Hepatitis E Virus RNA-Dependent RNA Polymerase Is Involved In

Hepatitis E Virus RNA-Dependent RNA Polymerase is Involved in RNA Replication and Infectious Particle Production Noémie Oechslin, Nathalie da Silva, Dagmara Szkolnicka, François-xavier Cantrelle, Xavier Hanoulle, Darius Moradpour, Jérôme Gouttenoire

To cite this version:

Noémie Oechslin, Nathalie da Silva, Dagmara Szkolnicka, François-xavier Cantrelle, Xavier Hanoulle, et al.. Hepatitis E Virus RNA-Dependent RNA Polymerase is Involved in RNA Replication and In- fectious Particle Production. Hepatology, Wiley-Blackwell, 2021, 10.1002/hep.32100. hal-03326608

HAL Id: hal-03326608 https://hal.archives-ouvertes.fr/hal-03326608 Submitted on 26 Aug 2021

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Copyright This article isprotected rights by All copyright. reserved 10.1002/HEP.32100 differencesversion and the between this throughcopyediting,typesetting, the proofreading pagination and process, whichmay lead to This article undergone has for buthas and accepted been been not publication review fullpeer the synthesis of viral RNA replicase viral the (ORF) Its worldwide. hepatitis Background [email protected] CH 48, Bugnon du Rue Vaudois, UniversitaireHospitalier Centre Hepatology, and Gastroenterology # * Keywords: 3 France 2 Lausanne 1 orsodn authors Corresponding CNRS,ERL9002 UMR1167 Division of Gastroenterology and Hepatology, The Noémie Oechslin

Accepted Article HepatitisE Virus se .

ORF1 is translated in translated is ORF1 authors 1011 Lausanne,Switzerland1011 , , Lausanne, RNA

Univ HEV| particle production| RdRp |reverse genetics| thumbsubdomain. XavierHanoulle and contributed ersity of

. 1 - Replication Aims *

The , Integrative StructuralBiology

NathalieSilva Da Switzerland

: RNA

positive

Lille, INSERM, Lille University Hospital, Institut Pasteur de Lille Hepatitis E virus(HEV)is E Hepatitis : . R

Approach equally thestudy to NA

or ais Moradpour Darius - to dependent RNA polymerase (RdRp) domain is responsible is domain (RdRp) polymerase RNA dependent 2 ,3

[email protected] , a large protein composed of composed protein large a - - DariusMoradpour DependentRNA Polymerase is Involvedin tad RNA strand

and .

Phone +41 21 314 07 49, 07 314 21+41 Phone and 1 * ABSTRACT

, Version of Record Version

InfectiousParticle DagmaraSzkolnicka

Results genome

Lausanne Unive

, , Lille,France

: 1 r éôe Gouttenoire Jérôme or one of the most common causes of acuteofmostcommonthe causesof one

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This article isprotected rights by All copyright. reserved fecal Orthohepevirus A ferret ( (FEDER),French Research Ministryand Univ the by funded were facilities NMR The as well as ( Support Financial CompetingInterest Statement: D.M.and J.G. wrote the N.O. research; performed Contributions: Author polymerase frame; reading open ORF, Abbrev RNAviruses, broader scale, bet interactions functional revealing HEV in genetics reverse of use successful the describes and protein ORF1 the of segment replication andinfectious particle production. This studyprovides structural insightsinto a key amphipathic release and/or assembly the in impaired including assembly and analys amphipathic resonance magnetic Nuclear subdomain. thumb RdRp the in located helix 1 31003A ) . Accepted(HEV) virus E Hepatitis Article

Members - s oral route and can cause large waterborne outbreaks waterborne large cause can and route oral

RdRp moe pig moose, , e s i ations: -

RNA replication 179424 revealed the .



 palm  is - French Agency for Research on AIDS and Viral Hepatitis - f t of it heli helix in the thumb the in helix

also involvedinproduction. virus aa, amino acid; amino aa, demonstrate

to DM and .

his x extending from amino acid sub

HEV : da rl o th of role dual a

hs ok a supported was work This

domain. family and

genotypes (gt) 1 and 2 are transmitted from humans to humans by the release manuscript;all authors reviewedmanuscript the All authors All . andresulted in the selection of

NDS, F. N.D.S., , Conclusions is NMR, nuclear magnetic resonance; magnetic nuclear NMR, CRSK human net diverse infect s a

that Other mutations Other . positive M gt, genotype gt,

Theauthors declare competingno interests. sub - utations on the hydrophobic side of the amphipathic 3_190706 s the

designed research; N.O., N.D.S., F. N.D.S., N.O., research; designed .

is INTRODUCTION en h RR tub n pl subdomains. palm and thumb RdRp the ween domain of the HEV RdRp HEV the of domain - ot ua ptoei sris eog o species to belong strains pathogenic human Most X.C., X.H., D.M. and J.G. analyzed data; N.O., X.H., N.O., data; analyzed J.G. and D.M. X.H., X.C.,

HEV replicase, similar to those of other positive - ei i HV N replication RNA HEV in helix strand RNA strand :

Nor tructure ot icuig aog tes fs, bird fish, others, among including, hosts

; ersity of ersity to to JG s

HCV, hepatitis C virus; C hepatitis HCV, th

enhanced 162 by

ein oni, NS Erpa Union European CNRS, Council, Region )

, ucin nlss dniid conserved a identified analyses function 8 h Sis ainl cec Foundation Science National Swiss the virus cla virus the Novartis Foundation (

to Lille.

164 a

RNA replication RNA second

ssified in the the in ssified

in resource in of the ORF1 protein

RdRp, RNA RdRp, with a dual role in viral RNA viral in role dual a with - site H

compensatorychange and (ECTZ101316 to E . - -

X.C., X.H. and J.G. and X.H. X.C., V, hepatitis V, limited settings limited

Hepeviridae

but impaired but demonstrated - production virus dependent RNA dependent

18C140 to DM) .

Functional s E - 

bat , strand

family

n a On virus; - virus helix XH).

(

2 an s ) . , , ,

This article isprotected rights by All copyright. reserved predicted a and thumb of subdomain palm selecta in element targeting the conserved residues and functional analyses revealed a dual role of this structural amphipathic studytook advantage of variantthis establish to arobust HEV cell culture system G1634R, RdRp the mapped for enzymatic the constituting RdRp of activity catalytic the three famil demonstrated in vitro as length and subgenomic (RdRp) polymerase a and of debate are domains primarily defined secretion virion corresponding the to protein (i) into translated are which individuals and manifestations,extrahepatic other as well as neurologic 3 a of undercooked HEV gt 3 and 4 represent a primarily porcine

major cause of acute hepatitis and methyltransferase, a tigr acute trigger can HEV has a 7.2 n h peet td, we study, present the In The

Accepteda Article the the ies template

distinctsubdomains

b and a high affinity of this domain for the 3’ non HEV RdRp has been poorly studied , it is, hypervariable le

( displays , 7 subgenomic replic subgenomic identified leadingto , the E RNA HEV   expected 9 processed into individual proteins or act as one large polyproteinlarge one as processedact individualintoor proteins

- forpositive ) helix in the the in helix . (

11 HEV RdRpHEVh by While While meat products ( 7

- kb positive -

n the on ) 9 . infcnl increase significantly

) the ( B - . n patients in 10

the functions of the chronic The sd on ased a uncertain are region that  positive replication )  - RdRp . hoi lvr alr i ptet wt pexsig iroi, cause cirrhosis, preexisting with patients in failure liver chronic

- macro - , namely, strand RNAsynthesis The ai o s of basis helix in the palm subdomains palm the in helix ORF1 it core thumb

- the ORF1 protein representing protein ORF1 the adopts capsid strand RNA on s hepatitis , revealing a functional interaction between the between interaction functional a revealing ,

, RdRp also in resource -

omology referred strand h hg cnevto of conservation high the we could we region of the polymerase. the of region ih hoi hptts E hepatitis chronic with domain

report protein is composed of multiple domains multiple of composed is protein sub the and ,

a closed

qec hmlg wt ohr viruses other with homology equence and (iii) and a growing global health concern catalyze oan f h HV RdR HEV the of domain

palm,thumb andConservedfingers. involvedmotifs in RNAs

with potentialrapid evolution

, iu production. virus d - to h three the based analysbased genome encoding three open reading frames (ORF), Y

an identify oyeae activity polymerase , other ,

thus domain, as zoonosis ORF3, as well as " s N helicase RNA righthand - A to D, reside in the central palm subdomain palm central the in reside D, to A

rich settings

. the synthesis of viral RNA viral of synthesis the far

domains a . . P -

dimensional structure of of structure dimensional the compensatory a small a -

olymerase activity of recombinant RdRp coding region of the genome and e

the the negative putative s .

By homology By ( three

n rbvrn ramn failure treatment ribavirin and are 10 oevr tkn avnae f a of advantage taking Moreover,

hs nye hogot l virus all throughout enzyme this have a have . Hence, HEV , These motifs have been have motifs These

the viral replicase, (ii) the the (ii) replicase, viral the palmitoylatedprotein , persist in immunocompromised in persist and transmitted by 12 - ( dimensionalorganization p 13 papain ) . . , Site Interestingly,a definedfunction an second -

strand 14 to worldwide

- ) RNA cirrhosis - modelling ietd mutagenesis directed . like cysteine protease hrfr, recent a Therefore, infection , -

RNA

site change in the in change site - including the full the including which have been have which . eedn RNA dependent the the W

is still a matter a stillis 

 conserved a

( ( ehr these hether which serves consumption 6 3 - with (

helix in the the in helix

) - mutation inmutation 15 involved in represent have been . 5 , including ,

) . )

partially

. HEV

RdRp ORF2 i.e. , with gt s s -

This article isprotected rights by All copyright. reserved Accepted Article production. subdomain subdomain thumb the in identified positive other of

and thatit mayregulatethe - tad N vrss or idns ugs ta te amphipathic the that suggest findings our viruses, RNA strand

s ey ls t te rdce first predicted the to close very is act ivity the of HEVRdRp

in RNA repli  - ei o te palm the of helix cation and  - virus helix This article isprotected rights by All copyright. reserved the GraphPadPrism 6 software package (GraphPad Software). Berthold additionof at stored and HEV83 with transfected cells RainerUlrichG. post Eagle medium supplemented with 2% low (200 extracellular total the form x were subjected to three freeze extra the in infectivity determine full transcribed are Material. study 10%bovine fetal serum. (ATCC Emerson,National Institutesof Health, Bethesda, MD) and HepG2/C3A hepatoblastoma cell g Nuclear magnetic resonance ( resonancemagnetic Nuclear Plasmids lines. Cell AcceptedStatisticalanalyses. Luciferase assays. ArticleVirusdetermination infectionand ofinfectivity.

described - ation to prepare the intracellular sample. Infectivity was determined in duplicate by focus focus by duplicate in determined was Infectivity sample. intracellular the prepare to inoculationand stained usinganti an as well as

CRL

luminometer

assay after inoculation of HepG2/C3Aofafterinoculation 24 assay a into seededcells

60 - 10741 ,

µ in vitro in in - S10 - 20°C until 20°C L egh E RA clue uentns n S10 and supernatants culture RNA, HEV length

( the coelenterazinesubstrate ( Friedrich LoefflerInstitute, vitro - ) were maintained in Dulbecco 3 human 3 SupplementaryMaterial

(Bad Germany).Wildbad,

transcription and RNA transfection RNA and transcription

transcription and RNA transfection are described Gaussia

Significancevalues were calculated by using theunpaired ttestwith measurement  µ ) r intracellular or L) - hepatocellular carcinoma hepatocellular 2_Gluc MATERIALS ANDMATERIALS METHODS - and

luciferase activity was measured in culture supernatant -

- and intracellular compartments, respectively. Cell pellets pellets Cell respectively. compartments, intracellular and NMR thaw cycles, followed by centrifugati - derived constructs. Culture medium was collected was medium Culture constructs.derived . Luciferase activity was measured in duplicate after duplicate in measured was activity Luciferase . ) structur ) - - IgG fetal bovine serum ORF2 rabbitpolyclonal antibody kindly provided by 0.8 Greifswald, .

µ (50

M) ’ s modified Eagle medium supplemented with al

µ to 10to

L) virus sample and culture in modified in culture and sample virus L) analys

Five days after transfectionFivedaysafter with cell

Germany). µ L s

ofculture medium for5 s using a (kindly provided by Suzanne U. Suzanne by provided (kindly . s.

All constructs All

The The ( - 15 cls ee avse to harvested were cells 3

) NMR structural analyses NMRstructural . C - on for 15 well plate with 1/10 of1/10 with wellplate ells were fixed 5 days in the the prepar Supplementary min ed in this in ed at 3,000 in vitroin s

daily from s

This article isprotected rights by All copyright. reserved residues of structure representative the for howeverpeptideextremitieshighlyare of flexible the the (Fig. 2C). 1644 to 1628 best revealed an structurepropensity coil for values random shift the chemical and experimental the between difference the to corresponding dCB and S5 ( trifluoroethanol peptide 1647] NMR variant C E Gly Hepeviridae a with virusesstrand to the thumb prediction in the palm (Fig. solved 1703 conserved (Fig. 1) especially prediction structure secondary i.e. Alignment of the RdRp a onservation of a of onservation and

) The Acceptedof structure NMR Article segment . HEV 1634, which

13 a ) conformers .

S3)

n aa n C secondary NMR chemical shifts (dCA , Tested in different conditions, different in Tested ribavirin treatment

n ohr positive other and G1634R prompted us to to us prompted G1634R thumb e gt Pro ,

The per g.

of ofrs h pstos f motifs of positions the confirms 1 sequence



family members family 1630 subdomain, 1628 to Gly and , palm owl virus Norwalk -

produced in produced helical fold of the peptide spanning from Pro (Fig as reveal as sub TFE has been found to be obtained 3 - - . n residue backbone 1645

sub as well as A

domain of domain S  of the peptideat each position (Fig ) lignment of the , 3 - subdomain identity

ORF1 helix prediction throughout the the throughout prediction helix

) doma allowing determination of its of determination allowing sequences from phylogenetically distant

ed but but

shows

1644 is predicted to display failure - tad N vrss o wih a which for viruses RNA strand 13

show E. by

C appears

in motifs in

protein ( avian,  of coli despite Caliciviridae clearly exhibits an amp the nearly the s

the

a strong a

ed (G1634Rvariant) and only revealed

solve (16) yielded

a regular a ORF1

HEV RdRp sequence ferret, ferret, bat, moose and

to be to root 13

a 30 . segment

A relatively

substituted by arginine T C the 15 - - he C  D 50%

N and N RESULTS - and conserved and

RdRp perfect , [1 mean

well

conserved within the the withinconserved acid amino where structu respectively

622 family; high -

terminal region of  - (Fig - A resolved NMR spectra spectra NMRresolved dCB)

- - a structure helical conformation extending from extending conformation helical - 1622 13 low 1647 square deviation (RMSD) values comprises

with D backbone C doubly labelled doubly C degree of structural of degree , s. re

Norovirus

aa three 1 s

obtained for of - hipathic character ,te eta pr o te ei including helix the of part central the ], located

1647

from and Hepeviridae

sre t dtrie h s the determine to served , .

ORF1 sequence conservation. sequence

2A epc t te eodr structure secondary the to respect prediction for a for prediction (Fig. 2D) (Fig. -

dimensional structure dimensional

different trout S

1628 to Gly HEV gt

reveals an reveals

super and 2 a

polymerase

at end the of t

in ) (aa) protein . conserved genus the

Within Within

HEV (Fig. S1) patients with chronic hepatitis Hepeviridae Hepeviridae B). Thelatter analysis clearly

ORF1[1622 impo

sequences are also highly also are sequences 3 (83 HEV RdRp from family and the location of location the and family As illustrated in FigillustratedinAs recombinant ; in the presence of 20% of presence the in segment conservation sition

th Ast6139/01/Sp 1644 (Fig with amphipathic - that n e 2 tutr hs been has structure

-  thumb subdomain thumb predicted 27 hissegment most -

with

- heli family of family

1647] strain; aa , ,

combined with corresponding other 1622

cal fold in all in fold cal in th in . Interestingly (Fig. S4 (Fig. ORF1[1622 hydrophobic

showed that 2B). The ,

, , with dCA members

(Fig. S2) (Fig. econdary however, - is residues  positive 1647  

ure - - . region strain 1115 helix heli

and

2 20 by x E - - - ) . , , , , .

This article isprotected rights by All copyright. reserved they provided a replication advantage in the subgenomic replicon context (Fig. 3) infectivities. full replication, consequence functional replication hydrophilic demonstrate they residues conserved capacity replication the G_R, Chim_ferretwas Mut and D_A Mut alike from constructs were prepared by swapping conservation, sequence aa to compared as structural of importance the explore To in fold charged residue Asp by replication Ala of and alanine by comparable Arg amphipathiccharacter of residue which satisfies the physicochemicalproperties of this exception of Ala subs were residues luciferase Gaussia of expression the allowing replicon subgenomic 164 HEV the in and buffer aqueouspresenceof TFE. in peptide the of behaviors observed the by supported further as as well as the the HEV gt

The conserved hydrophobic face of face hydrophobic conserved The R1 protein ORF1 Taken Alanine

Accepted Article side opposite the on residues polar

4 (Fig 1635 (Ins +1A) (Ins 1635

, moose ( RNA replication

a . panel of panel

3A)

oehr tee results these together, . neutral

usiuin f Leu of substitution

face to As shown in Figure 4, Figure in shown As . GenBank accession number

3 T - those of bu 30 about length HEV genomes were prepared and analysed for intra for analysed and prepared were genomes HEV length his phenotype resembles the onephenotypehis resembles the Kernow

, 1637

increased

mutations targeting the strictly conserved residues conserved strictly the targeting mutations

residues that some mutations within this segment this within mutations some that nldn te aual ocrn vrat 13R ehne E RNA HEV enhance G1634R, variant occuring naturally the including  on - t

strongly impaired strongly 1640 by ituted ei 162 helix

. which was substituted by a

In order to characterize the functional role of the - its hydrophobic face for HEV RNA replication (Fig replication RNA HEV for face hydrophobic its - C1 p6 67 y ecn ( leucine by 1637 s od Srrsnl, lnn sbttto o the of substitution alanine Surprisingly, fold. 

(Mut D_A - n iu pouto o te muta the of production virus on replication exposed on as compared helix

clone 1634 8 alanine

- 1628 164 ofr te motne of importance the confirm all of all ) ) did

( 4 - Mt _) r Phe or L_A) (Mut 13 - deficient

164 , HEV helix the of pa one is involved in in involved is ) which

not not to wildto the and confirmed here in the 83 these rt of KF951328)

4 u RA n Mt A_L Mut and R_A Mut ORF1 protein ORF1

side byof alanineinsertion an between Leu RNA replication RNA impair but increased the control

should mutations (upper part of leucine (Mut A_L), a HEV gt of

(lower part) (lower - typeconstruct variantG1634R

GAD preserve

iu production virus 1641 ferret

 reduced

162 (Fig. 3A) - , especially , , f hmrs hmmoe and Chim_moose chimeras of helix

tions face as shown by luciferase values luciferase by shown as HEV Mt F_A) (Mut .  the panel

8 This amphipathic character ischaracteramphipathic This -

- helix 164 the replication

ofthe 162

(Fig respectively , amphipathic (JN998607) virus produ virus not do that . , as , (17) 4

Substitution of Arg  was - larger 162 8 segment with the one . 3 - osre negatively conserved - eia fold helical )

3A) those those - 164 previously

 27 clone and (Fig. 3) (Fig. r d or

. 8 -

. prepared in a in prepared helix - Furthermore, 3B). - .

164 4 To evaluate the the evaluate To and hydrophobic and extracellularand approximately

charged isruption of the the of isruption is involved in in involved is located on the on located ction . . . Indeed, total 4 

Inter ) . Conserved . impair

(Mut G_R) helix and of the the of and decreased

described

1634and although chimeric with the the with e stingly, as well

162

1630 RNA gt

2 8 3 s - - .

This article isprotected rights by All copyright. reserved restore the to response in mutation compensatory Therefore, itself wild the to compared ascapacity replication substitution E1451K the by compensated le alone R_A Mut with combination in or alone replicon construct preceding motifA appearance 1370 3A) (Fig. assays replication transient ( showed of selection yield not did F_A Mut and L_A cassette RNA replication, were introduced into a subgenomic replicon harboring a neomycin resistance palm assemblyas well as and S6).(Fig. Mut G_R did not show any fold particle release,reveal intra type, suggesting infectivityat measured 7.4 Taken together, these results demonstrate that the ORF1 protein, i.e. the viral replicase viral the i.e. protein, ORF1 the that demonstrate results these together, Taken AcceptedTo evaluate To ArticleReverse genetics indicate a functional interaction between the HEV RdRp thumb and two ,

(Fig - x -

doesnot

oe specifically more 1703 sub 10 n etaellr infectivities extracellular and confirm virus production virus positions . a reduced efficiency reduced a 4

(17)

yielded

4 domains vs

). the from thefrom

G418

. of of A

2.5

in order to select for second for select to order in s expected, a expected, s the replication advantage replication the fully whether E1451K a

impaired assembly larger larger foci than infectious particle x in thethumb andpalm Glu s - d ( resistant

. 10 compensate expressing a luciferase reporter and to a to Mutations Mut R_A, Mut L_A, Mut A_L and Mut G418 1451 the the - 5 to

ed , clones per µg RNA, respectively) d

- n approximately 30 approximately n adaptive change usiuin nte dp am udmi my e osdrd s a as considered be may subdomain palm RdRp the in substitution Lys release ofinfectious particle of Mut R_A Mut of h RR domain RdRp the 5 post 5

K that secretionthat - resistant ;

clones

seeFig. 1

subs full Mut of colony formation colony of - any - transfection

for the the other length construct length t R_A itu

after

secretion , the 30 the cell populationcell of infectious virus G418 , tio

hc poie a uniaie vlain f infectious of evaluation quantitative a provides which . the corresponding the )

.No compensatorychange was identified for n S were Mut R_A Mut 1 of

subdomains equencing of equencing E1451K in the in conveyed 3 - -

mutants

MutG_R (Mut R_A/Palm E_K) R_A/Palm (Mut cells, resistant fold decrease fold - - days site , fold -

was

type was observed for observed was type prepared and assayed. and prepared ( s involved is 8

) first

compensatory

decrease

of antibiotictreatmentof

, indicating a functional interaction between interaction functional a indicating ,

Interestingly, we observe at least at in the palm subdomain ( with a mutated a with , , indicating more efficient cell of Mut R_A Mut of

revealed

by predicted and s .

. (Fig

the the the the

harboring the

(Fig. 5B) Chim

substitutions were introduced into a into introduced were substitutions observedreplicationin 40% . Mut A_L and A_L Mut n iu production virus in in RNA replication RNA in

region E1451K 4 the ). Furthermore, the ratio between

_  lower

changes moose ismoose as compared to the wild the to compared as - . retentionandMuttheR_A of helix of the of helix

, ORF3 An corresponding to corresponding in line with the results from

F_A, which impaired HEV Palm substitution as compared to the to compared as about 3 about As shown in Figure 5C, Figure in shown As substitution .

Crystal violet stainingCrystalviolet

Mut R_A Mut start codon start (Fig. 5A) (Fig. reduc

Palm E_K alone E_K d

that infection palm subdomain palm -

fold which was fully fully was which ,

ed - E_K , subgenomic , to

including for

. - allowed ( by2 about increase in increase

cell spread Palm While Mut While

) Mut R_ Mut RdRp MutA_L.

(

can also which  ORF3)

- E_K) wild type with

the the aa A in - - .

This article isprotected rights by All copyright. reserved recent predicted proximity close likely and production particle infectious in RNAessential replication for to 1628 RdRp the of subdomain amphipathic in interaction surface the core structure of the enzyme. This implies that the less conserved hydrophilic side may be as its amphipathic character suggest that the hydrophobic side of the (Fig the to we can structure RdRp wherenucleic the acid is synthesizedtheon basis of its template. chanaofformation contribute enzyme the theactiveofand to completely the encircle site viruses between ( subdomain structura HEVproduction. of importance the confirm (Fig the E_K 5D, Figure in shown As full 18 - C AcceptedS functionally and structurally we study, present the In Article ) length HEV genome and genome HEV length combination of these mutations (Mutmutations theseofcombination . . . tructural information tructural onserved

showed 6) 5 However,

first C crystal structure crystal . - predict

), show ), exposed, with residue Asp The contribution of the conserved residues of this of residues conserved the of contribution The 164

l   

lmn regulating element is known - -  4 helix

 helix of the palm subdomainpalmhelix the of s a 2 a .

with other partners. helix in the C that s osre rsde o h hdohbc ie f hs ei wr fud o be to found were helix this of side hydrophobic the on residues Conserved ed  dp so a overall an show RdRps , notably , - ,

fold decrease fold -

in the palmsubdomain

helix only ih neatos ewe te igr ad hm sboan which subdomains thumb and fingers the between interactions with a to be the mphipathic

very of th of .

poorly replicating Mut R_A Mut replicating poorly 162 for HEV for NMR the one from one the ewe the between c - onserved e terminal region of 8 poor while r

analysed RdRp region with - putative 164 . analyses revealed an amphipathic an revealed analyses

 Reverse genetics allowed to identify a functional interaction functional a identify to allowed genetics Reverse HEV

, -

capacity to produce infectious virus. These findings further findings These virus. infectious produce to capacity

4 total helix 1640 and in particular for the ORF1 protein ORF1 the for particular in and esidues on

n h RdRp the in  N rpiain n vrs production. virus and replication RNA DISCUSSION papain for Norwalk virus Norwalk

infectiv , 162 -

amphipathic . helix in R_A/

three inresideswhich the

volved intra 8 - 164 - HEV Palm 162 like cysteine protease cysteine like it

- - the hydrophilic the highest dimensional y as compared to the wild the to compared as y

and extracellula and 4 in 8

ORF1 protein in -

virus assembly E_K), known to replicate as replicate to knownE_K), 164  did not yield infectious virus infectious yield not did thumb

 thethumb that we used as template as used we that

- helix 4

structural variability 

raiain ht s ey similar very is that organisation the the - n the in s helix to R to helix E1451K side ubdomain characterized subdomain r in r RdRp , more precisely in the thumb

Hence, ,



werefound being fectious virus production virus fectious thumb  -

helix extending from aa from extending helix domain revealed a high a revealed domain -

helix is oriented toward compensatory N thumb subdomain thumb A replication as well as replication A potentially engaged in respectto -

, is very limited very is , type subdomain s n important an is

islocated closely

among . Interesting . to to conserved a in this study, this in the wildthe

be involvedbe h t The while

change

viruses

known and humb Palm - type . nel for l y, A a .

This article isprotected rights by All copyright. reserved subdomain palm wehypothesize 162 cellular membranes or proteins Amphipathic helices are usually engaged in hydrophobic interaction polymeraseto maintain efficientcatalytic activity. interactions electrostatic th of compensate likely which R_A) (Mut maybenefit thisfrom functional a indicating R_A), (Mut subdomain subdomain s ourknowledge, to time first the for applied,we protein structural proteins nonstructural and structural between proteins, viralbetween links functionalof mapping the as well as changes adaptive in advances rapid a of development the a them, of study allowing agent of determinants However production analyses segment studies. domain domains binding acid fatty to homology structural e lectable V AcceptedInter a ofsubstitution Strikingly, Article 2 iral evolution iral e twoe

to to 164 s.

A a not has remarkable example remarkable stingly, the stingly, , diverse the loe u to us allowed mphipathic f h OF protein ORF1 the of

 ,

subgenomic s el s fntoa itrcin ewe te hm ad am subdomains. palm and thumb the between interaction functional a as well as 7 - led to the appearance of a positively charged residue, i.e. Lys at position at Lys i.e. residue, charged positively a of appearance the to led Pl E_K) (Palm

helicesinthe

dniiain of identification complete segment

thatthe hydrophobicof theside , nyai activity enzymatic

positive yet 5A inhibitors as potent antivirals for the treatment of hepatitis C hepatitis of treatment the for antivirals potent as inhibitors 5A can hl te yrpii sd a b sur be may side hydrophilic the while the

identified en prove been  approach

serve to to serve selectable subgenomic replicon system replicon subgenomic selectable or of the understanding -

helix 1628 tutr of structure replicon s for s dniy da rl o te E RR in RdRp HEV the of role dual a identify - n epne o mtto itoue i the in introduced mutation a to response in strand viruses strand three ewe tee se these between

second the loss of loss the is  conserved positively charged residue charged positivelyconserved

. . identify mutations which mutationsidentify entire Although . -

- we , helix As we the dimensional n - th , 164

and hepatitis C virus (HCV) virus C hepatitis

- the RdRp domain with cellular membranes (data not shown), in site e dniid a identified the of 4

did

, e.g. , structure the thumb subdomain ha subdomain thumb the

te functions other described here positive E RdRp HEV compensatory not not observe an ersnig short a representing virus life cycle by the identification of cell culture cell of identification the by cycle life virus

structure

alpha and likely structural interaction structural likely and

reverse genetics to the study of HEV studyof the to genetics reverse g (  ments

charge. Given the Given charge. provide opnaoy mutation compensatory - helixfaces - ) , (19) .

flavi n diin i alwd o dniy non identify to allowed it addition, In

domain ofRdRpthe ,

favor favor

stabilize the conformation of the the of conformation the stabilize is s el as well as face changes s - y While While

only the second or picorna or interaction of the a

and the opportunities offered by offered opportunities the and -

rmwr f framework virus fitness. virus the exposed. (23, 24) (23, will an

s , lmn, structure element, core of the enzyme,coreof the i.e. the

N rpiain n virus and replication RNA

an s have been applied to to applied been have (Fig. 6)(Fig.

, i.e. Arg i.e. , e require be nyai activity enzymatic predicted

o design to with viruses amphipathic .   This system This This other - or ei i te thumb the in helix , it isplausibleit,

n h RdRp the in Reverse geneticsReverse structurally solved

ORF1 protein future at position 1630 position at (20 . implies that implies

close proximity close d Future studies Future direct element

- o a the map to 22) ( 26 character functional . Using . including

- . enabled function antiviral ) Among . of of s

1451, Here, , , e.g. palm

that this the the the the aa a - . ,

This article isprotected rights by All copyright. reserved and replication, several hypothesis, this the production virus raise production virus and replication RNA between equilibrium an helix virus assembly enhanc greatly whichNS5B two of selection to led the viral capsid to believed n example, For be to shown been have viruses of number increasing an of state of the enzyme the of multimerization for centralits region, suchas the factor subdomain human factor host a to compared as likely species. animal diverses encapsidation in involved be molecular mechanism residue of and in involved subdomain,the most conserved region of the of side replicatio RNA in involved residues conserved absolutely ae o tee observations these on Based AcceptedS Tak The t Article

HEV

urprisingly, w urprisingly, G1634R

1628 the possibility that that possibility the en s humb subdomain .

polymerase activity

C together -

164 , ontribution 

virus production virus including contribute to virus assembly by assembly virus to contribute to

- in theRdRp helix .

The a Asp (reviewed in iu assembly virus (

onstructural 30 increasing RNA replication to pcfc protein specific e did not only annot observe did e and

, , i.e. Arg i.e. , ORF2protein.

)

the functional collecteddata in studythis maintain . Hepeviridae determines 1640

insertion

a plausiblepartner interaction of connecting of changes the the the the aual occu naturally throughout all throughout of the RdRp from Hence, (reviewedin polymerase

, ed ( Leu 1630,

has the poten HEV RdRp HEV 29 efficient viral propagation viral efficient protein would be surface be would HEV s

specific infspecific

) its contribution to

a, hrfr, e eitd hog itrcin ih viral a with interaction through mediated be therefore, may, )

in the the in represent a family of viruses . - in the viroporin p7 and the the and p7 viroporin the in ,

Indeed,t rti itrcin ih viral a with interaction protein Moreover, cell culture adaptation of interaction of residue Asp residue of interaction RNA synthesis and Asp RdRp

which would have to be conserved be to have would which A a esnil opnn o the of component essential an 5A, . It .

hypervariable region hypervariable (

1634, Ala 1634, 31 1640, rring Hepeviridae regulates the regulates , and especially of of especially and , is ectivity, revealing a direct role of role direct a revealingectivity, the the ) tial he capsid protein is highlyisespeciallyconserved,capsid protein he in

). other vir effect also affect

transferring

to to uain hv be fud o nac RNA enhance to found been have mutations RdRp hc is which - exposed. be enhanced

plausible

virus production.

1637 of mutations in theofin mutations virus production (Fig. S7). HEV has to keep u .

ses, including family members family virus as

. Furthermore, our results our Furthermore, .

balance the the and

located newly , therefore, , Conservation n and located on the hydrophobic the on located and n RdRp

Phe

as well as well as demonstrate thatmutations  with involved in virus assembly virus in involved fingers subdomain of the RdRp the of subdomain fingers 1640 with a viral factor is more is factor viral a with 1640

s by genetic changes -

embly Or observation Our . helix synthesized RNA genome RNA synthesized between RNA replication and replication RNA between

n h hdohlc side hydrophilic the on

1641 or host factor host or inviral RNA encapsidation

a T poliovirus broad h he replication machiner 1628

.  an that the oli the that , R point

- interact helix esidue substitutions of of

HCV from fish or bird or fish from - 164  ost range s HCV the polymerase the

within toward a toward helix (

27 infectious clone 4

Asp

with the palm the with

to to ) in the thumb the in go replicase , therefore, s, indicate , .

is Supporting 1628

favor

merization the thumb the 1640

essential infecting Y1320H shared -

164

RNA ( in may 28 s and and that s is , ies

 to to in . ) 4

- .

This article isprotected rights by All copyright. reserved production aswell as subdomain may ribavirin treatment and the lack of direct antiviral the curren to respond to fail who E hepatitis chronic direct importance the production,releaseassemblyincludingand particle demonstrates spread have to address the mechanism(s) by which implication important study our in as well as their in observed as al also to compared p6 with the G1634R mutation hypothesis. varianta of selection likely to patients in pressure selective ( isolates 3 inposition this in ribavirin treatment failure the additionin secretion not impair RNA replication subdomain 32 .

o conclude, To Tod

Accepted( Article or ) s 15

G1634R significant election of viral genomes with improved fitness, e.g. affect virus assembl virusaffect and

otiue to contribute 83 antivirals ) t .

maybe a consequence of the et al induce -

its ( - . 13 27

on . S d variant recently mutagenic )

. uch that the viral replicase has a dual role in viral RNA replication and infectious and replication RNA viral in role dual a has replicase viral the that infectious

. . In the case of HEV infection, virus assembly but also but assembly virus 7 mutations of the genome which favor replication intracellular of The antiviral effect of r of effectantiviral The

in the study in Todt by the most

u study our functionallyand structurally

a dru to to

s selected withincreased fitness taey a alw o nefr wt bt vrl N rpiain and replication RNA viral both with interfere to allow may strategy dev (

dsg treig th targeting design g 15 Hepeviridae for the for reduced . . Interestingly, a positively charged residue, either Arg clone y. properties ) eloped eloped a robust infectio . , , i.e. Mut

In Hence, th Hence, accumulation of virus of accumulation

provide

accordance with chronic hepatitis E hepatitis chronic with s in understand

( infectious virus production. Of note, Mut G_R correspondsMutnote,virusproduction.G_R to infectious Of 15 immunocompromised

G_R and Chim ) familymembers . ( s RNAreplicationcapacity

14 e increase in intracellular infectivityobservedintracellular in increase e et al et By measuring By

more ibavirin the , on 31

ing of ing .), with our study

particle first efficient )

. a mutation in the RdRp domain we could showwecould is there is an unmet need

(Fig. S6) (Fig. characterizing eas HV os o fc srn immune strong face not does HEV Because

is mediated is

conserved agent t structural ribavirin n

treatmen harboring the harboring

system using secretion .

moose, showed

cell buta glycineispresent in 80%ofHEV gt infectivity Lessons from other virusesotherLessonsfrom s to treat chronic hepatitis E , . suboptimal - ains ih hoi hptts and E hepatitis chronic with patients ,

to harboring

While th While they treatment failure, treatment

- t. through cell spread  insight

that - Hence, t Hence, viral the polymerase (Fig. 4) (Fig. helix of the the of helix , i.e. G1634R,i.e. , observed a at an at

to escape drug pressure

G1634R HEV the e

s the the ribavirin in depletion of depletion e

especially a earlier G1634R .

aking into consideration into aking observation gt gt 3 Two mutants Two

2 G1634R variant, during of the G1634R

- fold decrease in virus h HV RdRp HEV the

n impaired secretion change genomes harboring E RdRp HEV

future studies will studies future time point time is treatment

is in is favors substitution

or Lys, is found in patients with

the

haveproven line s s in o

todevelop

ur findings

cell Todtby may have may GTP pool GTP which did which either with this withthis

variant ( - d thumb to . more

- T and can cell the he as et ,

This article isprotected rights by All copyright. reserved (C conformers to correspond for propensity of presence the indicate values for values shift chemical coil random the 1622 (dCA shifts chemical NMR secondary 2. Figure sequence from is subdomain fully background blue and c grey yellow, light by denoted are subdomains inferredconvention (asterisk, invariant; colon, highly similar; dot, be similar). can and line bottom the on are above SOPM PREDATOR, SIMPA96, Cons.) members family Amino acid of analysis Sequence Figure whose passionvirology for inspired many. ImagingFacility theUniversityof Lausanne of Takaji Wakita onserved RdRp motifs A motifs RdRp onserved

Accepted Articleand scientist wonderful a Kunz, Stefan of memory the to dedicated is article This acknowledgegratefully authors The

not indicated not osre residues conserved - - 1647 dCB)

the alignment ( alignment the for 1 .

ORF1 protein amino acid proteinamino ORF1 A con A all sequences all ,with dCAand dCB are shown (aa) for reagents. highlighted by a black a byhighlighted   ORF1[1622 HEV gt

sequence traces) of of traces) s helix and and helix . (see

erved T he degree of aa physicochemical conservation at each position is shown is position each at conservationphysicochemical aa of degree he along the h, details in details

the 3strain

  was highli -

- - 

ORF1[1622 alignment D RdRp helix is predicted in the in predicted is helix helix; - Confocal laser scanning microscopy was - 1647] sheet, respectively.sheet, in

determined

ghted corresponding tothe difference between the experimentaland 83 the palm subdomain are indicated by colored by indicated are subdomain palm the peptide sequence Fig. S Fig.

ACKNOWLEDGMENTS e, n PHD and  of of domain . - 2 - oiie bak as ad eaie wie as values bars) (white negative and bars) (black Positive ei and helix FIGURE - extended strand extended ORF1[1622 27 (

square s of the RdR in the same color. The The color. same the in

- 2 162 1647] Suzanne U. Emerson, U. Suzanne ). AB740232).

13 on the basis of basis the on c A 8 from C . - available

the HEV open reading frame 1 ( 1 frame reading open HEV the 164

N  that have bee have that LEGENDS . onsensus secondary structure prediction structure secondary onsensus 

umbering - and

p he, respectively. sheet, - 4 1647] (C) h smlrt idx codn t ClustalW to according index similarity the

domains

corresponding to fold into an into fold

13 ). Random coil or coil Random ).

Superimposition of the 20 lowest energylowest 20 the ofSuperimposition

thumb subdomain thumb C at . The secondary NMR chemical shifts chemical NMR secondary The . 

, respectively. Positive and negative and Positive respectively. , of aa of https://npsa

the

from n generated by CS bygenerated n

algorithms

predicted Koji Ishii Koji is amphipathic a

The fingers, palm and thumb relative to the ORF1 protein ORF1 the to relative broad range ORF1

performed at the Cellular (B) - prabi.ibcp.fr discrepant prediction discrepant , Rainer G. Ulrich G. Rainer ,

 , respectively s, eodr structure Secondary DSC - protein of helix the  , GOR IV, HNN IV, GOR , ORF

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osetta Hepeviridae RdRp HEV amino acids as ,

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protein. (A) shown friend,

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This article isprotected rights by All copyright. reserved differences comparedto wt Statistical construct. each for 1 to normalized infectivity panel).(left experimentsindependent three of titrations two least at in determined SD) ± mean (ffu; units determined at protein 3 frame reading open produce respectively. controls, negative and Fig. replication carcinoma hepatocellular 4. Figure (right). profilein or (left)side theeither from presentedand top onhydrophilic andsidebeneath side basic orange, are ORF1 of model structure all among conserved are that residues HeliQuest by shown the denotes wt RdRp. the par of motif GDD the in substitution alanine an harboring replicon ± SD) indicated subgenomic in replication. RNA HEV in F ( scale hydrophobicity White and Wimley the to according colored is which structure, energy C C (N,atoms backbone the 1 33 H and igure .

 Accepted Article(E) na wild ental )

- hw wt ter aea can and chain lateral their with shown 3). ranging ei 1628 helix

determined at d 1, 2 and 3 post The amphipathic feature of the

on the right 3 13 A . C NMR chemical shifts.

in parental The conserved hydrophobic face of ORF1 protein ORF1 protein ORF1 - competent

The ratio ofe ratio The the sequence from hydrophilicfrom -

E rpios abrn a asi lcfrs rpre ad h mutations the and reporter luciferase Gaussia a harboring replicons HEV ye replicon. type ( d in blue in https://heliquest.ipmc.cnrs.fr)

- are 5 post 1644. , with the wild denoted

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by ) - (blue

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deviation (RMSD) values (Å) for

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HEV and on (Mann

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are

This article isprotected rights by All copyright. reserved amphipathic identified in shown Ast6139/01/Sp bou (RdR polymerase RNA dependent subdomain is located in close proximity to the first Figure Whitneytest) in foc by respectively ( deficient Mut mutation HEV full change E_K Palm the and R_A Mut harboring genomes the and R_A Mut (P) palm (F), fingersRdRp the units(RLU R_A HEVsubgenomic in mutations harboring replicons HEV left. the on crystal violet staining RNAs as negative and positive controls, respectively, cells sequence. sequenc and 1 least at was to used replicon subdomains palm and thumb Figure

Accepted least at Article

electroporated intoS10electroporated were electroporated with Mut or us

P 5 - 6

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form . alm 3 . in the  elw ge ad cy and grey yellow,

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r ± SD ±

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The number of electroporated cells plated in a 100 selected G418 selected to to primer

) -

for

determined at d 1, 2 and 3 postand23 1,ddeterminedat and extracellular infectivities were determined at _ change E_K

- 164 s harborings _. W E_K. The bars indicate bars The palmsubdomain is highlightedin andred the predicted second -

3 cells and maintained under s underandmaintained cells 3

served 4

-  an and in the thumb subdomain as template RNA - - ei o the of helix .

, resistant cells replicatingcells resistant R_A replicon RNA or GAD or wild ild comparedto wt - p)

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ceai rpeetto o the of representation Schematic Gaussia luciferasereporterandGaussia adaptive changes. adaptive - helix 1628 helix single focus forming units (ffu; mean ± SD) ± mean (ffu; units forming focus

, r ),

subdomains (

cells were then lysed for total RNA extraction RNA total for lysed then were cells 35 Te oiin f h compensatory the of position The . RNA mutations eplication ) . are

P Fingers, palm and thumb subdomains are . -

1644. - alm

(D) eedn RA oyeae thumb polymerase RNA dependent followed by G418 selection  denoted -

- helix

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of the

R_A (C) The bars indicate bars The ttsia dfeecs (Mann differences Statistical HEV Mut R_A replicon. S10 replicon. R_AMut HEV

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palm subdomain

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n te ORF3 the and dish is indicated µg for mutation /mL G418 forG418 /mL n h RdRp the in . -

relative lightrelative for for

transfection P determined location of

 10

0.0 change .

d s strain RNA HEV 0

Mut and 0 - 1 s, 3 - - - . ,

This article isprotected rights by All copyright. reserved 21. 20. 19. 18. 17. 16. 15. 14. 13. 12. 11. 10. 9. 8. 7. 6. 5. 4. 3. 2. 1.

Accepted Article 2018;1062:165 and Establishment BM. Kummerer CM. Rice Virolcells.J 1999;73:3854 S, Schlesinger M, Lippa BM, Pragai RNASelectionofcapablereplicons per of Jr., TA, Hoffman E, Agapov I, Frolov Virol2019;93 St Crystal First D. Bussiere Nonstructural AO, Frank M, Holdorf A, Hyrina A, Proudfoot D. Moradpour s J, Ferrer Gouttenoire D, Ferrero N, Oechslin N, 2019;93 Silva Da A, Recombinant Pollan D, Szkolnicka Origins and Iranzo D, Kazlauskas YI, Wolf Proc NatlAcadSci U S A2020;117:1731 al. T 2016;65:1733 ribavirin for transpl evidence organ in Todt D, Gisa A, Radonic A, Nitsche A, Behrendt P, Suneetha PV, Pischke S, et al. In vivo failure treatment 2014;147:1008 ribavirin with mutationhepatitisin the Y Debing A p P. Gong H, Jia RNAtemplate specificities, recruitmentand synthesis. J Gen Virol 2016;97:2231 Mahilkar S, Paingankar MS, Lole KS. Hepatitis Proc NatlAcadSci U S A positiveof additionalgroup an of delineation virus: assignmen assisted Koonin EV, Gorbalenya AE, Purdy MA, Rozanov MN, Reyes GR, Bradley DW. Computer RevNat Gastroenterol Hepatol 2018;15:96 Nimgaonkar I, Ding Q, Schwartz RE, Ploss A. Hepatitis E virus: advances and challenges. required forinfectious particle secretion.PLoS Pathog virology: al. is and et protein ORF3 J, virus EE Oppliger hepatitis of M, association membrane mediates Matter Palmitoylation hepatitis J, Mauron N, on Oechslin L, Abrami Update A, Pollan J, J. Gouttenoire Gouttenoire J, Neyts Implicationsclinical for practice. D, Moradpour Y, Debing organ in hepatitis2008;358:811 chronic and virus E Hepatitis JM Peron L, Ouezzani JM, Mansuy J, Selves N, Kamar on Guidelines hepatitisEvirus infection.J HepatolPractice 2018;68:1256 Clinical EASL Liver. the of Study the for Association European Evirus infection.Nat Rev Dis Primers 2017;3:17086. Kamar N, Izopet J, Pavio N, Aggarwal R, Labrique A, Wedemeyer H, Dalton HR. Hepatit HoofnagleJH, Nelson KE, Purcell RH. Hepatitis NEnglE. J Med 2012;367:1237 global The ST. hepatitisEvirus Wiersma genotypes1 and 2 in 2005. Hepatology 2012;55:988 JS, Wittenborn J, Theaker GA, Stevens DB, Rein al.et ICTVVirus TaxonomyProfile: Hepeviridae. GenJ Virol 2017;98:2645 PoelderMengS,VanOkamotoSmithDB,H,PurdyMA,XJ, Jameel Harrison TJ, WHM, tructural olymerases odt D odt Robust hepatitis E virus infection and transcriptional response in human hepatocytes.human intranscriptional responseand infection virus E hepatitisRobust , : Friesland M Friesland e00459 , o Gisa A Gisa verview.Subcell Biochem 2018;88:39 e : f volution of the - - e00170 rom the h 1743. 817. - - h ptts E epatitis 1011e1007. 173. - ptts E epatitis 19. - , Dallmeier K, Pischke S, Bremer B, Manns M, Wedemeyer H, et al. Aal. et H, M, Wedemeyer Manns B, Bremer S, Pischke K, Dallmeier ,

ra , edge N Vrl RNA Viral N. Verdaguer C, Orta t of functional domains in the nonstructural polyprotein of hepatitis E hepatitis of polyproteinnonstructural the in domains functional of t

, Moeller N, Praditya D, Kinast V, Bruggemann Y, Knegendorf L, et L, Knegendorf Y, Bruggemann V, Kinast D, Praditya N, Moeller , - s 19. p - tructure nue mtgnss f h hptts vrs genome. virus E hepatitis the of mutagenesis induced ositive E

1992;89:8259

v - virus RNApolymerase promotes its replication andassociates iral 3865. g

v lobalRNA iruses - - s

f p J, Lucia J, unction trand RNAtrand rotein J

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- - This article isprotected rights by All copyright. reserved Accepted Article

This article isprotected rights by All copyright. reserved Accepted Article