De Micco, P., De Lamballerie, X., 2004. Sequences of flavivirus•related RNA influenza virus. Antimicrob. Agents Chemother. 49, 981–986. viruses persist in DNA form integrated in the genome of Aedes spp. mosquitoes. Geiss, B.J., Thompson, A.A., Andrews, A.J., Sons, R.L., Gari, H.H., Keenan, S.M., Peersen, J. Gen. Virol. 85, 1971–1980. O.B., 2009. Analysis of flavivirus NS5 methyltransferase cap binding. J. Mol. Biol. Crotty, S., Cameron, C.E., Andino, R., 2001. RNA virus error catastrophe: direct molec• 385, 1643–1654. ular test by using ribavirin. Proc. Natl. Acad. Sci. U.S.A. 98, 6895–6900. Ghosh, D., Basu, A., 2008. Present perspectives on flaviviral chemotherapy. Drug Day, C.W., Smee, D.F., Julander, J.G., Yamshchikov, V.F., Sidwell, R.W., Morrey, J.D., Discov. Today 13, 619–624. 2005. Error•prone replication of West Nile virus caused by ribavirin. Antiviral Gorbalenya, A.E., Donchenko, A.P., Koonin, E.V., Blinov, V.M., 1989a. N•terminal Res. 67, 38–45. domains of putative helicases of flavi• and pestiviruses may be serine proteases. De Clercq, E., Neyts, J., 2009. Antiviral agents acting as DNA or RNA chain terminators. Nucleic Acids Res. 17, 3889–3897. Handbook Exp. Pharmacol. 189, 53–84. Gorbalenya, A.E., Koonin, E.V., Donchenko, A.P., Blinov, V.M., 1989b. Two related de Lamballerie, X., Crochu, S., Billoir, F., Neyts, J., de Micco, P., Holmes, E.C., Gould, superfamilies of putative helicases involved in replication, recombination, E.A., 2002. Genome sequence analysis of Tamana bat virus and its relationship repair and expression of DNA and RNA genomes. Nucleic Acids Res. 17, 4713– with the genus Flavivirus. J. Gen. Virol. 83, 2443–2454. 4730. Dong, H., Ray, D., Ren, S., Zhang, B., Puig•Basagoiti, F., Takagi, Y., Ho, C.K., Li, H., Gorbalenya, A.E., Pringle, F.M., Zeddam, J.L., Luke, B.T., Cameron, C.E., Kalmakoff, Shi, P.Y., 2007. Distinct RNA elements confer specificity to flavivirus RNA cap J., Hanzlik, T.N., Gordon, K.H., Ward, V.K., 2002. The palm subdomain•based methylation events. J. Virol. 81, 4412–4421. active site is internally permuted in viral RNA•dependent RNA polymerases of Dong, H., Ren, S., Zhang, B., Zhou, Y., Puig•Basagoiti, F., Li, H., Shi, P.Y., 2008. West an ancient lineage. J. Mol. Biol. 324, 47–62. Nile virus methyltransferase catalyzes two methylations of the viral RNA cap Grard, G., Lemasson, J.J., Sylla, M., Dubot, A., Cook, S., Molez, J.F., Pourrut, X., Charrel, through a substrate•repositioning mechanism. J. Virol. 82, 4295–4307. R., Gonzalez, J.P., Munderloh, U., Holmes, E.C., de Lamballerie, X., 2006. Ngoye Egloff, M.P., Benarroch, D., Selisko, B., Romette, J.L., Canard, B., 2002. An RNA cap virus: a novel evolutionary lineage within the genus Flavivirus. J. Gen. Virol. 87, (nucleoside•2 ′•O•)•methyltransferase in the flavivirus RNA polymerase NS5: 3273–3277. crystal structure and functional characterization. EMBO J. 21, 2757–2768. Grard, G., Moureau, G., Charrel, R.N., Holmes, E.C., Gould, E.A., de Lamballerie, X. Egloff, M.P., Decroly, E., Malet, H., Selisko, B., Benarroch, D., Ferron, F., Canard, B., Genomics and evolution of Aedes•borne flaviviruses and related viruses, J Gen 2007. Structural and functional analysis of methylation and 5 ′•RNA sequence Virol (2009) in press. requirements of short capped RNAs by the methyltransferase domain of dengue Grard, G., Moureau, G., Charrel, R.N., Lemasson, J.J., Gonzalez, J.P., Gallian, P., Gritsun, virus NS5. J. Mol. Biol. 372, 723–736. T.S., Holmes, E.C., Gould, E.A., de Lamballerie, X., 2007. Genetic characterization Eldrup, A.B., Prhavc, M., Brooks, J., Bhat, B., Prakash, T.P., Song, Q., Bera, S., Bhat, of tick•borne flaviviruses: new insights into evolution, pathogenetic determi• N., Dande, P., Cook, P.D., Bennett, C.F., Carroll, S.S., Ball, R.G., Bosserman, M., nants and taxonomy. Virology 361, 80–92. Burlein, C., Colwell, L.F., Fay, J.F., Flores, O.A., Getty, K., LaFemina, R.L., Leone, Grassmann, C.W., Isken, O., Behrens, S.E., 1999. Assignment of the multifunctional J., MacCoss, M., McMasters, D.R., Tomassini, J.E., Von Langen, D., Wolanski, B., NS3 protein of bovine viral diarrhea virus during RNA replication: an in vivo and Olsen, D.B., 2004. Structure–activity relationship of heterobase•modified 2 ′•C• in vitro study. J. Virol. 73, 9196–9205. methyl ribonucleosides as inhibitors of hepatitis C virus RNA replication. J. Med. Greig, J.R., Brownlee, A., Wilson, D.R., Gordon, W.S., 1931. The nature of louping ill. Chem. 47, 5284–5297. Vet. Record 11, 325. Ellis, B.R., Barrett, A.D., 2008. The enigma of yellow fever in East Africa. Rev. Med. Gritsun, T.S., Venugopal, K., Zanotto, P.M., Mikhailov, M.V., Sall, A.A., Holmes, E.C., Virol. 18, 331–346. Polkinghorne, I., Frolova, T.V., Pogodina, V.V., Lashkevich, V.A., Gould, E.A., 1997. Emonet, S., Grard, G., Brisbarre, N., Moureau, G., Temmam, S., Charrel, R., de Lam• Complete sequence of two tick•borne flaviviruses isolated from Siberia and the ballerie, X., 2006. LoPPS: a long PCR product sequencing method for rapid UK: analysis and significance of the 5 ′ and 3 ′•UTRs. Virus Res. 49, 27–39. characterisation of long amplicons. Biochem. Biophys. Res. Commun. 344, Gubler, D.J., Clark, G.G., 1995. Dengue/dengue hemorrhagic fever: the emergence of 1080–1085. a global health problem. Emerg. Infect. Dis. 1, 55–57. Emonet, S.F., Grard, G., Brisbarre, N.M., Moureau, G.N., Temmam, S., Charrel, R.N., de Guyatt, K.J., Westaway, E.G., Khromykh, A.A., 2001. Expression and purification of Lamballerie, X., 2007. Long PCR Product Sequencing (LoPPS): a shotgun•based enzymatically active recombinant RNA•dependent RNA polymerase (NS5) of the approach to sequence long PCR products. Nat. Protoc. 2, 340–346. flavivirus Kunjin. J. Virol. Methods 92, 37–44. Erbel, P., Schiering, N., D’Arcy, A., Renatus, M., Kroemer, M., Lim, S.P., Yin, Z., Keller, Guzman, M.G., Kouri, G., 2008. Dengue haemorrhagic fever integral hypothesis: con• T.H., Vasudevan, S.G., Hommel, U., 2006. Structural basis for the activation of firming observations, 1987–2007. Trans. R. Soc. Trop. Med. Hyg. 102, 522–523. flaviviral NS3 proteases from dengue and West Nile virus. Nat. Struct. Mol. Biol. Hahn, Y.S., Galler, R., Hunkapiller, T., Dalrymple, J.M., Strauss, J.H., Strauss, E.G., 1988. 13, 372–373. Nucleotide sequence of dengue 2 RNA and comparison of the encoded proteins Ergonul, O., 2008. Treatment of Crimean•Congo hemorrhagic fever. Antiviral Res. 78, with those of other flaviviruses. Virology 162, 167–180. 125–131. Hoshino, K., Isawa, H., Tsuda, Y., Yano, K., Sasaki, T., Yuda, M., Takasaki, T., Kobayashi, Falgout, B., Markoff, L., 1995. Evidence that flavivirus NS1•NS2A cleavage is mediated M., Sawabe, K., 2007. Genetic characterization of a new insect flavivirus isolated by a membrane•bound host protease in the endoplasmic reticulum. J. Virol. 69, from Culex pipiens mosquito in Japan. Virology 359, 405–414. 7232–7243. Huggins, J.W., 1989. Prospects for treatment of viral hemorrhagic fevers with rib• Falgout, B., Miller, R.H., Lai, C.J., 1993. Deletion analysis of dengue virus type 4 avirin, a broad•spectrum antiviral drug. Rev. Infect. Dis. 11 (Suppl. 4), S750–761. nonstructural protein NS2B: identification of a domain required for NS2B•NS3 Huggins, J.W., Robins, R.K., Canonico, P.G., 1984. Synergistic antiviral effects of protease activity. J. Virol. 67, 2034–2042. ribavirin and the C•nucleoside analogs tiazofurin and selenazofurin against Falgout, B., Pethel, M., Zhang, Y.M., Lai, C.J., 1991. Both nonstructural proteins NS2B togaviruses, bunyaviruses, and arenaviruses. Antimicrob. Agents Chemother. 26, and NS3 are required for the proteolytic processing of dengue virus nonstruc• 476–480. tural proteins. J. Virol. 65, 2467–2475. Hurrelbrink, R.J., Nestorowicz, A., McMinn, P.C., 1999. Characterization of infectious Farfan•Ale, J.A., Lorono•Pino, M.A., Garcia•Rejon, J.E., Hovav, E., Powers, A.M., Murray Valley encephalitis virus derived from a stably cloned genome•length Lin, M., Dorman, K.S., Platt, K.B., Bartholomay, L.C., Soto, V., Beaty, B.J., cDNA. J. Gen. Virol. 80 (Pt 12), 3115–3125. Lanciotti, R.S., Blitvich, B.J., 2009. Detection of RNA from a novel West Nile• Ingrosso, D., Fowler, A.V., Bleibaum, J., Clarke, S., 1989. Sequence of the d• like virus and high prevalence of an insect•specific flavivirus in mosquitoes aspartyl/ l•isoaspartyl protein methyltransferase from human erythrocytes. in the Yucatan Peninsula of Mexico. Am. J. Trop. Med. Hygiene 80, 85– Common sequence motifs for protein, DNA, RNA, and small molecule 95. S•adenosylmethionine•dependent methyltransferases. J. Biol. Chem. 264, Fauman, E.B., Blumenthal, R.M., Cheng, X., 1999. Structure and evolution of 20131–20139. AdoMet•dependent methyltransferases. In: Cheng, X., Blumental, R.M. (Eds.), Irie, K., Mohan, P.M., Sasaguri, Y., Putnak, R., Padmanabhan, R., 1989. Sequence anal• S•adenosylmethionine•Dependent Methyltransferases. World Scientific Pub• ysis of cloned dengue virus type 2 genome (New Guinea•C strain). Gene 75, lishing, Singapore, pp. 1–38. 197–211. Ferrer•Orta, C., Arias, A., Escarmis, C., Verdaguer, N., 2005. A comparison of viral Jansson, A.M., Jakobsson, E., Johansson, P., Lantez, V., Coutard, B., de Lamballerie, X., RNA•dependent RNA polymerases. Curr. Opin. Struct. Biol.. Unge, T., Jones, T.A., 2009. Structure of the methyltransferase domain from the Ferrer•Orta, C., Arias, A., Perez•Luque, R., Escarmis, C., Domingo, E., Verdaguer, N., Modoc virus, a flavivirus with no known vector. Acta Crystallogr. Section D: Biol. 2004. Structure of foot•and•mouth disease virus RNA•dependent RNA poly• Crystallogr.. merase and its complex with a template•primer RNA. J. Biol. Chem. 279, Johansson, M., Brooks, A.J., Jans, D.A., Vasudevan, S.G., 2001. A small region of the 47212–47221. dengue virus•encoded RNA•dependent RNA polymerase, NS5, confers inter•

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

22 M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx

action with both the nuclear transport receptor importin•beta and the viral Lim, S.P., Wen, D., Yap, T.L., Yan, C.K., Lescar, J., Vasudevan, S.G., 2008. A scintilla• helicase, NS3. J. Gen. Virol. 82, 735–745. tion proximity assay for dengue virus NS5 2 ′•O•methyltransferase•kinetic and Johnson, H.N., 1957. The Rio Bravo virus: virus identified with group B arthropod• inhibition analyses. Antiviral Res. 80, 360–369. borne viruses by hemagglutination•inhibition and complement•fixation tests. Lindenbach, B.D., Rice, C.M., 2003. Molecular biology of flaviviruses. Adv. Virus Res. In: Proceedings of the Ninth Pacific Science Congress, vol. 17, p. 39. 59, 23–61. Jonsson, C.B., Hooper, J., Mertz, G., 2008. Treatment of hantavirus pulmonary syn• Liu, W.J., Sedlak, P.L., Kondratieva, N., Khromykh, A.A., 2002. Complementation anal• drome. Antiviral Res. 78, 162–169. ysis of the flavivirus Kunjin NS3 and NS5 proteins defines the minimal regions Julander, J.G., Shafer, K., Smee, D.F., Morrey, J.D., Furuta, Y., 2009. Activity of T•705 essential for formation of a replication complex and shows a requirement of NS3 in a hamster model of yellow fever virus infection in comparison with that in cis for virus assembly. J. Virol. 76, 10766–10775. of a chemically related compound, T•1106. Antimicrob. Agents Chemother. 53, Luo, D., Xu, T., Hunke, C., Gruber, G., Vasudevan, S.G., Lescar, J., 2008a. Crystal struc• 202–209. ture of the NS3 protease•helicase from dengue virus. J. Virol. 82, 173–183. Junglen, S., Kopp, A., Kurth, A., Pauli, G., Ellerbrok, H., Leendertz, F.H., 2009. A new Luo, D., Xu, T., Watson, R.P., Scherer•Becker, D., Sampath, A., Jahnke, W., Yeong, S.S., flavivirus and a new vector: characterization of a novel flavivirus isolated from Wang, C.H., Lim, S.P., Strongin, A., Vasudevan, S.G., Lescar, J., 2008b. Insights into uranotaenia mosquitoes from a tropical rain forest. J. Virol. 83, 4462–4468. RNA unwinding and ATP hydrolysis by the flavivirus NS3 protein. EMBO J. 27, Kaptein, S., Jacobs, M., Gamarnik, A., de Lamballerie, X., De Clercq, E., Sztaricskai, F., 3209–3219. Neyts, J. A derivate of the antibiotic doxorubicin is a selective in vitro inhibitor Luzhkov, V.B., Selisko, B., Nordqvist, A., Peyrane, F., Decroly, E., Alvarez, K., Karlen, A., of dengue and yellow fever virus replication, submitted for publication. Canard, B., Qvist, J., 2007. Virtual screening and bioassay study of novel inhibitors Khan, S.H., Goba, A., Chu, M., Roth, C., Healing, T., Marx, A., Fair, J., Guttieri, M.C., for dengue virus mRNA cap (nucleoside•2 ′O)•methyltransferase. Bioorg. Med. Ferro, P., Imes, T., Monagin, C., Garry, R.F., Bausch, D.G., 2008. New opportunities Chem. 15, 7795–7802. for field research on the pathogenesis and treatment of Lassa fever. Antiviral Maag, D., Castro, C., Hong, Z., Cameron, C.E., 2001. Hepatitis C virus RNA•dependent Res. 78, 103–115. RNA polymerase (NS5B) as a mediator of the antiviral activity of ribavirin. J. Biol. Kim, D.Y., Guzman, H., Bueno Jr., R., Dennett, J.A., Auguste, A.J., Carrington, C.V., Chem. 276, 46094–46098. Popov, V.L., Weaver, S.C., Beasley, D.W., Tesh, R.B., 2009. Characterization of Mackenzie, J., 2005. Wrapping things up about virus RNA replication. Traffic 6, Culex Flavivirus (Flaviviridae) strains isolated from mosquitoes in the United 967–977. States and Trinidad. Virology 386, 154–159. Mackenzie, J.M., Khromykh, A.A., Jones, M.K., Westaway, E.G., 1998. Subcellular Kim, Y.G., Yoo, J.S., Kim, J.H., Kim, C.M., Oh, J.W., 2007. Biochemical characterization localization and some biochemical properties of the flavivirus Kunjin nonstruc• of a recombinant Japanese encephalitis virus RNA•dependent RNA polymerase. tural proteins NS2A and NS4A. Virology 245, 203–215. BMC Mol. Biol. 8, 59–70. Mackow, E., Makino, Y., Zhao, B.T., Zhang, Y.M., Markoff, L., Buckler White, A., Klumpp, K., Leveque, V., Le Pogam, S., Ma, H., Jiang, W.R., Kang, H., Granycome, C., Guiler, M., Chanock, R., Lai, C.J., 1987. The nucleotide sequence of dengue type Singer, M., Laxton, C., Hang, J.Q., Sarma, K., Smith, D.B., Heindl, D., Hobbs, C.J., 4 virus: analysis of genes coding for nonstructural proteins. Virology 159, 217– Merrett, J.H., Symons, J., Cammack, N., Martin, J.A., Devos, R., Najera, I., 2006. 228. The novel nucleoside analog R1479 (4 ′•azidocytidine) is a potent inhibitor of Malet, H., Egloff, M.P., Selisko, B., Butcher, R.E., Wright, P.J., Roberts, M., Gruez, A., NS5B•dependent RNA synthesis and hepatitis C virus replication in cell culture. Sulzenbacher, G., Vonrhein, C., Bricogne, G., Mackenzie, J.M., Khromykh, A.A., J. Biol. Chem. 281, 3793–3799. Davidson, A.D., Canard, B., 2007. Crystal structure of the RNA polymerase domain Koonin, E.V., 1991. The phylogeny of RNA•dependent RNA polymerases of positive• of the West Nile virus non•structural protein 5. J. Biol. Chem. 282, 10678– strand RNA viruses. J. Gen. Virol. 72 (Pt 9), 2197–2206. 10689. Koonin, E.V., 1993. Computer•assisted identification of a putative methyltransferase Malet, H., Masse, N., Selisko, B., Romette, J.L., Alvarez, K., Guillemot, J.C., Tolou, H., domain in NS5 protein of flaviviruses and lambda 2 protein of reovirus. J. Gen. Yap, T.L., Vasudevan, S., Lescar, J., Canard, B., 2008. The flavivirus polymerase as Virol. 74 (Pt 4), 733–740. a target for drug discovery. Antiviral Res. 80, 23–35. Kroeger, A., Nathan, M., Hombach, J., 2004. Dengue. Nat. Rev. Microbiol. 2, 360–361. Malinoski, F.J., Hasty, S.E., Ussery, M.A., Dalrymple, J.M., 1990. Prophylactic rib• Kroschewski, H., Lim, S.P., Butcher, R.E., Yap, T.L., Lescar, J., Wright, P.J., Vasude• avirin treatment of dengue type 1 infection in rhesus monkeys. Antiviral Res. van, S.G., Davidson, A.D., 2008. Mutagenesis of the dengue virus type 2 NS5 13, 139–149. methyltransferase domain. J. Biol. Chem. 283, 19410–19421. Mancini, E.J., Assenberg, R., Verma, A., Walter, T.S., Tuma, R., Grimes, J.M., Owens, R.J., Kuno, G., Chang, G.J., 2005. Biological transmission of arboviruses: reexamination Stuart, D.I., 2007. Structure of the Murray Valley encephalitis virus RNA helicase of and new insights into components, mechanisms, and unique traits as well as at 1.9 Angstrom resolution. Protein Sci. 16, 2294–2300. their evolutionary trends. Clin. Microbiol. Rev. 18, 608–637. Mancini, E.J., Kainov, D.E., Grimes, J.M., Tuma, R., Bamford, D.H., Stuart, D.I., 2004. Kuno, G., Chang, G.J., 2006. Characterization of Sepik and Entebbe bat viruses closely Atomic snapshots of an RNA packaging motor reveal conformational changes related to yellow fever virus. Am. J. Trop. Med. Hyg. 75, 1165–1170. linking ATP hydrolysis to RNA translocation. Cell 118, 743–755. Kuno, G., Chang, G.J., 2007. Full•length sequencing and genomic characterization of Mandl, C.W., Heinz, F.X., Stockl, E., Kunz, C., 1989. Genome sequence of tick•borne Bagaza, Kedougou, and Zika viruses. Arch. Virol.. encephalitis virus (Western subtype) and comparative analysis of nonstructural Kuno, G., Chang, G.J., Tsuchiya, K.R., Karabatsos, N., Cropp, C.B., 1998. Phylogeny of proteins with other flaviviruses. Virology 173, 291–301. the genus Flavivirus. J. Virol. 72, 73–83. Mandl, C.W., Holzmann, H., Kunz, C., Heinz, F.X., 1993. Complete genomic sequence Lamarre, D., Anderson, P.C., Bailey, M., Beaulieu, P., Bolger, G., Bonneau, P., Bös, M., of Powassan virus: evaluation of genetic elements in tick•borne versus et al., 2003. An NS3 protease inhibitor with antiviral effects in humans infected mosquito•borne flaviviruses. Virology 194, 173–184. with hepatitis C virus. Nature 426, 186–189. Manns, M.P., Foster, G.R., Rockstroh, J.K., Zeuzem, S., Zoulim, F., Houghton, M., 2007. Lesburg, C.A., Cable, M.B., Ferrari, E., Hong, Z., Mannarino, A.F., Weber, P.C., 1999. The way forward in HCV treatment—finding the right path. Nat. Rev. Drug Discov. Crystal structure of the RNA•dependent RNA polymerase from hepatitis C virus 6, 991–1000. reveals a fully encircled active site. Nat. Struct. Biol. 6, 937–943. Margulies, M., Egholm, M., Altman, W.E., Attiya, S., Bader, J.S., Bemben, L.A., Berka, J., Lescar, J., Luo, D., Xu, T., Sampath, A., Lim, S.P., Canard, B., Vasudevan, S.G., 2008. Braverman, M.S., Chen, Y.J., Chen, Z., Dewell, S.B., Du, L., Fierro, J.M., Gomes, X.V., Towards the design of antiviral inhibitors against flaviviruses: the case for the Godwin, B.C., He, W., Helgesen, S., Ho, C.H., Irzyk, G.P., Jando, S.C., Alenquer, M.L., multifunctional NS3 protein from Dengue virus as a target. Antiviral Res. 80, Jarvie, T.P., Jirage, K.B., Kim, J.B., Knight, J.R., Lanza, J.R., Leamon, J.H., Lefkowitz, 94–101. S.M., Lei, M., Li, J., Lohman, K.L., Lu, H., Makhijani, V.B., McDade, K.E., McKenna, Leung, D., Schroder, K., White, H., Fang, N.X., Stoermer, M.J., Abbenante, G., Martin, M.P., Myers, E.W., Nickerson, E., Nobile, J.R., Plant, R., Puc, B.P., Ronan, M.T., Roth, J.L., Young, P.R., Fairlie, D.P., 2001. Activity of recombinant dengue 2 virus NS3 G.T., Sarkis, G.J., Simons, J.F., Simpson, J.W., Srinivasan, M., Tartaro, K.R., Tomasz, protease in the presence of a truncated NS2B co•factor, small peptide substrates, A., Vogt, K.A., Volkmer, G.A., Wang, S.H., Wang, Y., Weiner, M.P., Yu, P., Begley, and inhibitors. J. Biol. Chem. 276, 45762–45771. R.F., Rothberg, J.M., 2005. Genome sequencing in microfabricated high•density Leveque, V.J., Johnson, R.B., Parsons, S., Ren, J., Xie, C., Zhang, F., Wang, Q.M., picolitre reactors. Nature 437, 376–380. 2003. Identification of a C•terminal regulatory motif in hepatitis C virus RNA• Mastrangelo, E., Bollati, M., Milani, M., Selisko, B., Peyrane, F., Canard, B., Grard, G., de dependent RNA polymerase: structural and biochemical analysis. J. Virol. 77, Lamballerie, X., Bolognesi, M., 2007a. Structural bases for substrate recognition 9020–9028. and activity in Meaban virus nucleoside•2 ′•O•methyltransferase. Protein Sci. 16, Lewis, J.A., Chang, G.J., Lanciotti, R.S., Kinney, R.M., Mayer, L.W., Trent, D.W., 1993. 1133–1145. Phylogenetic relationships of dengue•2 viruses. Virology 197, 216–224. Mastrangelo, E., Milani, M., Bollati, M., Selisko, B., Peyrane, F., Pandini, V., Sor• Leyssen, P., Balzarini, J., De Clercq, E., Neyts, J., 2005. The predominant mechanism rentino, G., Canard, B., Konarev, P.V., Svergun, D.I., de Lamballerie, X., Coutard, by which ribavirin exerts its antiviral activity in vitro against flaviviruses and B., Khromykh, A.A., Bolognesi, M., 2007b. Crystal structure and activity of Kunjin paramyxoviruses is mediated by inhibition of IMP dehydrogenase. J. Virol. 79, virus NS3 helicase; protease and helicase domain assembly in the full length 1943–1947. NS3 protein. J. Mol. Biol. 372, 444–455. Leyssen, P., Charlier, N., Lemey, P., Billoir, F., Vandamme, A.M., De Clercq, E., de Matusan, A.E., Pryor, M.J., Davidson, A.D., Wright, P.J., 2001. Mutagenesis of the Lamballerie, X., Neyts, J., 2002. Complete genome sequence, taxonomic assign• Dengue virus type 2 NS3 protein within and outside helicase motifs: effects ment, and comparative analysis of the untranslated regions of the Modoc virus, on enzyme activity and virus replication. J. Virol. 75, 9633–9643. a flavivirus with no known vector. Virology 293, 125–140. May, F.J., Lobigs, M., Lee, E., Gendle, D.J., Mackenzie, J.S., Broom, A.K., Conlan, J.V., Leyssen, P., De Clercq, E., Neyts, J., 2006. The anti•yellow fever virus activity of rib• Hall, R.A., 2006. Biological, antigenic and phylogenetic characterization of the avirin is independent of error•prone replication. Mol. Pharmacol. 69, 1461–1467. flavivirus Alfuy. J. Gen. Virol. 87, 329–337. Li, H., Clum, S., You, S., Ebner, K.E., Padmanabhan, R., 1999. The serine protease Medeiros, D.B., Nunes, M.R., Vasconcelos, P.F., Chang, G.J., Kuno, G., 2007. Complete and RNA•stimulated nucleoside triphosphatase and RNA helicase functional genome characterization of Rocio virus (Flavivirus: Flaviviridae), a Brazilian fla• domains of dengue virus type 2 NS3 converge within a region of 20 amino acids. vivirus isolated from a fatal case of encephalitis during an epidemic in Sao Paulo J. Virol. 73, 3108–3116. state. J. Gen. Virol. 88, 2237–2246.

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx 23

Milani, M., Mastrangelo, E., Bollati, M., Selisko, B., Decroly, E., Bouvet, M., Canard, Robin, G., Chappell, K., Stoermer, M.J., Hu, S.H., Young, P.R., Fairlie, D.P., Martin, B., Bolognesi, M., 2009. Flaviviral methyltransferase/RNA interaction: structural J.L., 2009. Structure of West Nile virus NS3 protease: ligand stabilization of the basis for enzyme inhibition. Antiviral Res. 83, 28–34. catalytic conformation. J. Mol. Biol. 385, 1568–1577. Miller, S., Kastner, S., Krijnse•Locker, J., Bühler, S., Bartenschlager, R., 2007. The non• Sampath, A., Xu, T., Chao, A., Luo, D., Lescar, J., Vasudevan, S.G., 2006. Structure• structural protein 4A of dengue virus is an integral membrane protein inducing based mutational analysis of the NS3 helicase from dengue virus. J. Virol. 80, membrane alterations in a 2K•regulated manner. J. Biol. Chem. 282, 8873–8882. 6686–6690. Mittl, P.R., Grutter, M.G., 2006. Opportunities for structure•based design of protease• Sang, R.C., Gichogo, A., Gachoya, J., Dunster, M.D., Ofula, V., Hunt, A.R., Crabtree, M.B., directed drugs. Curr. Opin. Struct. Biol. 16, 769–775. Miller, B.R., Dunster, L.M., 2003. Isolation of a new flavivirus related to cell fusing Morales•Betoulle, M.E., Monzon Pineda, M.L., Sosa, S.M., Panella, N., Lopez, M.R., agent virus (CFAV) from field•collected flood•water Aedes mosquitoes sampled Cordon•Rosales, C., Komar, N., Powers, A., Johnson, B.W., 2008. Culex flavivirus from a dambo in central Kenya. Arch. Virol. 148, 1085–1093. isolates from mosquitoes in Guatemala. J. Med. Entomol. 45, 1187–1190. Selisko, B., Dutartre, H., Guillemot, J.C., Debarnot, C., Benarroch, D., Khromykh, A., Morrey, J.D., Taro, B.S., Siddharthan, V., Wang, H., Smee, D.F., Christensen, A.J., Furuta, Despres, P., Egloff, M.P., Canard, B., 2006. Comparative mechanistic studies of de Y., 2008. Efficacy of orally administered T•705 pyrazine analog on lethal West novo RNA synthesis by flavivirus RNA•dependent RNA polymerases. Virology. Nile virus infection in rodents. Antiviral Res. 80, 377–379. Selisko, B., Peyrane, F.F., Canard, B., Alvarez, K., Decroly, E. Biochemical characterisa• Moureau, G., Ninove, L., Izri, A., Cook, S., De Lamballerie, X., Charrel, R.N., 2009. tion of the (nucleoside•2 ′O)•methyltransferase activity of dengue virus protein Flavivirus RNA in phlebotomine sandflies. Vector Borne Zoon. Dis.. NS5 using purified capped RNA oligonucleotides 7Me ± GpppACn, J Gen Virol Murthy, H.M., Clum, S., Padmanabhan, R., 1999. Dengue virus NS3 serine protease. (2009), in press. Crystal structure and insights into interaction of the active site with substrates Shiryaev, S.A., Kozlov, I.A., Ratnikov, B.I., Smith, J.W., Lebl, M., Strongin, A.Y., 2007. by molecular modeling and structural analysis of mutational effects. J. Biol. Cleavage preference distinguishes the two•component NS2B•NS3 serine pro• Chem. 274, 5573–5580. teinases of Dengue and West Nile viruses. Biochem. J. 401, 743–752. Murthy, H.M., Judge, K., DeLucas, L., Padmanabhan, R., 2000. Crystal structure of Speight, G., Coia, G., Parker, M.D., Westaway, E.G., 1988. Gene mapping and positive Dengue virus NS3 protease in complex with a Bowman•Birk inhibitor: impli• identification of the non•structural proteins NS2A, NS2B, NS3, NS4B and NS5 of cations for flaviviral polyprotein processing and drug design. J. Mol. Biol. 301, the flavivirus Kunjin and their cleavage sites. J. Gen. Virol. 69 (Pt 1), 23–34. 759–767. Speroni, S., De Colibus, L., Mastrangelo, E., Gould, E., Coutard, B., Forrester, N.L., Blanc, Nair, V., Chi, G., Shu, Q., Julander, J., Smee, D.F., 2009. A heterocyclic molecule S., Canard, B., Mattevi, A., 2008. Structure and biochemical analysis of Kokobera with significant activity against dengue virus. Bioorg. Med. Chem. Lett. 19, virus helicase. Proteins 70, 1120–1123. 1425–1427. Staples, J.E., Monath, T.P., 2008. Yellow fever: 100 years of discovery. JAMA 300, Nall, T.A., Chappell, K.J., Stoermer, M.J., Fang, N.X., Tyndall, J.D., Young, P.R., Fairlie, 960–962. D.P., 2004. Enzymatic characterization and homology model of a catalyti• Streeter, D.G., Witkowski, J.T., Khare, G.P., Sidwell, R.W., Bauer, R.J., Robins, R.K., cally active recombinant West Nile virus NS3 protease. J. Biol. Chem. 279, Simon, L.N., 1973. Mechanism of action of 1• d•ribofuranosyl•1,2,4•triazole•3• 48535–48542. carboxamide (Virazole), a new broad•spectrum antiviral agent. Proc. Natl. Acad. Ng, K.K., Parra, F., 2004. Crystal structure of Norwalk virus polymerase reveals the Sci. U.S.A. 70, 1174–1178. carboxyl terminus in the active site cleft. J. Biol. Chem.. Sumiyoshi, H., Mori, C., Fuke, I., Morita, K., Kuhara, S., Kondou, J., Kikuchi, Y., Naga• Nomaguchi, M., Ackermann, M., Yon, C., You, S., Padmanabhan, R., 2003. De novo syn• matu, H., Igarashi, A., 1987. Complete nucleotide sequence of the Japanese thesis of negative•strand RNA by Dengue virus RNA•dependent RNA polymerase encephalitis virus genome RNA. Virology 161, 497–510. in vitro: nucleotide, primer, and template parameters. J. Virol. 77, 8831–8842. Tajima, S., Takasaki, T., Matsuno, S., Nakayama, M., Kurane, I., 2005. Genetic charac• Nomaguchi, M., Teramoto, T., Yu, L., Markoff, L., Padmanabhan, R., 2004. Require• terization of Yokose virus, a flavivirus isolated from the bat in Japan. Virology ments for West Nile virus ( −)• and (+)•strand subgenomic RNA synthesis in 332, 38–44. vitro by the viral RNA•dependent RNA polymerase expressed in Escherichia coli . Takegami, T., Sakamuro, D., Furukawa, T., 1995. Japanese encephalitis virus non• J. Biol. Chem. 279, 12141–12151. structural protein NS3 has RNA binding and ATPase activities. Virus Genes 9, Nowak, T., Farber, P.M., Wengler, G., 1989. Analyses of the terminal sequences of 105–112. West Nile virus structural proteins and of the in vitro translation of these pro• Takhampunya, R., Ubol, S., Houng, H.S., Cameron, C.E., Padmanabhan, R., 2006. Inhi• teins allow the proposal of a complete scheme of the proteolytic cleavages bition of dengue virus replication by mycophenolic acid and ribavirin. J. Gen. involved in their synthesis. Virology 169, 365–376. Virol. 87, 1947–1952. O’Farrell, D., Trowbridge, R., Rowlands, D., Jager, J., 2003. Substrate complexes of Tan, B.H., Fu, J., Sugrue, R.J., Yap, E.H., Chan, Y.C., Tan, Y.H., 1996. Recombinant dengue hepatitis C virus RNA polymerase (HC•J4): structural evidence for nucleotide type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA•dependent import and de•novo initiation. J. Mol. Biol. 326, 1025–1035. RNA polymerase activity. Virology 216, 317–325. Osatomi, K., Sumiyoshi, H., 1990. Complete nucleotide sequence of dengue type 3 Tao, Y., Farsetta, D.L., Nibert, M.L., Harrison, S.C., 2002. RNA synthesis in a virus genome RNA. Virology 176, 643–647. cage—structural studies of reovirus polymerase lambda3. Cell 111, 733–745. Petersen, L.R., Hayes, E.B., 2008. West Nile virus in the Americas. Med. Clin. North Theiler, M., 1930. Studies on the action of yellow fever virus in mice. Ann. Trop. Med. Am. 92, 1307–1322, ix. 24, 249–272. Peyrane, F., Selisko, B., Decroly, E., Vasseur, J.J., Benarroch, D., Canard, B., Alvarez, K., Tsantrizos, Y.S., 2004. The design of a potent inhibitor of the hepatitis C virus NS3 2007. High•yield production of short GpppA• and 7MeGpppA•capped RNAs and protease: BILN2061 • from the NMR tube to the clinic. Biopolymers 76, 309–323. HPLC•monitoring of methyltransfer reactions at the guanine•N7 and adenosine• Umareddy, I., Chao, A., Sampath, A., Gu, F., Vasudevan, S.G., 2006. Dengue virus NS4B 2′O positions. Nucleic Acids Res. 35, e26. interacts with NS3 and dissociates it from single•stranded RNA. J. Gen. Virol. 87, Pletnev, A.G., Yamshchikov, V.F., Blinov, V.M., 1990. Nucleotide sequence of the 2605–2614. genome and complete amino acid sequence of the polyprotein of tick•borne Vajpai, N., Strauss, A., Fendrich, G., Cowan•Jacob, S.W., Manley, P.W., Grzesiek, S., encephalitis virus. Virology 174, 250–263. Jahnke, W., 2008. Solution conformations and dynamics of ABL kinase•inhibitor Poch, O., Sauvaget, I., Delarue, M., Tordo, N., 1989. Identification of four conserved complexes determined by NMR substantiate the different binding modes of motifs among the RNA•dependent polymerase encoding elements. EMBO J. 8, imatinib/nilotinib and dasatinib. J. Biol. Chem. 283, 18292–18302. 3867–3874. Vasilakis, N., Weaver, S.C., 2008. The history and evolution of human dengue emer• Porterfield, J.S., 1980. Antigenic characteristics and classification of Togaviridae. In: gence. Adv. Virus Res. 72, 1–76. Schlesinger, R.W. (Ed.), The Togaviruses. Academic Press, New York, pp. 13–46. Vliegen, I., Paeshuyse, J., De Burghgraeve, T., Lehman, L.S., Paulson, M., Shih, I.H., Price, J.L., 1978. Isolation of Rio Bravo and a hitherto undescribed agent, Tamana bat Mabery, E., Boddeker, N., De Clercq, E., Reiser, H., Oare, D., Lee, W.A., Zhong, W., virus, from insectivorous bats in Trinidad, with serological evidence of infection Bondy, S., Purstinger, G., Neyts, J., 2009. Substituted imidazopyridines as potent in bats and man. Am. J. Trop. Med. Hyg. 27, 153–161. inhibitors of HCV replication. J. Hepatol. 50, 999–1009. Pryor, M.J., Rawlinson, S.M., Butcher, R.E., Barton, C.L., Waterhouse, T.A., Vasudevan, Walker, J.E., Saraste, M., Runswick, M.J., Gay, N.J., 1982. Distantly related sequences S.G., Bardin, P.G., Wright, P.J., Jans, D.A., Davidson, A.D., 2007. Nuclear localization in the alpha• and beta•subunits of ATP synthase, myosin, kinases and other of dengue virus nonstructural protein 5 through its importin alpha/beta• ATP•requiring enzymes and a common nucleotide binding fold. EMBO J. 1, 945– recognized nuclear localization sequences is integral to viral infection. Traffic 951. 8, 795–807. Westaway, E.G., Mackenzie, J.M., Kenney, M.T., Jones, M.K., Khromykh, A.A., 1997. Rankin Jr., J.T., Eppes, S.B., Antczak, J.B., Joklik, W.K., 1989. Studies on the mecha• Ultrastructure of Kunjin virus•infected cells: colocalization of NS1 and NS3 with nism of the antiviral activity of ribavirin against reovirus. Virology 168, 147– double•stranded RNA, and of NS2B with NS3, in virus•induced membrane struc• 158. tures. J. Virol. 71, 6650–6661. Rawlinson, S.M., Pryor, M.J., Wright, P.J., Jans, D.A., 2006. Dengue virus RNA poly• Whitehead, S.S., Blaney, J.E., Durbin, A.P., Murphy, B.R., 2007. Prospects for a dengue merase NS5: a potential therapeutic target? Curr. Drug Targets 7, 1623–1638. virus vaccine. Nat. Rev. Microbiol. 5, 518–528. Ray, D., Shah, A., Tilgner, M., Guo, Y., Zhao, Y., Dong, H., Deas, T.S., Zhou, Y., Li, H., Shi, Wu, J., Bera, A.K., Kuhn, R.J., Smith, J.L., 2005. Structure of the flavivirus helicase: P.Y., 2006. West Nile virus 5 ′•cap structure is formed by sequential guanine N•7 implications for catalytic activity, protein interactions, and proteolytic process• and ribose 2 ′•O methylations by nonstructural protein 5. J. Virol. 80, 8362–8370. ing. J. Virol. 79, 10268–10277. Reed, W., 1901a. Propagation of yellow fever: observations based on recent research. Xu, T., Sampath, A., Chao, A., Wen, D., Nanao, M., Chene, P., Vasudevan, S.G., Lescar, J., Med. Records 60, 201–209. 2005. Structure of the Dengue virus helicase/nucleoside triphosphatase catalytic Reed, W., 1901b. Propagation of yellow fever: observations based on recent domain at a resolution of 2.4 Å. J. Virol. 79, 10278–10288. researches. Med. Record 60, 201–209. Yamashita, T., Unno, H., Mori, Y., Tani, H., Moriishi, K., Takamizawa, A., Agoh, M., Rice, C.M., Lenches, E.M., Eddy, S.R., Shin, S.J., Sheets, R.L., Strauss, J.H., 1985. Tsukihara, T., Matsuura, Y., 2008. Crystal structure of the catalytic domain of Nucleotide sequence of yellow fever virus: implications for flavivirus gene Japanese encephalitis virus NS3 helicase/nucleoside triphosphatase at a resolu• expression and evolution. Science 229, 726–733. tion of 1.8 Å. Virology 373, 426–436.

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2583; No. of Pages 24 ARTICLE IN PRESS

24 M. Bollati et al. / Antiviral Research xxx (2009) xxx–xxx

Yap, T.L., Xu, T., Chen, Y.L., Malet, H., Egloff, M.P., Canard, B., Vasudevan, S.G., Lescar, Zhang, B., Dong, H., Zhou, Y., Shi, P.Y., 2008. Genetic interactions among the West J., 2007. Crystal structure of the dengue virus RNA•dependent RNA polymerase Nile virus methyltransferase, the RNA•dependent RNA polymerase, and the 5 ′ catalytic domain at 1.85•angstrom resolution. J. Virol. 81, 4753–4765. stem•loop of genomic RNA. J. Virol. 82, 7047–7058. Yon, C., Teramoto, T., Mueller, N., Phelan, J., Ganesh, V.K., Murthy, K.H., Padmanab• Zhang, L., Padmanabhan, R., 1993. Role of protein conformation in the processing han, R., 2005. Modulation of the nucleoside triphosphatase/RNA helicase and of dengue virus type 2 nonstructural polyprotein precursor. Gene 129, 197– 5′•RNA triphosphatase activities of Dengue virus type 2 nonstructural protein 205. 3 (NS3) by interaction with NS5, the RNA•dependent RNA polymerase. J. Biol. Zhao, B., Mackow, E., Buckler White, A., Markoff, L., Chanock, R.M., Lai, C.J., Makino, Y., Chem. 280, 27412–27419. 1986. Cloning full•length dengue type 4 viral DNA sequences: analysis of genes Yusof, R., Clum, S., Wetzel, M., Murthy, H.M., Padmanabhan, R., 2000. Purified coding for structural proteins. Virology 155, 77–88. NS2B/NS3 serine protease of dengue virus type 2 exhibits cofactor NS2B depen• Zhou, H., Singh, N.J., Kim, K.S., 2006. Homology modeling and molecular dynamics dence for cleavage of substrates with dibasic amino acids in vitro. J. Biol. Chem. study of West Nile virus NS3 protease: a molecular basis for the catalytic activity 275, 9963–9969. increased by the NS2B cofactor. Proteins 65, 692–701. Zanotto, P.M., Gould, E.A., Gao, G.F., Harvey, P.H., Holmes, E.C., 1996. Population Zhou, Y., Ray, D., Zhao, Y., Dong, H., Ren, S., Li, Z., Guo, Y., Bernard, K.A., Shi, P.Y., Li, dynamics of flaviviruses revealed by molecular phylogenies. Proc. Natl. Acad. H., 2007. Structure and function of flavivirus NS5 methyltransferase. J. Virol. 81, Sci. U.S.A. 93, 548–553. 3891–3903.

Please cite this article in press as: Bollati, M., et al., Structure and functionality in flavivirus NS•proteins: Perspectives for drug design. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.11.009 G Model AVR•2510; No. of Pages 14 ARTICLE IN PRESS

Antiviral Research xxx (2009) xxx–xxx

Contents lists available at ScienceDirect

Antiviral Research

journal homepage: www.elsevier.com/locate/antiviral

Review Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control

E.A. Gould a,e,∗, B. Coutard b, H. Malet b, B. Morin b, S. Jamal b, S. Weaver c, A. Gorbalenya d, G. Moureau a, C. Baronti a, I. Delogu a, N. Forrester c, M. Khasnatinov g, T. Gritsun f, X. de Lamballerie a, B. Canard b a Institut de Recherche pour le Développement UMR190/Unité des Virus Emergents, Université de la Méditerranée, Marseille, France b Architecture et Fonction des Macromolécules Biologiques, CNRS and Universités d’Aix•Marseille I et II, UMR 6098, ESIL Case 925, 13288 Marseille, France c Department of Pathology, University of Texas Medical Branch, Galveston, TX, United States d Department of Medical Microbiology, Leiden University Medical Center, Leiden, The Netherlands e Centre for Ecology and Hydrology, Mansfield Road, Oxford OX1 3SR, UK f School of Biological Sciences (AMS Building), University of Reading, Berks RG6 6AJ, UK g Laboratory of Transmissive Infections, Institute of Epidemiology and Microbiology, 664025, Irkutsk, Karl Marks Str., 3, Russia article info abstract

Article history: The alphaviruses were amongst the first arboviruses to be isolated, characterized and assigned a taxo• Received 17 May 2009 nomic status. They are globally very widespread, infecting a large variety of terrestrial animals, insects Received in revised form 7 July 2009 and even fish, and circulate both in the sylvatic and urban/peri•urban environment, causing consid• Accepted 11 July 2009 erable human morbidity and mortality. Nevertheless, despite their obvious importance as pathogens, there are currently no effective antiviral drugs with which to treat humans or animals infected by any Keywords: of these viruses. The EU•supported project—VIZIER (Comparative Structural Genomics of Viral Enzymes Alphavirus Involved in Replication, FP6 Project: 2004•511960) was instigated with an ultimate view of contribut• Genomics Structure/function studies ing to the development of antiviral therapies for RNA viruses, including the alphaviruses [Coutard, B., Replicative enzymes Gorbalenya, A.E., Snijder, E.J., Leontovich, A.M., Poupon, A., De Lamballerie, X., Charrel, R., Gould, E.A., Antivirals Gunther, S., Norder, H., Klempa, B., Bourhy, H., Rohayemj, J., L’hermite, E., Nordlund, P., Stuart, D.I., Owens, Evolution R.J., Grimes, J.M., Tuckerm, P.A., Bolognesi, M., Mattevi, A., Coll, M., Jones, T.A., Åqvist, J., Unger, T., Hilgen• Classification feld, R., Bricogne, G., Neyts, J., La Colla, P., Puerstinger, G., Gonzalez, J.P., Leroy, E., Cambillau, C., Romette, Biogeography J.L., Canard, B., 2008. The VIZIER project: preparedness against pathogenic RNA viruses. Antiviral Res. VIZIER 78, 37–46]. This review highlights some of the major features of alphaviruses that have been investi• gated during recent years. After describing their classification, epidemiology and evolutionary history and the expanding geographic distribution of Chikungunya virus, we review progress in understanding the structure and function of alphavirus replicative enzymes achieved under the VIZIER programme and the development of new disease control strategies. © 2009 Elsevier B.V. All rights reserved.

Contents

1. Introduction ...... 00 1.1. Classification ...... 00 1.2. Structure, genome strategy and replication ...... 00 2. The alphaviruses as human pathogens ...... 00 3. Molecular epidemiology and biogeography ...... 00 3.1. Evolutionary origins of the alphaviruses ...... 00 3.2. Alphavirus emergence in the New World ...... 00 3.3. Recent emergence of Chikungunya virus ...... 00 4. Studies on the active domains of alphavirus replicative enzymes ...... 00 4.1. Alphavirus nsP1 protein ...... 00 4.2. Alphavirus nsP2 protein ...... 00

∗ Corresponding author at: Centre for Ecology and Hydrology, Mansfield Road, Oxford OX1 3SR, UK. E•mail address: [email protected] (E.A. Gould).

0166•3542/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi: 10.1016/j.antiviral.2009.07.007

Please cite this article in press as: Gould, E.A., et al., Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.07.007 G Model AVR•2510; No. of Pages 14 ARTICLE IN PRESS

2 E.A. Gould et al. / Antiviral Research xxx (2009) xxx–xxx

4.3. Alphavirus nsP3 protein ...... 00 4.4. Alphavirus nsP4 protein ...... 00 5. Progress towards therapy and prevention ...... 00 5.1. Search for antiviral drugs ...... 00 5.1.1. Chloroquine ...... 00 5.1.2. Quinine ...... 00 5.1.3. Ribavirin...... 00 5.1.4. Interferon and ribavirin ...... 00 5.1.5. Inhibitors of alphavirus entry and maturation ...... 00 5.1.6. Carbodine ...... 00 5.1.7. Small RNA molecules...... 00 5.1.8. Plant compounds...... 00 5.1.9. nsP1 active domains as a target for inhibition...... 00 5.1.10. nsP2 active domains as a target for inhibition...... 00 5.1.11. nsP3 active domains as a target for inhibition ...... 00 5.1.12. nsP4 active domains as a target for inhibition...... 00 5.2. Antiviral vaccines ...... 00 6. Conclusion ...... 00 Acknowledgements...... 00 References ...... 00

1. Introduction The four non•structural proteins are defined as nsP1, nsP2, nsP3 and nsP4. The genomic RNA is positive•stranded and serves as the 1.1. Classification mRNA for translation of the polyprotein precursor that is auto• catalytically processed to the four non•structural viral proteins by The VIIIth edition of the International Committee for the Tax• the virus•encoded protease in nsP2 ( Fig. 1 ). The non•structural onomy of Viruses (ICTV) currently lists 29 species in the genus proteins form the transcription/replication complex that medi• Alphavirus that together with the genus Rubivirus forms the family ates the synthesis of diverse viral RNAs of both polarities. The Togaviridae (Weaver et al., 2005 ). The alphaviruses are arthropod• nsP1 protein was implicated in capping of viral RNAs ( Ahola and borne (arboviruses), whereas the rubiviruses are transmitted via Kääriäinen, 1995; Scheidel et al., 1989 ) and in initiation of negative• the respiratory tract. All arthropod•borne alphaviruses are anti• strand RNA synthesis ( Sawicki and Sawicki, 1994 ). It is bound to genically related but most can be distinguished in cross•reactivity the cytoplasmic membrane via a central amphipathic alpha helix tests ( Chanas et al., 1976; Clarke and Casals, 1958; Karabatsos, located in the middle of the protein ( Lampio et al., 2000 ). The nsP2 1975; Porterfield, 1961 ) with which they have been divided into gene encodes a putative helicase domain at the 5 ′end and a pro• 8 antigenic complexes: Eastern, Western, and Venezuelan equine tease domain at the 3 ′end, which presents a unique fold distantly encephalitis, Trocara (complex assigned based only on genetic related to that of known cysteine proteases ( Russo et al., 2006 ). divergence), Middelburg, Ndumu, Semliki Forest and Barmah For• This protease domain is linked to the downstream domain of the est. In addition, there are two non•arthropod•borne species, Salmon O′•methyltransferase fold, that may be enzymatically active only in pancreatic disease virus and Southern elephant seal virus. Based on non•arthropod•borne alphaviruses ( Feder et al., 2003 ). In its free comparative sequence analysis, the arthropod•borne alphaviruses form, the nsP2 protein induces cytotoxicity and is responsible for share a minimum of about 40% amino acid identity in the more transcriptional shut•off, which is dependent on the integrity of the divergent structural proteins and 60% in the non•structural pro• carboxy•terminal peptide located downstream of its helicase and teins. protease domains. The nsP3 protein is required for RNA replica• tion. It carries three domains: the N•terminal sequence reveals a macro domain, the crystal structures of which have recently been 1.2. Structure, genome strategy and replication determined for Venezuelan equine encephalitis virus (VEEV) and Chikungunya virus (CHIKV) ( Malet et al., 2009 ). It is followed by a Alphavirus virions are approximately 70 nm in diameter. They serine/threonine•rich sequence that may be phosphorylated. The are spherical with a lipid bilayer containing heterodimeric protein C•terminal region is poorly conserved in both size and sequence spikes composed of two envelope glycoproteins E1 and E2. Many (Strauss and Strauss, 1994 ). The nsP4 protein carries the viral RNA alphaviruses also contain a third envelope protein E3. The het• polymerase motif ( Kamer and Argos, 1984 ). During RNA replica• erodimers are organized in a T = 4 icosahedral lattice consisting of tion, a negative•stranded copy is produced and used as a template 80 trimers of E1–E2 complex. The enclosed nucleocapsid core con• for the synthesis of genome•sized positive•strand RNA and subge• sists of 240 copies of capsid protein and a single copy of the genomic nomic 26S mRNA corresponding to the 3 ′ third of the viral genome RNA, although Aura virus is reported also to enclose the 26S subge• and encoding the viral structural proteins ( Garmashova et al., 2006; nomic RNA ( Rumenapf et al., 1995 ). The one•to•one relationship Hahn et al., 1988; Lemm et al., 1994; Mukhopadhyay et al., 2006; between glycoprotein heterodimers and nucleocapsid proteins is Weaver, 2005 ). important in virus assembly. E1 is the fusion protein for virus entry The non•structural proteins function in the cytoplasm of into the acidic cytoplasmic endosomes. The structure of the E1 infected cells in association with membrane surfaces, and attach• glycoprotein of Semliki Forest virus has been determined by crystal• ment appears to be mediated by nsP1 palmitoylation ( Ahola et al., lography ( Lescar et al., 2001 ), revealing a fold closely related to the 2000 ). For reasons not yet explained, the nsP2 protein migrates to flavivirus envelope protein. The E2 glycoprotein extends outwards the infected cell nuclei and this has also recently been shown to be from the envelope and forms the petals of the spike that cover the the case for the nsP3 protein ( Gorchakov et al., 2008 ). The capsid underlying E1 protein fusion peptide at neutral pH ( Mukhopadhyay protein is cotranslationally cleaved from the structural polyprotein et al., 2006 ). by its own protease activity and assembles with the viral genomic

Please cite this article in press as: Gould, E.A., et al., Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.07.007 G Model AVR•2510; No. of Pages 14 ARTICLE IN PRESS

E.A. Gould et al. / Antiviral Research xxx (2009) xxx–xxx 3

Fig. 1. Alphavirus genome coding strategy (adapted and updated from Strauss and Strauss, 1994 ). Open•reading frame (ORF) represented as open box, and untranslated regions as solid black lines; sg (subgenomic), asterisk between nsP3 and nsP4 identifies the position of the stop codon that is present in some alphaviruses and is read through to produce the precursor nsP1,2,3,4 polyprotein, Me•tr (methyltransferase), Hel (helicase), Pro (protease), MD (macro domain—exhibits adenosine di•phosphoribose 1′′ •phosphate phosphatase activity), RdRp (RNA•dependent RNA polymerase), C (capsid), E (envelope).

RNA to form the viral nucleocapsids in the cytosol. Glycoproteins antigenic complex, which is divided into six distinct antigenic sub• are translocated, via the Golgi apparatus, to the plasma membrane types ( Walton and Grayson, 1988; Young, 1972; Young and Johnson, and assembled nucleocapsids bud through these membranes, thus 1969 ). Subtypes IAB and IC are associated with major epidemics and acquiring a lipid envelope containing the integral membrane gly• equine epizootics during which equine mortality due to encephali• coproteins, E1 and E2. tis can reach 83%. In 1995, a major outbreak in Venezuela and In this review, we analyse (a) progress in genomics, towards Colombia, was associated with the VEEV subtype IC. This epidemic understanding alphavirus evolution, taxonomy, and vector–host resulted in roughly 100,000 human cases, with more than 300 relationships in the context of their epidemiology and pathogene• fatal encephalitis cases ( Diaz et al., 1997 ). Other epidemics indicate sis, and (b) the structures of active domains in alphavirus replicative that VEEV still represents a serious public health problem (Weaver enzymes with the ultimate objective of identifying suitable targets et al., 1996 ). In humans, whilst the overall mortality rate is low for molecules that can inhibit their function and thereby serve as (<1%), neurological disease, including disorientation, ataxia, men• antivirals. tal depression, and convulsions, can be detected in up to 14% of infected individuals, especially children ( Johnson and Martin, 1974 ). 2. The alphaviruses as human pathogens Neurological sequelae in humans are also common ( Leon, 1975 ). However, most human infections present as a non•specific febrile Ten of the arthropod•borne alphaviruses are considered to be illness or aseptic meningitis. In rare cases, the fever and headache of significant importance in terms of public health. Indeed with the may progress through nausea and vomiting to somnolence or delir• recent emergence of chikungunya fever (see later) as a major human ium and coma with seizures, impaired sensorium, and paralysis disease in Asia and potentially globally, the alphavirus profile has being commonly observed. The severity of neurologic involvement been significantly raised. Alphaviruses that circulate in the Old and sequelae is greater with decreasing age. World most commonly cause febrile illness and painful arthralgias Horses are more susceptible than humans to neurological dis• or polyarthralgias, particularly in the small joints. A characteristic ease caused by these VEEV subtypes IAB and IC, but are considered macular•papular rash often appears 3–5 days after illness onset. to be dead•end hosts for EEEV and WEEV. Moreover, veterinary vac• In severe cases the joints are swollen and tender, and rheumatic cines are available to reduce the risk of clinical disease. EEEV and signs and symptoms may persist for weeks or months following WEEV, are widespread throughout the eastern and western regions the acute illness. In general, infections with Old World alphaviruses of North America, including Canada, and both are also found in such as CHIKV, or O’nyong nyong virus (ONNV) in Africa/Asia, Sind• South America and Cuba. In North America, they are transmitted bis virus (SINV) and closely related viruses (Ockelbo, Whataroa) to horses by infected ornithophilic (bird•biting) mosquitoes that which are widespread throughout the Old World, or Ross River thrive in wetland habitats. Highlands J virus (HJV), a close relative (RRV), and Barmah Forest virus (BFV), which are confined to Aus• of WEEV, is not known to be pathogenic for humans but appears to tralia, are rarely fatal and only infrequently result in encephalitic be an important pathogen of several domestic bird species. VEEV disease ( Lewthwaite et al., 2009 ). also causes encephalitic disease in horses and, occasionally, humans In contrast to these Old World diseases, the New World bitten by mosquitoes normally associated with the horses ( Weaver, alphaviruses VEEV, Eastern equine encephalitis virus (EEEV), and 2005; Weaver and Barrett, 2004; Weaver et al., 1997 ). The natu• Western equine encephalitis virus (WEEV) present a different epi• ral life cycle of VEEV involves small mammals, particularly rodents demiological and clinical picture. VEEV is one species in the VEE in forest environments more frequently found in South America.

Please cite this article in press as: Gould, E.A., et al., Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.07.007 G Model AVR•2510; No. of Pages 14 ARTICLE IN PRESS

4 E.A. Gould et al. / Antiviral Research xxx (2009) xxx–xxx

Other closely related alphaviruses are recognised in the Americas over, before or after this recombination event, it must also be but in most cases they are not known to cause disease in humans assumed that one or more of these ancestral alphaviruses (not the or animals. recombinant virus) was then dispersed to the Old World, for the alphaviruses to establish in the new environments and to continue their global dispersal. Whilst this scenario may be theoretically 3. Molecular epidemiology and biogeography possible, it is hard to imagine that these transoceanic movements would occur very frequently more than 1000 years ago, since little 3.1. Evolutionary origins of the alphaviruses if any commercial movement of ships took place during the first millennium AD. In 1988, nucleotide sequence comparisons of several If one argues that birds could have moved the viruses across the alphaviruses revealed that WEEV was a recombinant virus oceans more than 1000 years ago, then one is inevitably obliged to that had apparently arisen through mixed infection of cells by ask why is this not a common occurrence today? On the other hand, ancestral EEEV•related and SINV•related viruses ( Hahn et al., 1988 ). during the past 500–600 years, transoceanic crossings by ships for Subsequent phylogenetic analyses confirmed this interpretation commercial and/or immigration and slave•trading purposes have (Levinson et al., 1990 ) and it was estimated that recombination been the major factor for the introduction into the Americas of many had occurred prior to the divergence of EEEV to produce the extant different Old World mosquito species, including Aedes aegypti , the South American lineages ( Weaver et al., 1993 ). These observations Culex pipiens complex, Aedes albopictus (Calder and Laird, 1994 ) were important milestones in our understanding of alphavirus and African arboviruses including Yellow fever virus, Dengue virus, diversity and raised an interesting issue that remains to be satisfac• St. Louis encephalitis virus, West Nile virus and Powassan virus torily resolved, viz., the geographic origin of the arthropod•borne (reviewed in Bloom, 1993; de Lamballerie et al., 2008b; Gould et viruses in the genus Alphavirus . al., 2003; Lounibos, 2002; Strode, 1951; Tabachnick, 1991; Tatem et Phylogenetic trees based on alphavirus sequences that include al., 2006 ). Whilst in the modern era, introductions do occur in the the envelope (E1, E2) genes, are incongruent with those based reverse direction (New World to Old World), they are usually impor• solely on non•structural gene sequence. All trees based on E1 gene tations of recognised arboviruses by viraemic individuals returning sequence show that SINV, an Old World virus, diverges with the to their country of origin, following a business or leisure trip in WEEV•complex viruses, i.e. New World viruses ( Fig. 2 a), whereas the Americas. In temperate regions of the Old World, these individ• the other New World equine encephalitis viruses, i.e. EEEV and ual introductions from the New World rarely, if ever, result in the VEEV diverge independently of the WEEV/SINV complex and also establishment of endemic/epidemic arboviruses. There is a detailed independently of the indigenous Old World viruses that include record of YFV being introduced into South Wales (United Kingdom) Semliki Forest virus (SFV), Ross River virus (RRV) and CHIKV. How• from Cuba; the virus caused a small localised outbreak of yellow ever, trees based on non•structural, and/or capsid genes, show that fever, but rapidly disappeared from circulation ( Smith and Gibson, both SINV and the antigenically closely related New World virus, 1986 ) presumably, because the habitat lacked a suitable vector for Aura virus (AURAV) diverge from the New World viruses and are YFV to become established. included in a clade of Old World alphaviruses that contain Bebaru The alternative possibility, i.e. that the alphaviruses originated virus (BEBV), Getah virus (GETV), SFV, RRV and other related viruses in the Old World, is also recognised ( Lavergne et al., 2006; Powers et (Fig. 2 b). In other words, in trees based on non•structural genes, al., 2001; Weaver et al., 1997 ). In Africa, a wide range of alphaviruses SINV, and AURAV diverge independently from the WEEV•complex such as SFV, SINV, CHIKV, ONNV and others circulate in geo• viruses which are now seen to have diverged with EEEV and VEEV. graphically overlapping environments, where they have relatively Another important observation is that trees based on either E1 similar ecological requirements. Thus, the possibility of ancestral or non•structural genes show that the New World alphaviruses, alphaviruses in Africa causing mixed infections of vertebrates and Mayaro virus (MAYV) and Una virus (UNV), always cluster with the recombining to produce an ancestral lineage of a virus such as Old World alphaviruses, RRV, SFV, GETV in the SFV antigenic com• WEEV, is at least plausible. Secondly, during the centuries of inten• plex, confirming that transoceanic alphavirus introductions must sive commercial trading between Africa and the Americas, some or have occurred, as previously proposed ( Powers et al., 2001; Weaver manyofthesealphavirusescouldhavebeencarriedtotheAmericas, et al., 1997 ). In the case of MAYV the supposition of transoceanic in the way that many flaviviruses and mosquitoes were transported introductions is also supported by the evidence that this New World to the New World ( Bloom, 1993; Bryant et al., 2007; Gould et al., virus causes arthritic disease in humans, as is characteristic of the 2003, 2001; Lounibos, 2002; Tabachnick, 1991 ). Under this sce• Old World alphaviruses, rather than encephalitic disease as typi• nario, transoceanic alphavirus dispersal would not need to occur in fied by New World viruses such as EEEV and VEEV ( Poidinger et both directions. Moreover, the extremely low probability of a mixed al., 1997; Russell, 1998 ). Weaver et al. (1997) proposed that these infection occurring between an enzootic New World virus and an alphavirus dispersal patterns could be most readily explained if the Old World virus, introduced to the Americas more than 1000 years origin of these viruses was the New World. However, in order to ago, no longer needs to be explained. In addition, the geographi• support this argument, it was necessary to propose that several cally wide distribution of the alphaviruses in natural cycles in the transoceanic crossings (i.e. at least three) must have taken place, in Old World is entirely consistent with Old World anthropological both directions, i.e. westwards and eastwards ( Powers et al., 2001 ). distribution and commerce, and bird migratory patterns. Clearly, Moreover, it was estimated that recombination, as seen in WEEV more alphaviruses need to be isolated and studied in detail to fill and closely related descendant viruses, is likely to have occurred the gaps in our knowledge and to resolve the question of how these between 1300 and 1900 years ago ( Weaver et al., 1997 ). viruses have evolved and dispersed during the past few millennia. Assuming this to be a reasonable estimate for the time of the However, the ideas related above, appear to favour an Old World recombination event, and assuming a New World origin for the origin for the alphaviruses that are currently circulating. alphaviruses, it would have been necessary for an ancestral SINV• related virus to be present in the New World, either by evolutionary 3.2. Alphavirus emergence in the New World origination or by introduction from the Old World. This virus would then have to encounter an ancestral EEEV•related virus in the In the New World, prior to late 2004 or early 2005, alphaviruses same ecological habitat in the New World and then produce mixed were known to cause spasmodic human outbreaks in different infections in rodents, or other animal species such as birds. More• geographic regions of the world. For example, in equatorial South

Please cite this article in press as: Gould, E.A., et al., Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.07.007 G Model AVR•2510; No. of Pages 14 ARTICLE IN PRESS

E.A. Gould et al. / Antiviral Research xxx (2009) xxx–xxx 5

Fig. 2. Phylogenetic analyses of selected alphaviruses. (a) Midpoint rooted tree generated using partial E1 envelope glycoprotein amino acid sequences and the neighbour joining program implemented in PAUP 4.0 ( Swofford, 1998 ). Numbers indicate bootstrap values generated using 1000 resamplings. Scale indicates 5% amino acid sequence divergence. Gray box shows recombinant alphaviruses that were derived from ancestors of EEEV and SINV. Shaded background indicates recombinant viruses. (b) Most parsimonious alphavirus nsP4 tree, rooted with NSA/SPDV outgroup Bootstrap percentage numbers on nodes with 50% or more support from 1000 resamplings, are indicated. Asterisk (*) indicates studies in VIZIER; shaded background indicates New World viruses. and Central America, VEEV exists in two epidemiological forms: emergence. It was subsequently demonstrated that a Ser•Asn sub• (i) enzootic viruses that are transmitted continuously in sylvatic stitution in the E2 gene was the sole determinant of the increased habitats between mosquitoes and rodent reservoir hosts and (ii) vector infectivity ( Brault et al., 2004 ). Similar patterns of enzootic epidemic/epizootic viruses ( Weaver and Barrett, 2004; Weaver et and epizootic behaviour have also been observed with EEEV and al., 2004 ), that emerge periodically to cause outbreaks involv• WEEV although these viruses are not known to show increased ing large numbers of humans and horses ( Weaver et al., 1996 ). patterns of virulence during these outbreaks. A Mexican equine epizootic, involving a proven epizootic vector Ochlerotatus (formerly Aedes ) taeniorhynchus , serves as an excel• 3.3. Recent emergence of Chikungunya virus lent example. When compared with closely related enzootic strains, VEEV isolates from infected horses exhibited significantly greater In the Old World, spasmodic febrile/arthritic outbreaks are asso• infectivity for the vector that was associated with horses, suggest• ciated with infections due to SINV, CHIKV, ONNV, RRV, or BFV ing that adaptation to the appropriate vector contributed to disease in Africa, and/or Europe, Asia, South East Asia/Australasia. Prior

Please cite this article in press as: Gould, E.A., et al., Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.07.007 G Model AVR•2510; No. of Pages 14 ARTICLE IN PRESS

6 E.A. Gould et al. / Antiviral Research xxx (2009) xxx–xxx

Table 1 Number of non•structural alphavirus protein targets sequenced expressed and under further analysis in the VIZIER project.

nsP1 nsP2 nsP3 nsP4 Crystallized proteins Crystal structures

Aura virus 1 Barmah Forest virus 2 16 4 4 Chikungunya virus (2 strains) 0 10 9 11 2 3 Eastern equine encephalitis virus 2 17 4 2 1 1 Mayaro virus 0 14 4 3 1 O’nyong nyong virus 2 16 5 15 Salmon pancreatic disease virus 0 3 0 1 Semliki Forest virus 1 4 3 1 1 Sindbis virus 2 18 4 2 Venezuelan equine encephalitis virus 2 15 4 4 1 2 Western equine encephalitis virus 2 14 4 2 1

Total (227) 13 127 41 46 7 6

to 2005, these alphaviruses were considered to be of relatively et al., 2008b ). This virus is continuing to cause major epidemics minor importance by most government health departments, largely throughout Asia involving millions of humans. because they were associated with localised outbreaks involving ItisnowconsideredpossiblethatCHIKVcouldspreadevenmore relatively few individuals and in general they were considered widely, reaching and becoming firmly established in Europe, and to be insignificant agents of encephalitic disease or fatal infec• tropical America. This potential global emergence of CHIKV appears tions. Moreover, clinical misdiagnosis of many of these alphavirus to be at least partly attributable to its adaptation to the Asian tiger infections in the dengue•endemic locations where they circulate is mosquito A. albopictus (Calder and Laird, 1994; de Lamballerie et al., probably another reason that they have been underappreciated as 2008b; Schuffenecker et al., 2006; Tsetsarkin et al., 2007 ), which causes of febrile illness. However, this attitude changed, from 2005, has dispersed widely in the past two decades. These conclusions onwards when CHIKV unexpectedly arose as a major epidemic were derived from a combination of genomic, phylogenetic, and human pathogenic arbovirosis throughout, central and southern vector transmission studies, from which it was conclusively demon• Africa, the Islands of the southern Indian Ocean, India, Indonesia strated that the adaptation of CHIKV to high vector competence in A. and Malaysia. As the result of these spreading epidemics, CHIKV is albopictus resulted directly from the substitution of the amino acid now frequently being introduced into other regions of Asia, Europe, alanine by valine at position 226 (A226V) in the envelope gene (E1) Australia and the Americas by travellers returning from CHIKV epi• of the virus ( Schuffenecker et al., 2006; Tsetsarkin et al., 2007 ). Even demic areas of Asia. Indeed in Northern Italy the introduced virus more surprisingly, based on phylogenetic evidence and local knowl• became established in A. albopictus and caused localised outbreaks edge of CHIKV epidemiology, it is clear that this selective adaptation of chikungunya fever ( Rezza et al., 2007 ). Although case fatality for the tiger mosquito has occurred on several independent occa• rates resulting from CHIKV infection are considered to be low, sions ( de Lamballerie et al., 2008b ). the very large numbers of clinical cases in Asia (now unofficially The global invasion by A. albopictus , mainly facilitated by tyre estimated to be millions) have almost certainly resulted in signif• shipments, is regarded as the “third wave” of human aided disper• icantly more mortality than is currently recognised, particularly sal of mosquito vectors of human disease, following the previous in countries where health services and recording procedures are cosmotropical spread of A. aegypti and C. pipiens (Benedict et al., poorly developed. Moreover, it has now become apparent that thou• 2007; Calder and Laird, 1994 ). A. albopictus is now established in sands of excess deaths, with many involving neurological disease, virtually all tropical, sub•tropical and southern temperate regions have accompanied recent CHIKV outbreaks ( Josseran et al., 2006; (Lounibos, 2002 ). Moreover, local species of A. albopictus in Florida, Mavalankar et al., 2008; Robin et al., 2008 ). Additionally, recent USA, have now been shown to be susceptible to infection by CHIKV studies on children in India, reported that at least 14% of CHIKV and competent to transmit this virus ( Reiskind et al., 2008 ). Thus, infections may result in neurological complications ( Lewthwaite et it is almost certainly only a matter of time before CHIKV invades al., 2009 ). the Americas, being introduced by infected humans returning from Until 2005, CHIKV was usually described as a virus that cir• endemic areas of Africa or Asia, possibly causing greater problems in culated in a natural cycle amongst forest•associated simians and the tropical regions due to the behavioural advantages of A. aegypti . sylvatic mosquito species ( Aedes furcifer, Aedes luteocephalus, Aedes Clearly, there is a pressing need for effective antivirals and vaccines taylori ), in the central and west African jungles, spasmodically with which to develop CHIKV disease control strategies. causing outbreaks of varying size and intensity amongst the local populations living near the jungles. Human outbreaks were associ• 4. Studies on the active domains of alphavirus replicative ated with domestic mosquito species such as A. aegypti and it has enzymes always been assumed that the virus is transmitted down the chain of sylvatic/domestic mosquito species via humans and simians that Although crystallization of alphavirus proteins provided early come into contact with the virus at the edges of the jungles and data concerned with the mechanism of virus entry into suscepti• in the nearby local villages/towns ( Gould and Higgs, 2009 ). CHIKV ble cells ( Lescar et al., 2001 ) progress has been much slower in the was also known to cause spasmodic outbreaks in many parts of case of non•structural proteins. The major difficulty to overcome Asia, although a forest reservoir cycle has never been identified in has been the problem of expressing suitable recombinant target Asia ( Powers and Logue, 2007 ). However, as indicated above, this proteins. This is reflected by the fact that the literature reports spasmodic pattern of outbreaks changed dramatically from 2005 the structures of only two non•structural protein domains, viz., onwards, when a major CHIKV fever epidemic was first identified the amphipathic helix of nsP1, a 20 amino acid peptide ( Lampio et on La Réunion Island in the southern Indian Ocean ( Charrel et al., al., 2000 ) and the protease•methyltransferase subdomain of nsP2 2007; de Lamballerie et al., 2008b; Schuffenecker et al., 2006 ). Epi• (Russo et al., 2006 ). Therefore, alphavirus nsP enzyme domains demics were also reported on many neighbouring islands and the were identified as potentially important targets for structural stud• virus dispersed rapidly to India, and south•east Asia ( de Lamballerie ies in VIZIER, and Table 1 summarises the current numbers and

Please cite this article in press as: Gould, E.A., et al., Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.07.007 G Model AVR•2510; No. of Pages 14 ARTICLE IN PRESS

E.A. Gould et al. / Antiviral Research xxx (2009) xxx–xxx 7

Fig. 3. Known structures of alphavirus nsP2 (protease) and nsP3 ( macro domain). (a) Cartoon representation of the VEEV protease (C•terminal part of nsP2) ( Russo et al., 2006 ). The domain coloured in orange corresponds to the catalytic domain where the catalytic cysteine and histidine are highlighted respectively in yellow and red. (b) Cartoon representation of the CHIKV macro domain (in green) complex with ADP•ribose (in blue). (c) Cartoon representation of the VEEV macro domain (in pink) complex with ADP•ribose (in yellow) ( Malet et al., 2009 ). status (in terms of protein solubility and crystal formation) of inorganic pyrophosphate ( Ahola and Kääriäinen, 1995 ). However, expressed alphavirus nsP targets. The decision to produce and study guanylyltransfer of the me7 GMP onto RNA remains to be demon• at the structural level, non•structural protein domains active in strated. nsP1 activity is dependent on membrane association ( Ahola viral replication has proved to be highly beneficial since genomics et al., 1999 ). This interaction is mediated by an amphipathic helix progressed at a rapid rate, and most of the predicted (identified) located in the middle of the nsP1 sequence ( Spuul et al., 2007 ). This structural folds appear to be original ( Fig. 3 ). helix contains hydrophobic amino acids that can interact with acyl Four non•structural proteins (nsP1, 2, 3 and 4), corresponding groups of the membrane and a cluster of positively charged residues to the four domains resulting from maturation (post•translational that contact the phospholipid polar heads ( Lampio et al., 2000 ). It processing by protease enzymatic function of nsP2) of the polypro• remains unclear if palmitoylation of several cysteines contributes to tein ( Fig. 1 ), are associated with the virus replication complex. membrane binding ( Ahola et al., 2000 ), but this post•translational They are synthesized as a short polyprotein nsP123, the trans• modification plays a role in the cellular localization of the replica• lation termination of which is terminated at a UGA stop codon, tion complex ( Laakkonen et al., 1996 ). found in most alphavirus genomes, and located near the junction between nsP3 and nsP4 ( Fig. 1 ). Alternatively, the ribosome can 4.2. Alphavirus nsP2 protein “read•through” this codon, using a rare suppressor tRNA to produce the complete nsP1234 polyprotein ( Li and Rice, 1989 ) which is sub• The N•terminal region of the nsP2 gene was predicted to encode sequently processed to the mature products in regulated sequential a helicase domain ( Gorbalenya et al., 1988; Hodgman, 1988 ) that order ( Vasiljeva et al., 2003 ). The current state of our knowledge was later classified as the helicase Superfamily 1 ( Gorbalenya and concerning the structural and enzymatic properties of these four Koonin, 1993; Singleton et al., 2007 ). Using in vitro tests, the nsP2 processed non•structural alphavirus proteins is summarised in gene of SFV exhibits nucleoside triphosphatase activity utilizing Table 2 . GTP and ATP ( Rikkonen et al., 1994 ) and stimulated by RNA. This activity is stimulated in the presence of RNA. nsP2 could thus act at 4.1. Alphavirus nsP1 protein least at two stages in the infected cell. Firstly, as with the NS3 protein of flaviviruses, the nsP2 nucleotide triphosphatase may produce The indication that the nsP1 gene carries RNA methyltransferase 5′ diphosphate mRNA, the expected final acceptor of the nsP1• activity was derived using a SINV nsP1 mutant engineered to allow mediated guanylyltransferase reaction catalysed by nsP1 ( Vasiljeva replication in insect cells depleted of methionine ( Mi et al., 1989 ). et al., 2000 ). Secondly, the nsP2 ATPase activity is believed to be This finding was extended in studies in which in vitro methyltrans• necessary to fuel RNA helicase activity. The dsRNA unwinding is ferase activity of the SINV nsP1 ( Mi and Stollar, 1991 ) and the SFV magnesium•dependent in SFV ( Gomez de Cedrón et al., 1999 ). The nsP1 ( Laakkonen et al., 1994 ) was demonstrated. The active site C•terminal region of nsP2 is a cysteine protease which is responsi• residues of MT were identified by comparative sequence analysis ble for viral polyprotein processing ( Strauss et al., 1992; Vasiljeva which demonstrated that this enzyme is conserved in many virus et al., 2001 ). families forming an alpha•like supergroup ( Rozanov et al., 1992 ). Structural analysis of the VEEV nsP2 C•terminal region revealed The substrate of the reaction is GTP, which becomes methylated at two distinct domains ( Russo et al., 2006 ). The first, adopting an orig• its N7 position ( me7 GTP). The product me7 GTP is then covalently inal a/b•fold very distantly related to that of papain•like proteases, bound to nsP1 to generate a covalent me7 GMP•nsP1 adduct and whose two principal catalytic residues, a cystine and a histidine, are

Table 2 Summary of current enzymatic and structural data of the alphavirus proteins nsP1, nsP2, nsP3 and nsP4.

nsP1 nsP2 nsP3 nsP4

Domain MTase/GTase NTPase/helicase Protease Macro Cter domain RdRp/TATase domain/ADPribosePPase Structural data Yes (partly) ( Spuul et No Yes ( Russo et al., 2006 ) Yes ( Malet et al., 2009 ) No No al., 2007 ) Enzymatic data Yes/(partly) ( Ahola and Yes/yes ( Rikkonen et Yes ( Vasiljeva et al., Yes (partly) ( Malet et No Yes ( Lemm et al., 1994; Kääriäinen, 1995 ) al., 1994 ) 2001 ) al., 2009 ) Rubak et al., 2009; Tomar et al., 2006 ) Inhibition data Yes ( Lampio et al., No Yes ( Pastorino et al., No No No 1999 ) 2008 )

Please cite this article in press as: Gould, E.A., et al., Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.07.007 G Model AVR•2510; No. of Pages 14 ARTICLE IN PRESS

8 E.A. Gould et al. / Antiviral Research xxx (2009) xxx–xxx uniquely located in the N•terminal subdomain. The second domain 4.4. Alphavirus nsP4 protein has a classical methyltransferase fold although it does not exhibit methyltransferase activity. Structural and biochemical data on the The alphavirus nsP4 contains a canonical GDD motif and is con• proteases of VEEV and CHIKV nsP2 support the hypothesis that this sidered to be the RNA•dependent RNA polymerase (RdRp) ( Kamer second domain contributes to substrate presentation to the pro• and Argos, 1984 ). It is believed that this protein synthesizes both tease active site ( Pastorino et al., 2008; Russo et al., 2006 ). This negative• and positive•strand RNAs. In SINV, the switch between structural arrangement may also regulate protease activity through the production of antisense RNA to genomic and subgenomic RNA possible RNA binding. is mediated by processing of the nsP123 region of the polyprotein (Lemm et al., 1998, 1994; Shirako and Strauss, 1994 ). In vitro , this activity has been reported to be sequence•specific ( Lemm et al., 4.3. Alphavirus nsP3 protein 1998 ). A peptide sequence containing two key arginines is involved The N•terminal region of nsP3 was initially described as a con• in promoter sequence recognition to initiate the synthesis of subge• served domain with an unknown function (X domain) in large nomic RNA ( Li et al., 2004 ). The N•terminal region of nsP4 is poorly replicative proteins of alphaviruses, coronaviruses and rubiviruses conserved and natively unfolded. It could be involved in the recruit• (Gorbalenya et al., 1991 ). This 160 amino acid domain belongs to mentofRNAorproteinpartners.Alone,nsP4doesnotreplicate RNA. a large protein family including more than 1000 domains from all An N•terminally truncated version of SINV nsP4 carries out termi• kingdoms of life, dubbed the macro domain family after a unique nal adenylyltransferase (TATase) activity that might be involved in ′ domain in the human macroH2A1 histone ( Perhson and Fuji, 1998 ). maintenance of the 3 poly A tail ( Tomar et al., 2006 ). Very recently, This histone association was found only in a small fraction of RdRp activity was demonstrated in vitro using a purified full•length domains in the family. Several members of this family were shown SINV nsP4 protein ( Rubak et al., 2009 ). to possess a diphosphate•ribose 1 ′′ •phosphate activity and can bind ADP•ribose ( Kumaran et al., 2005; Shull et al., 2005 ). For some 5. Progress towards therapy and prevention others, as exemplified by the Infectious Bronchitis coronavirus X domain, ADP•ribose binding was not revealed in vitro (Piotrowski Chikungunya fever commonly presents as a painful febrile et al., 2009 ). The macro domains of VEEV and CHIKV are very illness that is quite often accompanied by relapsing and incapac• similar. Their structures were found to resemble the homologous itating polyarthralgia that may persist for several months. During Escherichia coli domain more closely than the coronavirus macro the past 4–5 years, CHIKV has dispersed widely throughout Africa domains ( Malet et al., 2009 ). and Asia causing morbidity amongst millions of infected patients. Both VEEV and CHIKV macro domains have a specific affinity However, this may be only the tip of the iceberg because the two for ADP•ribose, via a conserved aspartic acid and they also display most important vectors of this virus, A. aegypti and A. albopic• diphosphate•ribose 1 ′′ •phosphate activity. A positively charged tus , are continually expanding their geographic distribution and amino acid patch also enables the binding of oligonucleotides such density amongst domestic and peridomestic human populations. as poly•ADP•ribose (PAR) or RNA, but the common determinant Thus it is the strongly held belief that CHIKV will spread even of substrate binding is a conserved adenosine 5 ′•monophosphate more widely, and quite possibly into the Americas, during the binding site. It has now been shown that mutation of amino acids next few years. There are currently no recognised antiviral ther• 10 and 24 from asparagine to alanine in the ADP•ribose binding apies or human vaccines with which to control infections due to region of the SINV macro domain impairs replication and viral RNA CHIKV. synthesis particularly in mouse neurones without any alteration in poly(ADP•ribose) binding ( Park and Griffin, 2009 ). Moreover, 5.1. Search for antiviral drugs mutation at position 10 had the greatest effect and caused nsP3 instability in neurones, decreased SINV•induced death of mature, Failure to develop approved antiviral therapeutic agents or vac• but not immature neurones, and attenuated virulence in 2 weeks cines has been particularly exposed during the recent outbreaks of old, but not 5•day•old mice. Thus, the nsP3 macro domain appears CHIKV in the Indian Ocean. Thus, patient treatment has been based to be significantly involved in both SINV replication and age• on non•salicylate analgesics and non•steroidal anti•inflammatory dependent susceptibility to encephalomyelitis. As a part of the drugs ( Pialoux et al., 2007 ). However, the elaboration of mouse structural genomics section in VIZIER, we have also determined the (Couderc et al., 2008; Ziegler et al., 2008 ) and non•human pri• crystal structure of the EEEV macro domain. The level of amino mate models ( Roques et al., 2007 ) together with antivirals currently acid conservation of the entire nsP3 when compared with VEEV undergoing clinical trials, and new approaches involving natural and EEEV is 43% identity. However, the structural model for EEEV is products of plants ( Li et al., 2007 ) provide the stimulus for improv• still under refinement, and was therefore not included in our most ing development of antiviral candidates. recent publication ( Malet et al., 2009 ). Sequence analysis of the C• terminal region of the nsP3 protein does not suggest any putative 5.1.1. Chloroquine enzymatic activity. This region encodes a cluster of up to 16 serines Chloroquine was first reported to inhibit SINV and SFV infectiv• and threonines that can be phosphorylated ( LaStarza et al., 1994; ity in vitro more than 35 years ago ( Cassell et al., 1984; Coombs et Li et al., 1990 ) although it is not catalysed by nsP3 ( Vihinen et al., al., 1981; Helenius et al., 1982; Inglot, 1969; Shimizu et al., 1972 ) but 2001 ). Its decreased phosphorylation in SINV produces less minus studies in mice suggested that the drug might enhance viral repli• strand RNA compared to wild type ( Dé et al., 2003 ). Moreover, vari• cation and aggravate the disease ( Maheshwari et al., 1991 ). Recent ants of SFV that are poorly phosphorylated or have deletions in research on the efficacy of Chloroquine has focused on the dosage this hypervariable domain exhibit a low viral pathogenicity pro• used to treat acute CHIKV infections ( de Lamballerie et al., 2008a; file in mice and show reduced viral RNA synthesis in cell culture Savarino et al., 2007 ). Chloroquine phosphate has also been used (Galbraith et al., 2005; Vihinen et al., 2001 ). The absence of the nsP3 to treat chronic chikungunya arthritis ( Brighton, 1984 ) by utilising C•terminus also alters SFV neurovirulence ( Tuittila et al., 2000 ). the anti•inflammatory properties of the molecule, rather than pos• The C•terminal region of nsP3 has specific sequence features of sible antiviral effects. Based on experiments in cell culture, results natively unfolded proteins, suggesting that this domain is involved comparable with those obtained using CHIKV, were also observed in transcription regulation as proposed previously ( Wang et al., with the SARS coronavirus. Chloroquine was therefore proposed as 1994 ). a potential antiviral molecule for the treatment of humans infected

Please cite this article in press as: Gould, E.A., et al., Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.07.007 G Model AVR•2510; No. of Pages 14 ARTICLE IN PRESS

E.A. Gould et al. / Antiviral Research xxx (2009) xxx–xxx 9 with the SARS coronavirus ( Keyaerts et al., 2004; Leyssen et al., consistent with the hypothesis that ribavirin acts mainly as an IMP 2006 ). dehydrogenase inhibitor against alphaviruses. To advance the studies with Chloroquine and CHIKV, a double blind placebo•controlled randomized trial (see http://clinicaltrials. 5.1.4. Interferon and ribavirin gov/ct/show/NCT00391313 ) ( de Lamballerie et al., 2008a; Leyssen A combination of interferon•alpha and ribavirin shows a syn• et al., 2006 ) was conducted on Réunion Island (Indian Ocean). No ergistic effect on the in vitro inhibition of CHIKV ( Briolant et al., statistical difference was observed between the Chloroquine and 2004 ). However, for clinical application it is expensive and requires placebo groups, either in mean duration of febrile arthralgia or rate parenteral injection, making it unsuitable for large•scale use dur• of decrease of viraemia (between day 1 and day 3). At day 200 post• ing epidemics. Human infection with CHIKV appears to induce infection Chloroquine•treated patients declared more frequently immunological dysfunction. However, interferon usually boosts the (p < 0.01) that they still suffered from arthralgia than patients who immune response. Therefore caution in its usage is recommended had received the placebo. However, the number of patients included until more extensive non•human primate models have been stud• in the study was too small to draw definitive conclusions regarding ied. Pegylated alpha interferon appears to be an effective treatment the efficacy of the Chloroquine treatment. Recent experiments in against infection with VEEV and has profound effects on the host macaques ( Macaca fascicularis ) also failed to identify a detectable immune response to infection ( Lukaszewski and Brooks, 2000 ). antiviral effect of Chloroquine following experimental infections Thus, it might be justified to test pegylated alpha interferon on and similar doses of Chloroquine (Le Grand et al., manuscript in CHIKV and other pathogenic alphaviruses. preparation). Thus, the use of Chloroquine to treat CHIKV•infected patients does not appear to be justified. 5.1.5. Inhibitors of alphavirus entry and maturation Molecules that inhibit alphavirus entry into susceptible cells 5.1.2. Quinine have also been investigated with promising results. A study recently The antimalarial drug, Quinine, inhibits replication of CHIKV in reported the possible inhibition of the replication of VEEV using vitro . Quinine appears to be a more likely candidate for antiviral polyclonal antibodies to laminin•binding protein ( Bondarenko et therapy against CHIKV because the 50% inhibitory concentration al., 2004 ). CHIKV infection of cultured human cells was also shown (IC ) value is much lower than that of Chloroquine (0.1 ␮g/ml 50 to be inhibited by impairing the maturation of the CHIKV E2 surface for Quinine versus 1.1 ␮g/ml for Chloroquine, using Vero cells and glycoprotein using furin inhibitors ( Ozden et al., 2008 ). A similar 1 × 10 2 cell culture infectious doses (CCID )). Also, in contrast 50 observation was previously reported using the flavivirus Tick•borne with Chloroquine, resistant mutants were obtained by growing encephalitis virus ( Stadler et al., 1997 ). CHIKV in increasing concentrations of Quinine. The mutations were detected in the nsP1 protein, suggesting impairment of function of the viral guanylyltransferase enzyme [XdeL; personal observation]. 5.1.6. Carbodine This region of nsP1 has also been indirectly related to methyltrans• Recent studies on the carbocyclic analogue of cytidine ferase activity in SINV isolates ( Laakkonen et al., 1994; Mi et al., (cyclopentylcytosine or carbodine) suggest that it has potential as 1989; Mi and Stollar, 1991; Wang et al., 1996 ) which has been shown an antiviral agent against VEEV ( Julandera et al., 2008 ). Carbodine to be an essential component of SINV pathogenicity in mice ( Zhu et was shown to inhibit cellular cytidine triphosphate (CTP) syn• al., 2009 ). Thus, the alphavirus nsP1 might prove to be a good target thetase, which converts UTP to CTP, resulting in indirect inhibition for antiviral therapy. of virus replication through reduction of CTP pools ( de Clercq et al., 1990 ). In cell culture, carbodine is a broad•spectrum antiviral, with 5.1.3. Ribavirin activity against several unrelated viruses ( Andrei and De Clercq, Ribavirin shows wide in vitro inhibitory activity against RNA 1990; Neyts et al., 1996 ) although cytotoxicity has been demon• viruses with different modes of action depending on the virus strated. The addition of exogenous cytidine (cyd) or uridine results (Leyssen et al., 2006 ). In some cases it inhibits IMP dehydro• in reversal of antiviral activity of carbodine in various cell lines genase, depleting cellular GTP pools. In others it is used as a (Andrei and De Clercq, 1990 ). The natural nucleosides are dextroro• non•canonical substrate for RNA synthesis introducing numerous tatory (d), but both d• and laevorotatory (l)•analogues can inhibit mutations whose accumulation may lead to virus inactivation due metabolic enzymes ( Gumina et al., 2002 ) and the d•enantiomer to error catastrophe ( Crotty et al., 2001; Severson et al., 2003 ). In may show reduced cytotoxicity. Accordingly, the d•enantiomer was the case of some arenaviruses ribavirin 5 ′•tri•phosphate interacts recently used to test the inhibitory qualities of carbodine against with the viral polymerase ( Sun et al., 2007 ). It was also tested as an VEEV•challenged mice ( Julandera et al., 2008 ). Increased survival aerosol for the treatment of paediatric Respiratory Syncytial Virus rates, increased average time to death and reduced brain virus titres infections but was not approved and this method has now been dis• were observed when carbodine treatment was followed by chal• continued. Ribavirin is used in combination with alpha•interferon lenge of the mice with the attenuated vaccine strain (TC83) of VEEV. for the treatment of hepatitis C virus (HCV) infection but the mecha• Evidence of inhibitory effects was also detected if the carbodine was nism of action is multifactorial. The predominant direct mechanism administered up to 4 days post•infection. by which ribavirin exerts its antiviral activity in vitro against fla• viviruses and paramyxoviruses is mediated by inhibition of IMP 5.1.7. Small RNA molecules dehydrogenase ( Leyssen et al., 2005 ), because viral RNA synthe• In common with many other RNA viruses, inhibition of sis requires a high GTP concentration, and also because resistance alphavirus replication in cell culture has been demonstrated using mutations can be isolated that map on the NS5b polymerase ( Young interfering RNAs and antisense oligonucleotides. Inhibition of VEEV et al., 2003 ). An indirect mechanism via decreasing the GTP pools was observed using a mixture of four short interfering RNAs may also be important ( Zhou et al., 2003 ). (O’Brien, 2007 ), and antisense morpholino oligomers were suc• In the case of alphaviruses, resistance to ribavirin was first cessfully used for the inhibition of SINV infection, in cell culture reported following studies with SINV ( Scheidel et al., 1987 ). Resis• and in mice ( Paessler et al., 2008 ) strongly suggesting that sim• tant mutants were mapped to the nsP1 protein ( Scheidel and ilar approaches should be applied to other alphaviruses such as Stollar, 1991 ). It was proposed that the mutant encoded an altered CHIKV. Methods to overcome the difficulties encountered in deliv• RNA guanylyltransferase enzyme with increased affinity for GTP, ering these antiviral molecules are currently the subject of major enabling it to replicate in cells with reduced levels of GTP. This is research.

Please cite this article in press as: Gould, E.A., et al., Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.07.007 G Model AVR•2510; No. of Pages 14 ARTICLE IN PRESS

10 E.A. Gould et al. / Antiviral Research xxx (2009) xxx–xxx

5.1.8. Plant compounds the search for antivirals. The first demonstration of antiviral activ• Compounds extracted from plants have also shown very ity provided by a nucleoside analogue (reviewed in Leyssen et al., encouraging antiviral effects against certain RNA viruses. The 2008 ) has contributed significantly to the consideration of replica• seco•pregnane steroid glaucogenin C and its monosugar•glycoside tive enzymes as highly promising targets in the field of antiviral cynatratoside A extracted from Strobilanthes cusia , together with drug discovery and design. three new pantasugar•glycosides of glaucogenin C from Cynanchum Prior to the commencement of the VIZIER project ( Coutard et paniculatum , can suppress a range of positive•strand RNA viruses al., 2008 ), the alphaviruses had not attracted too much attention including Tobacco mosaic virus (TMV), and the alphaviruses, in the context of drug discovery. However, as discussed earlier, this SINV, EEEV, and Getah virus in cell culture. Moreover, mice situation began to change from 2005 onwards, following the sur• were protected from lethal SINV infections with no adverse prisingly large•scale outbreaks of chikungunya fever on many of effects of the compounds on the infected animals ( Li et al., the inhabited islands of the southern Indian Ocean. Consequently, 2007 ). the recent first demonstration that alphavirus RdRp activity is encoded in the nsP4 gene ( Rubak et al., 2009 ) provides a strong 5.1.9. nsP1 active domains as a target for inhibition incentive for vigorous studies to identify effective inhibitors of In addition to the research activities described above, recent alphavirus RdRps. In alphaviruses, assembly of the multi•subunit efforts have focused on the identification of specific inhibitors of replication complex is required for demonstration of RdRp activ• viral enzymes involved in replication. Enzyme production pro• ity. Thus, in vitro assays require at least nsP123 and nsP4 in the cesses, activity assays, and structural data are all available, and reaction mixture, and the assays are only effective if recombi• could therefore provide a basis for inhibitor screening. Since nsP1 is nant nsP4 is expressed with the N•terminal tyrosine ( Rubak et the main enzyme involved in RNA capping, an essential step in pro• al., 2009; Shirako et al., 2003 ). Whilst experimental conditions for moting viral RNA translation, nsP1 is an excellent target candidate the generation of authentic nsP4 have been defined, recombinant for antiviral therapy. SINV and SFV nsP1s can be produced to high nsP123 is still expressed exclusively in mammalian cells ( Rubak levels in E. coli or insect cells, and both methyltransferase activity et al., 2009 ), which could prove to be a limitation in the context on GTP and guanylyltransferase activity have been tested in vitro . of HT inhibitor screening. Clearly many problems remain to be Following this strategy, several GTP analogues have been reported resolved. to inhibit the two activities with Ki values below 100 ␮M ( Lampio et al., 1999 ). 5.2. Antiviral vaccines

An alternative approach to virus disease control involves the use 5.1.10. nsP2 active domains as a target for inhibition of vaccines Whilst some are available to immunise horses against It has also been demonstrated that helicases are good antivi• VEEV, WEEV and EEEV, and both live and inactivated vaccines have ral targets in flaviviruses ( Goodell et al., 2006 ), picornaviruses been used to immunise laboratory workers at risk of exposure to (De Palma et al., 2008 ) and hepaciviruses ( Borowski et al., 2002 ). encephalitic alphaviruses, there are no licensed human vaccines The helicase region of SFV nsP2 can be expressed in E. coli against alphaviruses. A live•attenutated vaccine against VEEV was but the protein is unstable after purification, requiring a high first developed in 1961 ( Berge et al., 1961 ) and has been admin• salt concentration ( Gomez de Cedrón et al., 1999 ). Nevertheless, istered to more than 8000 individuals ( Alevizatos et al., 1967; the protein is active and production yields could be compatible Burke et al., 1977; Pittman et al., 1996 ). As the result of adverse with existing helicase HTP assays using non•radioactive readouts effects in a significant proportion of these vaccinated individu• (Boguszewska•Chachulska et al., 2004; Tani et al., 2009 ). Currently, als, an inactivated vaccine was developed ( Pittman et al., 1996 ) the protease domain of nsP2 is the most promising target for and a more promising highly attenuated vaccine (V3526) based rational inhibitor screening. This protease is responsible for non• on an infectious cDNA clone of VEEV was also developed ( Davis structural polyprotein processing, an essential function for virus et al., 1995; Hart et al., 2000 ) but concerns over the possibility of replication ( Balistreri et al., 2007; de Groot et al., 1990; Mayuri reversion to virulence have not been totally alleviated. Hence, alter• et al., 2008 ). Inhibitors can be selected through a structure•based native approaches such as the development of chimaeric viruses method, using the VEEV protease structural data as a template with the replicative machinery from SINV, and the structural genes (Russo et al., 2006 ). This strategy could be combined with enzy• from VEEV are still being pursued ( Berge et al., 1961; Paessler matic assays already described for several alphavirus proteases et al., 2006 ). The first attempts to develop inactivated vaccines (Zhang et al., 2009 ). Interestingly, leupeptin, a broad•spectrum cys• against CHIKV were reported in 1970 ( Eckels et al., 1970 ). How• teine protease inhibitor, has no effect on the protease activity of ever, these early immunogens have not been developed as licensed CHIKV nsP2 ( Pastorino et al., 2008 ), suggesting that this protease vaccines. has structural and functional specificities that could be specifically Later, a live•attenuated vaccine strain was developed by serial, targeted. plaque•to•plaque passages of CHIKV ( Edelman et al., 2000 ). This vaccine proved highly immunogenic but some phase II volunteers 5.1.11. nsP3 active domains as a target for inhibition developed transient arthralgia. At least three different methods Structural data and enzymatic properties of macro domains of are currently under development to produce safe, effective vac• nsP3 in two alphaviruses have recently been reported ( Malet et al., cines against CHIKV. One method involves the preparation of 2009 ). On the basis of very recently published results ( Park and purified inactivated virus using methods similar to those that Griffin, 2009 ), the nsP3 macro domain appears to be significantly have proved successful for the development of an inactivated vac• involved in both SINV replication and age•dependent susceptibil• cine against Tick•borne encephalitis virus ( Pavlova et al., 2003 ). ity to encephalomyelitis and therefore may become an appropriate A second method involves the use of cDNA fragments represent• target for the development of antivirals. ing the important immunogenic regions of the CHIKV genome (Muthumani et al., 2008 ). Finally, chimaeric alphaviruses contain• 5.1.12. nsP4 active domains as a target for inhibition ing the genetic backbone of SINV, TC•83 or a naturally attenuated The RdRp is the crucial enzyme in RNA virus replication. Because EEEV strain, and the structural proteins of wild•type CHIKV, have viral RdRps have no close homologues amongst cellular replica• produced promising results in murine efficacy studies ( Wang et al., tive enzymes, they have been targeted since the earliest times in 2008 ).

Please cite this article in press as: Gould, E.A., et al., Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.07.007 G Model AVR•2510; No. of Pages 14 ARTICLE IN PRESS

E.A. Gould et al. / Antiviral Research xxx (2009) xxx–xxx 11

6. Conclusion Bryant, J.E., Holmes, E.C., Barrett, A.D.T., 2007. Out of Africa: a molecular perspec• tive on the introduction of yellow fever virus into the Americas. PLoS Pathog. 3, e75. At the commencement of the VIZIER project, alphaviruses were Burke, D.S., Ramsburg, H.H., Edelman, R.J., 1977. Persistence in humans of anti• given a relatively low profile because other, apparently more wor• body to subtypes of Venezuelan equine encephalomyelitis (VEE) virus after risome RNA viruses, in terms of their potential virulence such as immunization with attenuated (TC•83) VEE virus vaccine. J. Infect. Dis. 136, 354–359. SARS coronavirus, avian influenza virus, Nipah virus, rabies virus Calder, L., Laird, M., 1994. Mosquito travelers, arbovirus vectors and the used tire and West Nile virus were attracting much more attention globally. trade. Trav. Med. Int. 12, 3–12. However, in 2005, the relative level of importance of alphaviruses Cassell, S., Edwards, J., Brown, D.T., 1984. Effects of lysosomotropic weak bases on as a significant public health problem was raised when CHIKV infection of BHK•21 cells by Sindbis virus. J. Virol. 52, 857–864. Chanas, A.C., Johnson, B.K., Simpson, D.I.H., 1976. Antigenic relationships of unexpectedly emerged as a major human pathogen in Africa, the alphaviruses by a simple micro•culture cross•neutralization method. J. Gen. Indian Ocean, India and Malaysia. The continuing epidemic of Virol. 32, 295–300. chikungunya fever, which has now lasted for 4 years and shows Charrel, R.N., de Lamballerie, X., Raoult, D., 2007. Chikungunya outbreaks—the glob• alization of vectorborne diseases. N. Engl. J. Med. 356, 769–771. little sign of abating, has the potential to reach the Americas, Clarke, D.H., Casals, J., 1958. Techniques for haemagglutination and where if past history of other invading RNA viruses is predictive, haemagglutination•inhibition with arthropod•borne viruses. Am. J. Trop. could lead to high levels of human morbidity. Faced with this Med. Hyg. 7, 561–573. Coombs, K., Mann, E., Edwards, J., Brown, D.T., 1981. Effects of chloroquine and prospect and the possibility that as the result of human impact cytochalasin B on the infection of cells by Sindbis virus and vesicular stomatitis on habitat and global/local distribution of vectors and hosts, other virus. J. Virol. 37, 1060–1065. alphaviruses such as ONNV might also emerge to produce much Couderc, T., Chrétien, F., Schilte, C., Disson, O., Brigitte, M., Guivel•Benhassine, F., Touret, Y., Barau, G., Cayet, N., Schuffenecker, I., Desprès, P., Arenzana•Seisdedos, more extensive epidemics than they have done in the past, the F., Michault, A., Albert, M.L., Lecuit, M., 2008. A mouse model for Chikungunya: search for antivirals to combat these viruses clearly needs to be young age and inefficient type•I interferon signaling are risk factors for severe intensified. disease. PLoS Pathog. 4 (2), e29. Coutard, B., Gorbalenya, A.E., Snijder, E.J., Leontovich, A.M., Poupon, A., De Lambal• lerie, X., Charrel, R., Gould, E.A., Gunther, S., Norder, H., Klempag, B., Bourhy, H., Acknowledgements Rohayemj, J., L’hermite, E., Nordlund, P., Stuart, D.I., Owens, R.J., Grimes, J.M., Tuckerm, P.A., Bolognesi, M., Mattevi, A., Coll, M., Jones, T.A., Åqvist, J., Unger, T., Hilgenfeld, R., Bricogne, G., Neyts, J., La Colla, P., Puerstinger, G., Gonzalez, The work reported in this review was supported under J.P., Leroy, E., Cambillau, C., Romette, J.L., Canard, B., 2008. The VIZIER project: the project entitled VIZIER (Comparative Structural Genomics preparedness against pathogenic RNA viruses. Antiviral Res. 78, 37–46. of Viral Enzymes Involved in Replication)—Contract number Crotty, S., Cameron, C.E., Andino, R., 2001. RNA virus error catastrophe: direct molec• ular test by using ribavirin. Proc. Natl. Acad. Sci. 89, 6895–6900. (2004•511960). Professor Scott Weaver’s alphavirus research was Davis, N.L., Brown, K.W., Greenwald, G.F., Zajac, A.J., Zacny, V.L., Smith, J.F., Johnston, supported by National Institute of Health grants U54•AI057156, R.E., 1995. Attenuated mutants of Venezuelan equine encephalitis virus con• R01•AI071192 and R01•AI069145. taining lethal mutations in the PE2 cleavage signal combined with a second•site suppressor mutation in E1. Virology 212, 102–110. de Clercq, E., Bernaerts, R., Shealy, Y.F., Montgomery, J.A., 1990. Broad•spectrum References antiviral activity of carbodine, the carbocyclic analogue of cytidine. Biochem. Pharmacol. 39, 319–325. Ahola, T., Kääriäinen, L., 1995. Reaction in alphavirus mRNA capping: formation of a de Groot, R.J., Hardy, W.R., Shirako, Y., Strauss, J.H., 1990. Cleavage•site preferences covalent complex of nonstructural protein nsP1 with 7•methyl•GMP. Proc. Natl. of Sindbis virus polyproteins containing the non•structural proteinase. Evidence Acad. Sci. U.S.A. 92, 507–511. for temporal regulation of polyprotein processing in vivo. EMBO J. 9, 2632–2638. Ahola, T., Kujala, P., Tuittila, M., Blom, T., Laakkonen, P., Hinkkanen, A., Auvinen, P., Dé, I., Fata•Hartley, C., Sawicki, S.G., Sawicki, D.L., 2003. Functional analysis of nsP3 2000. Effects of palmitoylation of replicase protein nsP1 on alphavirus infection. phosphoprotein mutants of Sindbis virus. J. Virol. 77, 13106–13116. J. Virol. 74, 6725–6733. de Lamballerie, X., Boisson, V., Reynier, J.C., Enault, S., Charrel, R.N., Flahault, A., Ahola, T., Lampio, A., Auvinen, P., Kääriäinen, L., 1999. Semliki Forest virus mRNA Roques, P., Le Grand, R., 2008a. On Chikungunya acute infection and chloroquine capping enzyme requires association with anionic membrane phospholipids for treatment. Vector Borne Zoonotic Dis. 8, 837–840. activity. EMBO J. 18, 3164–3172. de Lamballerie, X., Leroy, E., Charrel, R.N., Ttsetsarkin, K., Higgs, S., Gould, E.A., 2008b. Alevizatos, A.C., McKinney, R.W., Feigin, R.D., 1967. Live, attenuated Venezuelan Chikungunya virus adapts to tiger mosquito via evolutionary convergence: a sign equine encephalomyelitis virus vaccine. I. Clinical effects in man. Am. J. Trop. of things to come? Virol. J. 5, 33. Med. 16, 762–768. De Palma, A.M., Vliegen, I., De Clercq, E., Neyts, J., 2008. Selective inhibitors of picor• Andrei, G., De Clercq, E., 1990. Inhibitory effect of selected antiviral compounds on navirus replication. Med. Res. Rev. 28, 823–824. arenavirus replication in vitro. Antiviral Res. 14, 287–299. Diaz, L.A., Cardenas, V.M., Daza, E., Bruzon, L., Alcala, A., De la Hoz, O., Caceres, F.M.G., Balistreri, G., Caldentey, J., Kääriäinen, L., Ahola, T., 2007. Enzymatic defects of the Aristizabal, G., Martinez, J.W., Revelo, D.F., De la Hoz, F., Boshell, J., Camacho, T., nsP2 proteins of Semliki Forest virus temperature•sensitive mutants. J. Virol. 81, Calderon, L., Olano, V.A., Villarreal, L.I., Roselli, D., Alvarez, G., Ludwig, G., Tsai, T., 2849–2860. 1997. Epidemic Venezuelan equine encephalitis in La Guajira, Colombia, 1995. J. Benedict, M., Levine, R., Hawley, W., Lounibos, L., 2007. Spread of the tiger: global Infect. Dis. 175, 828–832. risk of invasion by the mosquito Aedes albopictus . Vector Borne Zoonotic Dis. 7, Eckels, K., Harrison, V.R., Hetrick, F.M., 1970. Chikungunya virus vaccine prepared by 76–85. Tween•Ether extraction. Appl. Microbiol. 19, 321–325. Berge, T.O., Banks, I.S., Tigertt, W.D., 1961. Attenuation of Venezuelan equine Edelman, R., Tacket, C.O., Wasserman, S.S., Bodison, S.A., Perry, J.G., Mangiafico, J.A., encephalomyelitis virus by in vitro cultivation in guinea pig heart cells. Am. 2000. Phase II safety and immunogenicity study of live chikungunya virus vac• J. Hygiene 73, 209–218. cine TSI•GSD•218. Am. J. Trop. Med. Hyg. 62, 681–685. Feder, M., Pas, J., Wyrwicz, L.S., Bujnicki, J.M., 2003. Molecular phylogenetics of the Bloom, K.J., 1993. The Mississippi Valley’s great yellow fever epidemic of 1878. ′ Louisiana State University Press, Baton Rouge, Louisiana. RrmJ/fibrillarin superfamily of ribose 2 •O•methyltransferases. Gene, 302. Boguszewska•Chachulska, A.M., Krawczyk, M., Stankiewicz, A., Gozdek, A., Haenni, Galbraith, S.E., Sheahan, B.J., Atkins, G.J., 2005. Deletions in the hypervariable domain A.•L., Strokovskaya, L., 2004. Direct fluorometric measurement of hepatitis C of the nsP3 gene attenuate Semliki Forest virus virulence. J. Gen. Virol. 87, virus helicase activity. FEBS Lett. 567, 253–258. 937–947. Bondarenko, E.I., Protopopova, E.V., Surovtsev, I.V., Shvalov, A.N., Loktev, V.B., 2004. Garmashova, N., Gorchakov, R., Frolova, E., Frolov, I., 2006. Sindbis Virus non• Replication by polyclonal antibodies to laminin•binding protein. Voprosi Viru• structural protein nsP2 is cytotoxic and inhibits cellular transcription. J. Virol. sologii 49, 32–37. 80, 5686–5696. Borowski, P., Schalinski, S., Schmitz, H., 2002. Nucleotide triphosphatase/helicase of Gomez de Cedrón, M., Ehsani, N., Mikkola, M.L., García, J.A., Kääriäinen, L., 1999. RNA hepatitis C virus as a target for antiviral therapy. Antiviral Res. 55, 397–412. helicase activity of Semliki Forest virus replicase protein nsP2. FEBS Lett. 448, Brault, A.C., Powers, A.M., Ortiz, D., Estrada•Franco, J.G., Navarro•Lopez, R., Weaver, 19–22. S.C., 2004. Venezuelan equine encephalitis emergence: enhanced vector infec• Goodell, J.R., Puig•Basagoiti, F., Forshey, B.M., Shi, P.•Y., Ferguson, D.M., 2006. Iden• tion from a single amino acid substitution in the envelope glycoprotein. Proc. tification of compounds with anti•West Nile virus activity. J. Med. Chem. 49, Natl. Acad. Sci. U.S.A. 101, 11344–11349. 2127–2137. Brighton, S.W., 1984. Chloroquine phosphate treatment of chronic chikungunya Gorbalenya, A.E., Koonin, E.V., 1993. Helicases: amino acid sequence comparisons arthritis. S. Afr. Med. J. 66, 217–218. and structure–function relationships. Curr. Opin. Struct. Biol. 3, 419–429. Briolant, S., Garin, D., Scaramozzino, N., Jouan, A., Crance, J.M., 2004. In vitro Gorbalenya, A.E., Koonin, E.V., Donchenko, A.P., Blinov, V.M.F.L.A., 1988. A novel inhibition of Chikungunya and Semliki Forest viruses replication by antiviral superfamily of nucleoside triphosphate•binding motif containing proteins compounds: synergistic effect of interferon• ␣ and ribavirin combination. Antivi• which are probably involved in duplex unwinding in DNA and RNA replication ral Res. 61, 111–117. and recombination. FEBS Lett. 235, 16–24.

Please cite this article in press as: Gould, E.A., et al., Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.07.007 G Model AVR•2510; No. of Pages 14 ARTICLE IN PRESS

12 E.A. Gould et al. / Antiviral Research xxx (2009) xxx–xxx

Gorbalenya, A.E., Koonin, E.V., Lai, M.M., 1991. Putative papain•related thiol proteases Chikungunya virus and central nervous system infections in children, India. of positive•strand RNA viruses. Identification of rubi• and aphthovirus proteases Emerg. Infect. Dis. 15, 329–331. and delineation of a novel conserved domain associated with proteases of rubi•, Leyssen, P., Balzarini, J., De Clercq, E., Neyts, J., 2005. The predominant mechanism alpha• and coronaviruses. FEBS Lett. 288, 201–205. by which ribavirin exerts its antiviral activity in vitro against flaviviruses and Gorchakov, R., Elena Frolova, E., Sawicki, S., Atasheva, S., Sawicki, D., Ilya Frolov, I., paramyxoviruses is mediated by inhibition of IMP dehydrogenase. J. Virol. 79, 2008. A new role for ns polyprotein cleavage in Sindbis virus replication. J. Virol. 1943–1947. 82, 6218–6231. Leyssen, P., De Clercq, E., Neyts, J., 2006. The anti•yellow fever virus activity of rib• Gould, E.A., de Lamballerie, X., Zanotto, P.M., Holmes, E.C., 2003. Origins, evolution, avirin is independent of error•prone replication. Mol. Pharmacol. 69, 1461–1467. and vector/host coadaptations within the genus Flavivirus. Adv. Virus Res. 59, Leyssen, P., De Clercq, E., Neyts, J., 2008. Molecular strategies to inhibit the replication 277–314. of RNA viruses. Antiviral Res. 78, 9–25. Gould, E.A., de Lamballerie, X., Zanotto, P.M.A., Holmes, E.C., 2001. Evolution, epi• Li, G.P., La Starza, M.W., Hardy, W.R., Strauss, J.H., Rice, C.M., 1990. Phosphorylation demiology and dispersal of flaviviruses revealed by molecular phylogenies. Adv. of Sindbis virus nsP3 in vivo and in vitro. Virology 179, 416–427. Virus Res. 57, 71–103. Li, G.P., Rice, C.M., 1989. Mutagenesis of the in•frame opal termination codon pre• Gould, E.A., Higgs, S., 2009. Impact of climate change and other factors on emerging ceding nsP4 of Sindbis virus: studies of translational readthrough and its effect arbovirus diseases. Trans. R. Soc. Trop. Med. Hyg. 103, 109–121. on virus replication. J. Virol. 63, 1326–1337. Gumina, G., Chong, Y., Choo, H., Song, G.Y., Chu, C.K., 2002. L•Nucleosides: Li, M.L., Lin, Y.H., Simmonds, H.A., Stollar, V., 2004. A mutant of Sindbis virus which is antiviral activity and molecular mechanism. Curr. Top. Med. Chem. 2, able to replicate in cells with reduced CTP makes a replicase/transcriptase with 1056–1086. a decreased Km for CTP. J. Virol. 78, 9645–9651. Hahn, C.S., Lustig, S., Strauss, E.G., Strauss, J.H., 1988. Western equine encephalitis Li, Y., Wang, L., Li, S., Chen, X., Shen, Y., Zhang, Z., He, H., Xu, W., Shu, Y., Liang, G., virus is a recombinant virus. Proc. Natl. Acad. Sci. U.S.A. 85, 5997–6001. Fang, R., Hao, X., 2007. Seco•pregnane steroids target the subgenomic RNA of Hart, M.K., Caswell•Stephan, K., Bakken, R., Tammariello, R., Pratt, W., Davis, N., John• alphavirus•like RNA viruses. Proc. Natl. Acad. Sci. 104, 8083–8088. ston, R.E., Smith, J.K.S., 2000. Improved mucosal protection against Venezuelan Lounibos, L.P., 2002. Invasions by insect vectors of human disease. Annu. Rev. Ento• equine encephalitis virus is induced by the molecularly defined, live•attenuated mol. 47, 233–266. V3526 vaccine candidate. Vaccine 18, 3067–3075. Lukaszewski, R.A., Brooks, T.J., 2000. Pegylated alpha interferon is an effective treat• Helenius, A., Marsh, M., White, J., 1982. Inhibition of Semliki Forest virus penetration ment for virulent Venezuelan equine encephalitis virus and has profound effects by lysosomotropic weak bases. J. Gen. Virol. 58, 47–61. on the host immune response to infection. J. Virol. 74, 5006–5015. Hodgman, T.G., 1988. A new superfamily of replicative proteins. Nature 333, 22–23. Maheshwari, R.K., Srikantan, V., Bhartiya, D., 1991. Chloroquine enhances replica• Inglot, A.D., 1969. Comparison of the antiviral activity in vitro of some non•steroidal tion of Semliki Forest virus and encephalomyocarditis virus in mice. J. Virol. 65, anti•inflammatory drugs. J. Gen. Virol. 4, 203–214. 992–995. Johnson, K.M., Martin, D.M., 1974. Venezuelan equine encephalitis. Adv. Vet. Sci. Malet, H., Jamal, S., Coutard, B., Dutartre, H., Papageorgiou, N., Heinonen, M., Ahola, Comp. Med. 18, 79–116. T., Forrester, N., Gould, E.A., Laffitte, D., Ferron, F., Lescar, J., de Lamballerie, X., Josseran, L., Paquet, C., Zehgnoun, A., Caillere, N., Le Tertre, A., Solet, J.L., Ledrans, Canard, B., 2009. The crystal structures of Chikungunya and Venezuelan equine M., 2006. Chikungunya disease outbreak, Reunion Island. Emerg. Infect. Dis. 12, encephalitis virus nsP3 macro domains define a conserved adenosine binding 1994–1995. pocket. J. Virol. 83, 6534–6545. Julandera, J.G., Bowen, R.A., Raoc, J.R., Daya, C., Shafera, K., Smeea, D.F., Morreya, J.D., Mavalankar, D., Shastri, P., Bandyopadhyay, T., Parmar, J., Ramani, K.V., 2008. Chuc, C.K., 2008. Treatment of Venezuelan equine encephalitis virus infection Increased mortality rate associated with chikungunya epidemic, ahmedabad, with ( −)•carbodine. Antiviral Res. 80, 309–315. India. Emerg. Infect. Dis. 14, 412–415. Kamer, G., Argos, P., 1984. Primary structural comparison of RNA•dependent Mayuri, Todd, W.G., Smith, J.L., Kuhn, R.J., 2008. Role for conserved residues of Sindbis polymerases from plant, animal and bacterial viruses. Nucleic Acids Res. 12, virus nonstructural protein 2 methyltransferase•like domain in regulation of 7269–7282. minus•strand synthesis and development of cytopathic infection. J. Virol. 82, Karabatsos, N., 1975. Antigenic relationships of group A arboviruses by plaque reduc• 7284–7297. tion neutralization testing. Am. J. Trop. Med. Hyg. 24, 527–532. Mi, S., Durbin, R., Huang, H.V., Rice, C.M., Stollar, V., 1989. Association of the Sind• Keyaerts, E., Vijgen, L., Maes, P., Neyts, J., Van Ranst, M., 2004. In vitro inhibition of bis virus RNA methyltransferase activity with the nonstructural protein nsP1. severe syndrome coronavirus by chloroquine. Biochem. Biophys. Res. Commun. Virology 170, 385–391. 323, 264–268. Mi, S., Stollar, V., 1991. Expression of Sindbis virus nsP1 and methyltransferase activ• Kumaran, D., Eswaramoorthy, S., Studier, F.W., Swaminathan, S., 2005. Structure and ity in Escherichia coli . Virology 184, 423–427. mechanism of ADP•ribose•1 ′′ •monophosphatase (Appr•1 ′′ •pase), a ubiquitous Mukhopadhyay, S., Zhang, W., Gabler, S., Chipman, P.R., Strauss, E.G., Strauss, J.H., cellular processing enzyme. Protein Sci. 14, 719–726. Baker, T.S., Kuhn, R.J., Rossmann, M.G., 2006. Mapping the structure and function Laakkonen, P., Ahola, T., Kääriäinen, L., 1996. The effects of palmitoylation on mem• of the E1 and E2 glycoproteins in alphaviruses. Structure 14, 63–73. brane association of Semliki Forest virus RNA capping enzyme. J. Biol. Chem. 271, Muthumani, K., Lankaraman, K.M., Laddy, D.J., Sundaram, S.G., Chung, C.W., Sako, 28567–28571. E., Wu, L., Khan, A., Sardesai, N., Kim, J.J., Vijayachari, P., Weiner, D.B., 2008. Laakkonen, P., Hyvönen, M., Peränen, J., Kääriäinen, L., 1994. Expression of Semliki Immunogenicity of novel consensus•based DNA vaccines against Chikungunya Forest virus nsP1•specific methyltransferase in insect cells and in Escherichia coli . virus. Vaccine 26, 5128. J. Virol. 68, 7418–7425. Neyts, J., Meerbach, A., McKenna, P., De Clercq, E., 1996. Use of the yellow fever virus Lampio, A., Ahola, T., Darzynkiewicz, E., Stepinski, J., Jankowska•Anyszka, M., Kääriäi• vaccine strain 17D for the study of strategies for the treatment of yellow fever nen, L., 1999. Guanosine nucleotide analogs as inhibitors of alphavirus mRNA virus infections. Antiviral Res. 30, 125–132. caping enzyme. Antiviral Res. 42, 35–46. O’Brien, L., 2007. Inhibition of multiple strains of Venezuelan equine encephalitis Lampio, A., Kilpeläinen, I., Pesonen, S., Karhi, K., Auvinen, P., Somerharju, P., virus by a pool of four short interfering RNAs. Antiviral Res. 75, 20–29. Kääriäinen, L., 2000. Membrane•binding of Semliki Forest virus mRNA cap• Ozden, S., Lucas•Hourani, M., Ceccaldi, P.E., Basak, A., Valentine, M., Benjannet, S., ping enzyme—solution structure of the binding peptide and interaction with Hamelin, J., Jacob, Y., Mamchaoui, K., Mouly, V., Desprès, P., Gessain, A., Butler• liposomes. J. Biol. Chem. 275, 37853–37859. Browne, G., Chretien, M., Tangy, F., Vidalain, P.O., Seidah, N.G., 2008. Inhibition of LaStarza, M.W., Grakoui, A., Rice, C.M., 1994. Deletion and duplication mutations in Chikungunya virus infection in cultured human muscle cells by furin inhibitors: the C•terminal non•conserved region of Sindbis virus nsP3: effects on phospho• impairment of the maturation of the E2 surface glycoprotein. J. Biol. Chem. 283, rylation and on virus replication in vertebrate and invertebrate cells. Virology 21899–21908. 202, 224–232. Paessler, S., Ni, H., Petrakova, O., Fayzulin, R.Z., Yun, N., Anishchenko, M., Weaver, S.C., Lavergne, A., de Thoisy, B., Lacoste, V., Pascalis, H., Pouliquen, J.•F., Mercier, V., Tolou, Frolov, I., 2006. Replication and clearance of Venezuelan equine encephalitis H., Dussart, P., Morvan, J., Talarmin, A., Kazanji, M., 2006. Mayaro virus: complete virus from the brains of animals vaccinated with chimeric SIN/VEE viruses. J. nucleotide sequence and phylogenetic relationships with other alphaviruses. Virol. 80, 2784–2796. Virus Res. 117, 283–290. Paessler, S., Rijnbrand, R., Stein, D.A., Ni, H., Yun, N.E., Dziuba, N., Borisevich, V., Sere• Lemm, J.A., Bergqvist, A., Read, C.M., Rice, C.M., 1998. Template•dependent initiation gin, A., Ma, Y., Blouch, R., Iversen, P.L., Zacks, M.A., 2008. Inhibition of alphavirus of Sindbis virus RNA replication in vitro. J. Virol. 72, 6546–6553. infection in cell culture and in mice with antisense morpholino oliogmers. Virol• Lemm, J.A., Rümenapf, T., Strauss, E.G., Strauss, J.H., Rice, C.M., 1994. Polypeptide ogy 376, 357–370. requirements for assembly of functional Sindbis virus replication complexes: Park, E., Griffin, D.E., 2009. The nsP3 macro domain is important for Sindbis virus a model for the temporal regulation of minus• and plus•strand RNA synthesis. replication in neurons and neurovirulence in mice. Virology (April) (Epub ahead EMBO J. 13, 2925–2934. of print). Leon, C.A., 1975. Sequelae of Venezuelan equine encephalitis in humans: a four year Pastorino, B.A.M., Peyrefitte, C.N., Almeras, L., Grandadam, M., Rolland, D., Tolou, H.J., follow•up. Int. J. Epidemiol. 4, 131–140. Bessaud, M., 2008. Expression and biochemical characterization of nsP2 cysteine Lescar, J., Roussel, A., Wien, M.W., Navaza, J., Fuller, S.D., Wengler, G., Rey, F.A., protease of Chikungunya virus. Virus Res. 131, 293–298. 2001. The Fusion glycoprotein shell of Semliki Forest virus: an icosahe• Pavlova, B.G., Loew•Baselli, A., Fritsch, S., Poellabauer, E.•M., Vartian, N., Rinke, I., dral assembly primed for fusogenic activation at endosomal pH. Cell 105, Ehrlich, H.J., 2003. Tolerability of modified tick•borne encephalitis vaccine FSME• 137–148. IMMUN “NEW” in children: results of post•marketing surveillance. Vaccine 21, Levinson, R.S., Strauss, J.H., Strauss, E.G., 1990. Complete sequence of the genomic 742–745. RNA of o’nyong•nyong virus and its use in the construction of alphavirus phylo• Perhson, J.H., Fuji, R.N., 1998. Evolutionary conservation of histone macroH2A sub• genetic trees. Virology 174, 110–123. types and domains. Nucleic Acids Res. 26, 2837–2842. Lewthwaite, P., Vasanthapuram, R., Osborne, J.C., Begum, A., Plank, J.L.M., Shankar, Pialoux, G., Gaüzère, B.A., Jauréguiberry, S., Strobel, M., 2007. Chikungunya, an epi• M.V., Hewson, R., Desai, A., Beeching, N.J., Ravikumar, R., Solomon, T., 2009. demic arbovirosis. Lancet Infect. Dis. 7, 633.

Please cite this article in press as: Gould, E.A., et al., Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.07.007 G Model AVR•2510; No. of Pages 14 ARTICLE IN PRESS

E.A. Gould et al. / Antiviral Research xxx (2009) xxx–xxx 13

Piotrowski, Y., Hansen, G., Boomaars•van der Zanden, A.L., Snijder, E.J., Gorbalenya, Strauss, E.G., deGroot, R.J., Levinson, R., Strauss, J.H., 1992. Identification of the A.E., Hilgenfeld, R., 2009. Crystal structures of the X•domains of a Group•1 and active site residues in the nsP2 proteinase of Sindbis virus. Virology 191, a Group•3 coronavirus reveal that ADP•ribose•binding may not be a conserved 932–940. property. Protein Sci. 18, 6–16. Strauss, J.H., Strauss, E.G., 1994. The alphaviruses: gene expression, replication, and Pittman, P.R., Makuch, R.S., Mangiafico, J.A., Cannon, T.L., Gibbs, P.H.C.J., Peters, C.J., evolution. Microbiol. Rev. 58, 491–562. 1996. Long•term duration of detectable neutralizing antibodies after adminis• Strode, G.K., 1951. Yellow Fever. McGraw•Hill, New York. tration of live•attenuated VEE vaccine and following booster vaccination with Sun, Y., Chung, D.H., Chu, Y.K., Jonsson, C.B., Parker, W.B., 2007. Activity of ribavirin inactivated VEE vaccine. Vaccine 14, 337–343. against Hantaan virus correlates with production of ribavirin•5 ′•triphosphate, Poidinger, M., Roy, S., Hall, R.A., Turley, P.J., Scherret, J.H., Lindsay, M.D., Broom, A.K., not with inhibition of IMP dehydrogenase. Antimicrob. Agents Chemother. 51, Mackenzie, J.S., 1997. Genetic stability among temporally and geographically 84–88. diverse isolates of Barmah Forest virus. Am. J. Trop. Med. Hyg. 57, 230–234. Swofford, D.L., 1998. PAUP*. Phylogenetic Analysis Using Parsimony (* and Other Porterfield, J.S., 1961. Cross•neutralization studies with group A arthropod•borne Methods), version 4. Sinauer Associates, Sunderland, MA. viruses. Bull. World Health Org. 24, 735–741. Tabachnick, W.J., 1991. Evolutionary genetics and arthropod•borne diseases. The Powers, A.M., Brault, A.C., Shirako, Y., Strauss, E.G., Kang, W., Strauss, J.H., Weaver, S.C., yellow fever mosquito, Aedes aegypti . Am. J. Entomol. 37, 14–24. 2001. Evolutionary relationships and systematics of the alphaviruses. J. Virol. 75, Tani, H., Akimitsu, N., Fujita, O., Matsuda, Y., Miyata, R., Tsuneda, S., Igarashi, M., 10118–10131. Sekiguchi, Y., Noda, N., 2009. High•throughput screening assay of hepatitis C Powers, A.M., Logue, C.H., 2007. Changing patterns of chikungunya virus: re• virus helicase inhibitors using fluorescence•quenching phenomenon. Biochem. emergence of a zoonotic arbovirus. J. Gen. Virol. 88, 2363–2377. Biophys. Res. Commun. 379, 1054–1059. Reiskind, M.H., Pesko, K., Westbrook, C.J., Mores, C.N., 2008. Susceptibility of Florida Tatem, A.J., Hay, S.I., Rogers, D.J., 2006. Global traffic and disease vector dispersal. mosquitoes to Chikungunya virus. Am. J. Trop. Med. Hyg. 78, 422–425. Proc. Natl. Acad. Sci. U.S.A. 103, 6242–6247. Rezza, G., Nicoletti, L., Angelini, R., Romi, R., Finarelli, A.C., Panning, M., Cordioli, P., Tomar, S., Hardy, R.W., Smith, J.L., Kuhn, R.J., 2006. Catalytic core of alphavirus non• Fortuna, C., Boros, S., Magurano, F., Silvi, G., Angelini, P., Dottori, M., Ciufolini, structural protein nsP4 possesses terminal adenylyltransferase activity. J. Virol. M.G., Majori, G.C., Cassone, A., 2007. Infection with Chikungunya virus in Italy: 80, 9962–9969. an outbreak in a temperate region. Lancet, 1840–1846. Tsetsarkin, K.A., Vanlandingham, D.L., McGee, C.E., Higgs, S., 2007. A single muta• Rikkonen, M., Peränen, J., Kääriäinen, L., 1994. ATPase and GTPase activities associ• tion in Chikungunya virus affects vector specificity and epidemic potential. PLoS ated with Semliki Forest virus nonstructural protein nsP2. J. Virol. 68, 5804–5810. Pathog. 3, e201. Robin, S., Ramful, D., Le Seach, F., Jaffar•Bandjee, M.C., Rigou, G., Alessandri, J.L., 2008. Tuittila, M.T., Santagati, M.G., Röyttä, M., Määttä, J.A., Hinkkanen, A.E., 2000. Repli• Neurologic manifestations of pediatric Chikungunya infection. J. Child Neurol.. case complex genes of Semliki Forest virus confer lethal neurovirulence. J. Virol. Roques, P., Joubert, C., Malleret, B., Delache, B., Brochard, P., Calvo, J., Morin, 74, 4579–4589. J., Mannioui, A., Martinon, F., Lebon, P., Verrier, B., Lotteau, V., de Vasiljeva, L., Merits, A., Golubtsov, A., Sizemskaja, V., Kääriäinen, L., Ahola, T., 2003. Lamballerie, X., Cherel, Y., Larcher, T., Aumont, G., Le Grand, R., 2007. Chikun• Regulation of the sequential processing of Semliki Forest virus replicase polypro• gunya et autres arboviroses émergentes en milieu tropical. Reunion Island tein. J. Biol. Chem. 278, 41636–41645. http://www.invs.sante.fr/agenda/colloque chikungunya/ . Vasiljeva, L., Merits, A., Auvinen, P., Kääriäinen, L., 2000. Identification of a novel Rozanov, M.N., Koonin, E.V., Gorbalenya, A.E., 1992. Conservation of the puta• function of the alphavirus capping apparatus: RNA 5 ′•triphosphatase activity of tive methyltransferase domain: a hallmark of the ‘Sindbis•like’ supergroup of nsP2. J. Biol. Chem. 275, 17281–17287. positive•strand RNA viruses. J. Gen. Virol. 73, 2129–2134. Vasiljeva, L., Valmu, L., Kääriäinen, L., Merits, A., 2001. Site•specific protease activity Rubak, J.K., Wasik, B.R., Rupp, J.C., Kuhn, R.J., Hardy, R.W., Smith, J.L., 2009. Character• of the carboxyl•terminal domain of Semliki Forest virus replicase protein nsP2. ization of purified Sindbis virus nsP4 RNA•dependent RNA polymerase activity J. Biol. Chem. 276, 30786–30793. in vitro. Virology 384, 201–208. Vihinen, H., Ahola, T., Tuittila, M., Merits, A., Kääriäinen, L., 2001. Elimination of Rumenapf, T., Strauss, E.G., Strauss, J.H., 1995. Aura virus is a New World Represen• phosphorylation sites of Semliki Forest virus replicase protein nsP3. J. Biol. Chem. tative of Sindbis•like viruses. Virology 208, 621–633. 276, 5745–5752. Russell, R.C., 1998. Vectors vs. humans in Australia—who is on top down under? An Walton, T.E., Grayson, M.A., 1988. Venezuelan equine encephalomyelitis. In: Monath, update on vector•borne disease and research on vectors in Australia. J. Vector T.P. (Ed.), The Arboviruses: Epidemiology and Ecology. CRC Press, Boca Raton, FL, Ecol. 23, 1–46. pp. 203–231. Russo, A.T., White, M.A., Watowich, S.J., 2006. The crystal structure of the Venezuelan Wang, E., Volkova, E., Adams, A.P., Forrester, N., Xiao, S.Y., Frolov, I., Weaver, S.C., equine encephalitis alphavirus nsP2 protease. Structure 14, 1449–1458. 2008. Chimeric alphavirus vaccine candidates for chikungunya. Vaccine 26, Savarino, A., Cauda, R., Cassone, A., 2007. On the use of chloroquine for Chikungunya. 5030–5039. Lancet Infect. Dis. 7, 633. Wang, H.L., O’Rear, J., Stollar, V., 1996. Mutagenesis of the Sindbis virus nsP1 pro• Sawicki, D.L., Sawicki, S.G., 1994. Alphavirus positive and negative strand RNA syn• tein: effects on methyltransferase activity and viral infectivity. Virology 217, thesis and the role of polyproteins in formation of viral replication complexes. 527–531. Arch. Virol. 9 (Suppl.), 393–405. Wang, Y.F., Sawicki, S.G., Sawicki, D.L., 1994. Alphavirus nsP3 functions to form repli• Scheidel, L.M., Durbin, R., Stollar, V., 1987. Sindbis virus mutants resistant to cation complexes transcribing negative•strand RNA. J. Virol. 68, 6466–6475. mycophenolic acid and ribavirin. Virology 158, 1–7. Weaver, S.C., 2005. Host range, amplification and arboviral disease emergence. Arch. Scheidel, L.M., Durbin, R., Stollar, V., 1989. SVLM21, a Sindbis virus mutant resistant Virol. Suppl., 33–44. to methionine deprivation, encodes an altered methyltransferase. Virology 173, Weaver, S.C., Barrett, A.D., 2004. Transmission cycles, host range, evolution and 408–414. emergence of arboviral disease. Nat. Rev. Microbiol. 2, 789–801. Scheidel, L.M., Stollar, V., 1991. Mutations that confer resistance to mycophenolic acid Weaver, S.C., Ferro, C., Barrera, R., Boshell, J., Navarro, J.•C., 2004. Venezuelan equine and ribavirin on Sindbis virus map to the nonstructural protein nsP1. Virology encephalitis. Annu. Rev. Entomol. 49, 141–174. 181, 490–499. Weaver, S.C., Frey, T.K., Huang, H.V., Kinney, R.M., Rice, C.M., Roehrig, J.T., Shope, Schuffenecker, I., Iteman, I., Michault, A., Murri, S., Frangeul, L., Vaney, M.C., Lavenir, R.E., Strauss, E.G., 2005. Togaviridae. In: Fauquet, C.M., Mayo, M.A., Maniloff, R., Pardigon, N., Reynes, J.M., Pettinelli, F., Biscornet, L., Diancourt, L., Michel, S., J., Desselberger, U., Ball, L.A. (Eds.), Virus Taxonomy VIIIth Report of the ICTV. Duquerroy, S., Guigon, G., Frenkiel, M.P., Brehin, A.C., Cubito, N., Despres, P., Kunst, Elsevier/Academic Press, London, pp. 999–1008. F., Rey, F.A., Zeller, H., Brisse, S., 2006. Genome microevolution of chikungunya Weaver, S.C., Hagenbaugh, A., Bellew, L.A., Netesov, S.V., Volchkov, V.E., Chang, G.•J.J., viruses causing the Indian ocean outbreak. PLoS Med. 3, e263. Clarke, D.K., Gousset, L., Scott, T.W., Trent, D.W., Holland, J.J., 1993. A comparison Severson, W.E., Schmaljohn, C.S., Javadian, A., Jonsson, C.B., 2003. Ribavirin causes of the nucleotide sequences of eastern and western equine encephalomyelitis error catastrophe during Hantaan virus replication. J. Virol. 77, 481–488. viruses with those of other alphaviruses and related RNA viruses. Virology 197, Shimizu, Y., Yamamoto, S., Homma, Ishida, M., 1972. Effect of chloroquine on the 375–390. growth of animal viruses. Arch. Gesamte Virusforsch. 36, 93–104. Weaver, S.C., Kang, W., Shirako, Y., Rumenapf, T., Strauss, E.G., Strauss, J.H., Shirako, Y., Strauss, E.G., Strauss, J.H., 2003. Modification of the 5 ′ terminus of Sindbis 1997. Recombinational history and molecular evolution of western equine virus genomic RNA allows nsP4 RNA polymerases with nonaromatic amino acids encephalomyelitis complex alphaviruses. J. Virol. 71, 613–623. at the N terminus to function in RNA replication. J. Virol. 77, 2301–2309. Weaver, S.C., Salas, R., Rico•Hesse, R., Ludwig, G.V., Oberste, M.S., Boshell, J., Tesh, Shirako, Y., Strauss, J.H., 1994. Regulation of Sindbis virus RNA replication: uncleaved R.B., 1996. Re•emergence of epidemic Venezuelan equine encephalomyelitis in P123 and nsP4 function in minus•strand RNA synthesis, whereas cleaved prod• South America. Lancet 348, 436–440. ucts from P123 are required for efficient plus•strand RNA synthesis. J. Virol. 68, Young, K.C., Lindsay, K.L., Lee, K.J., Liu, W.C., Jian•Wen, H.J., Milstein, S.L., Lai, M.M., 1874–1885. 2003. Identification of a ribavirin•resistant NS5B mutation of hepatitis C virus Shull, N.P., Spinelli, S.L., Phizicky, E.M., 2005. A highly specific phosphatase that acts during ribavirin monotherapy. Hepatology 38, 869–878. on ADP•ribose 1 ′′ •phosphate, a metabolite of tRNA splicing in Saccharomyces Young, N.A., 1972. Serologic differentiation of viruses of the Venezuelan encephali• cerevisiae . Nucleic Acids Res. 33, 650–660. tis (VE) complex. In: Proceedings of the workshop•symposium on Venezuelan Singleton, M.R., Dillingham, M.S., Wigley, D.B., 2007. Structure and mechanism of encephalitis virus, scientific publication 243. Pan American Health Organization, helicases and nucleic acid translocases. Annu. Rev. Biochem. 76, 23–50. Washington, DC, pp. 84–89. Smith, C.E., Gibson, M.E., 1986. Yellow fever in South Wales. Med. Hist. 30, 322–340. Young, N.A., Johnson, K.M., 1969. Antigenic variants of Venezuelan equine encephali• Spuul, P., Salonen, A., Merits, A., Jokitalo, E., Kääriäinen, L., Ahola, T., 2007. Role of the tis virus: their geographic distribution and epidemiologic significance. Am. J. amphipathic peptide of Semliki Forest virus replicase protein nsP1 in membrane Epidemiol. 89, 286–307. association and virus replication. J. Virol. 81, 872–883. Zhang, D., Tozser, J., Waugh, D.S., 2009. Molecular cloning, overproduction, purifi• Stadler, K., Allison, S.L., Schalich, J., Heinz, F.X., 1997. Proteolytic activation of tick• cation and biochemical characterization of the p39 nsp2 protease domains borne encephalitis virus by furin. J. Virol. 71, 8475–8481. encoded by three alphaviruses. Protein Expr. Purif. 64, 89–97.

Please cite this article in press as: Gould, E.A., et al., Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.07.007 G Model AVR•2510; No. of Pages 14 ARTICLE IN PRESS

14 E.A. Gould et al. / Antiviral Research xxx (2009) xxx–xxx

Zhou, S., Liu, R., Baroudy, B.M., Malcolm, B.A., Reyes, G.R., 2003. The effect of ribavirin ence the infectivity and pathogenicity of XJ•160 virus. Arch. Virol. 154, and IMPDH inhibitors on hepatitis C virus subgenomic replicon RNA. Virology 245–253. 310, 332–342. Ziegler, S.A., Lu, L., da Rosa, A.P., Xiao, S.Y., Tesh, R.B., 2008. An animal model for Zhu, W.•Y., Yang, Y.•L., Fu, S.•H., Wang, L.•H., Zai, Y.•G., Tang, Q., Liang, G.•D., studying the pathogenesis of chikungunya virus infection. Am. J. Trop. Med. Hyg. 2009. Substitutions of 169Lys and 173Thr in nonstructural protein 1 influ• 79, 133–139.

Please cite this article in press as: Gould, E.A., et al., Understanding the alphaviruses: Recent research on important emerging pathogens and progress towards their control. Antiviral Res. (2009), doi: 10.1016/j.antiviral.2009.07.007
                                  
 
 
                                                R        
R         R                                         R  "            R          
R                #$#%&'( )            *  +       R-&. .  R /               /               R                                   .            +                     0                          R           2  3  
   R      R            /                           R$     /  R          $35         R
R         R     R   R R /        R        /       R  M                             7  "