Available online at www.sciencedirect.com
ScienceDirect
Formation and working mechanism of the picornavirus
VPg uridylylation complex
1,2,6 3,6 2,4,5
Yuna Sun , Yu Guo and Zhiyong Lou
The initiation of picornavirus replication is featured by the initiated by a protein primer is the most unique one, in
uridylylation of viral protein genome-linked (VPg). In this which the virus encodes a protein, that is, viral protein
process, viral RNA-dependent RNA polymerase (RdRp) genome-linked (VPg), that acts as the primer to initiate
catalyzes two uridine monophosphate (UMP) molecules to the replication.
hydroxyl group of the third tyrosine residue of VPg.
0
Subsequently, the uridylylated VPg (VPg-pUpU) functions as VPg was first discovered to be covalently linked to the 5
the protein primer to initiate the replication of the viral genome. end of viral genomes extracted from mature virions of
Although a large body of functional and structural works has several single-stranded positive-sense RNA (+ssRNA)
been performed to define individual snapshots for particular viruses and subsequently was demonstrated to be
stages of the VPg uridylylation process, the formation, encoded by the viral genome [1 ,2]. So far, VPg has only
dynamics and mechanism of the whole VPg uridylylation been experimentally demonstrated in the Picornaviridae
complex still requires further elucidation. We would like to [3 ,4,5] and Caliciviridae [2] families, and its existence
provide an overview of the current knowledge of the been computationally predicted in Astroviridae [6,7 ].
picornaviral VPg uridylylation complex in this paper. Caliciviral VPg has a fairly large molecular weight, rang-
ing from 13 to 15 kDa, and has been demonstrated to play
Addresses
1 multiple roles in the viral life cycle, for example, func-
National Laboratory of Macromolecules, Institute of Biophysics,
Chinese Academy of Science, Beijing 100101, China tioning as a proteinaceous cap substitute when interacting
2
School of Medicine and MOE Key Laboratory of Protein Sciences, with host eIF4E for viral protein translation [8], binding
Tsinghua University, Beijing 100084, China
with RNA through the basic residue enriched N-terminus
3
College of Pharmacy and State Key Laboratory of Medicinal Chemical
[9], initiating genome replication, and coordinating the
Biology, Nankai University, Tianjin 300071, China
4 viral encapsidation process [10]. The multiple functions
Collaborative Innovation Center for Biotherapy, Tsinghua University,
Beijing 100084, China of caliciviral VPgs have been summarized in another
5
Collaborative Innovation Center for Biotherapy, State Key Laboratory of review [7 ] and warrant further study.
Biotherapy and Cancer Center, West China Hospital, West China
Medical School, Sichuan University, Chengdu, China
By contrast to the growing understanding of caliciviral
Corresponding author: Lou, Zhiyong ([email protected],
VPg, extensive studies have well characterized the mech-
anism of picornavirus-encoded VPg in viral replication.
6 Picornaviral VPgs have smaller size comprising 19–26
These authors contribute equally to this work.
0
amino acids (Table 1). The presence of VPg on the 5
end of the viral genome may help the virus antagonize the
0
Current Opinion in Virology 2014, 9:24–30 host innate immune response by shielding the 5 tripho-
sphate group of the viral genome (reviewed in Ref. [7 ]);
This review comes from a themed issue on Virus replication in
animals and plants however, VPg must be removed by a host enzyme follow-
ing picornavirus genome release into the cytoplasm
Edited by C Cheng Kao and Olve B Peersen
[11,12]. Previous results revealed that the removal of
For a complete overview see the Issue and the Editorial
VPg from the viral genome abolished the infectivity of
Available online 19th September 2014
the calicivirus, vesicular exanthema virus (VESV) [13],
http://dx.doi.org/10.1016/j.coviro.2014.09.003 but did not affect poliovirus (PV, a representative virus
1879-6257/# 2014 Elsevier B.V. All rights reserved. of Picornaviridae family) [1 ,5,14]. Further studies
demonstrated that the VPg of caliciviruses acts as a
cap substitution and plays an essential role in viral
protein translation [15], but picornaviruses use an
0
internal ribosome entry site (IRES) within the 5 UTR
of the viral genome, rather than VPg, to initiate viral
Introduction protein translation [16]. These observations led to the
The replication of RNA viruses is initiated by distinct identification of one of the most important discrepancies
mechanisms, for example, (1) de novo replication, such as between the functions of picornavirus and calicivirus
hepatitis C virus (HCV), (2) RNA primer-primed replication, VPgs: picornaviral VPgs predominantly coordinate the
used by numerous viruses, and (3) protein primer-primed initiation of virus replication but do not participate in
replication. Among these mechanisms, the replication viral protein translation. In this paper, we will focus on
Current Opinion in Virology 2014, 9:24–30 www.sciencedirect.com
Picornavirus VPg uridylylation complex Sun, Guo and Lou 25
Table 1
A complete summary of VPg sequences of picornaviruses.
Genus Virus Sequence of VPg
VPg1 GPYAGPLERQKPLKVKAKLQQQE Aphthoviru s FMDV VPg2 GPYAGPMERQKPLKVKVKAPVAKE
VPg3 GPYEGPVKKPVALKVKAKNLIVTE
SAYEGCSTRKTARQLARSVVGEGAYDGNVKRTTAREL Aquamavirus Seal picornavirus type 11 ARKAIPSEQ
Avihepatovirus Duck hepatitis A virus2
Avisivirus Avisivirus NLYSGEPTRAKVTRVTREFQ
Cardiovirus Encephalomyocarditis virus GPYNETARVKPKTLQLLDVQ
Cosavirus Cosavirus A GPYNGPTKKEIKTLKLKAQ
Dicipivirus Cadicivirus A SPYDSLYMNKMKKNARKPLNKVALHE
PV GAYTGLPNKKPNVPTIRTAKVQ
CVA16 GAYSGAPKQALKKPVLRTATVQ
CVB3 GAYTGVPNQKPRVPTLRQAKVQ Enterovirus EV71 GAYSGAPKQVLKKPALRTATVQ
Rhinovirus A GPYSGEPKPKSKAPERRVVTQ
Erbovirus Equine rhinitis B virus RAYNIPNVRQRLRKQLAVRAE Gallivirus Gallivirus A2
Hepatovirus HAV GVYHGVTKPKQVIKLDADPVESQ
Hunnivirus Hunnivirus A AYSGNAVVRDKKKPNGLKVIDIASLQ
Kobuvirus Aichivirus A AAYSAISHQKPKPKSQKPVPTRHIQRQ
Megrivirus Melegrivirus A2 Mischivirus Mischivirus A2
Mosavirus Mosavirus A2
Oscivirus Oscivirus A2
Parechovirus Human parechovirus RAYNPTLPVVKPKGTFPVS
Pasivirus Pasivirus A RPYNQTAHKMPVTKLTRGRRVLVSQ
Passerivirus Passerivirus A2
Rosavirus Rosavirus A2
Salivirus Salivirus A GAYSGTPVPKPRKKDLPKQPVYSGPVRRQ Sapelovirus Porcine sapeloviru s GAYSGAPRPETRKPVLRKAVVQ
Senecavirus Seneca Valley virus2
Teschovirus Porcine teschovirus GSYEATAIKPTKPNRQSLLKLVEMQ
Avian encephalomyelitis Tremovirus SAYSAAIKPLRVVRLEQSDAQ virus2
1
Seal picornavirus type 1 is a highly divergent picornavirus.
2
Indicates no experimental evidence to support the existence or sequence of VPg.
Highlighted indicate the conserved Tyr residues in the third position.
www.sciencedirect.com Current Opinion in Virology 2014, 9:24–30
26 Virus replication in animals and plants
the current knowledge of the major function of picorna- replication. The precise roles of 3BCD and 3BC in viral
viral VPg, functioning as a protein primer to initiate virus replication remain controversial.
replication.
Picornaviral VPg exists in variable states Picornaviral VPg uridylylation
The genome of picornaviruses contains a polyadenylated Picornaviral VPg (in the context of 3BC/3BCD) must be
+ssRNA genome and a single open-reading-frame encod- modified to VPg-pUpU before it can function as the
ing a polyprotein of approximately 250 kDa [17–21]. This protein primer to initiate genome replication. This modi-
pol
polyprotein is initially processed into one structural (P1) fication to VPg is accessed by picornaviral RdRp (3D )
and two non-structural (P2 and P3) regions and then covalently linking two uridine monophosphate (UMP)
undergoes proteolytic cleavage, generating various pre- molecules to the hydroxyl group of the third tyrosine
cursors and 11 mature proteins, that is, VP1–4, needed to residue of VPg, which is strictly conserved within the
pro
assemble virus capsid, and 2A, 2B, 2C, 3A, 3B, 3C , and Picornaviridae family (Table 1) [27]. This process is
pol
3D , necessary for virus replication in host cells known as VPg uridylylation, whereas calicivirus genomes
(Figure 1a). begin with a GU sequence, and the linkage of VPg to
calicivirus +ssRNA occurs via guanylation.
P3 region contains the most essential components for viral
pro pol
replication and is processed through two pathways: the Both viral proteins (i.e. VPg, 3C , 3D , and/or 3CD)
major and minor pathways [22,23]. P3 is cleaved into 3AB and RNA elements in viral genome comprise a so-called
and 3CD proteins via the major pathway; while the minor ‘VPg uridylylation complex’ to accomplish VPg uridyly-
pathway produces 3A and 3BCD proteins first, and then lation. Notably, the VPg uridylylation process in picor-
pol
3BCD is further processed into 3BC and 3D , and naviruses acts in a template-dependent manner by using
pro pol
eventually 3B (VPg), 3C and 3D . Within the major a small stem loop structure (cis-acting replication
pathway, PV 3AB was demonstrated to remodel RNA as a element, CRE) as the natural template, although
result of its helix-destabilizing activity [24] and to stimu- poly(rA) can also be used as the template for the VPg
pol
late the RdRp activity of 3D [25]. 3AB intermediate uridylylation reaction in vitro [28 ,29]. In this process,
can be further cleaved, generating 3A and VPg, with VPg similar structures within the CRE have been demon-
acting as the protein primer for the initiation of genome strated to be essential for both VPg uridylylation and
replication in vivo [26]. Although the generation of 3BCD virus replication [30–33], despite variable CRE position-
and 3BC has been described as part of the minor pathway, ing in different picornavirus genomes [34,35]. For
these proteins do not accumulate to significant levels example, the CRE within the PV genome was identified
during PV replication [22]. However, 3BCD and 3BC in the 2C coding region [30], but the CRE of FMDV was
0
are capable of initiating viral genome replication in vitro, located within the 5 UTR[36], suggesting a substantial
in place of VPg [22], suggesting a distinct model in which level of complexity associated with the picornaviral VPg
3BC(D), instead of VPg, serves as the primer for genome uridylylation complex.
Figure 1
IRES VPg (3B) oriL P1 P2 P3 poly(rA)
oriR
VP4 VP2 VP3 VP1 2A 2B 2C 3A 3B 3C 3D
5′ UTR 3′ UTR oriL
Current Opinion in Virology
0
Genome organization of picornavirus. VPg, encoded by the 3B region, is covalently linked to the 5 end of the viral genome. The internal ribosome entry
0 0 0
site (IRES) is located at the 5 UTR. Three cis-acting replication elements (CREs), oriL, oriI and oriR, are found in the 5 UTR, 2C coding region and 3
UTR, respectively. The polyprotein encoded by the viral genome is initially processed to P1, P2 and P3 precursors and eventually to 11 mature viral
proteins. Notably, although picornaviruses share fairly high sequence similarities, there are still many variations. For instance, VP4 of the hepatitis A
virus is very small and could not be detected in virions [54]. VP1 to VP4 assemble into the virus particle. 2A is a protease that cleaves the P1 and P2
regions. 2B and 3A interact with membranes for the formation of the viral replication complex. 2C is an ATPase or helicase involved in the function of
pro
the viral replication complex. 3B is VPg, 3C is a viral protease that cleaves the linkage between each part of the polyprotein except the 2C cleavage
pol
site, and 3D is an RNA-dependent RNA polymerase.
Current Opinion in Virology 2014, 9:24–30 www.sciencedirect.com
Picornavirus VPg uridylylation complex Sun, Guo and Lou 27
Diversity of VPg binding sites in picornaviral VPg uridylylation. In the crystal structure of FMDV
pol pol
3D s VPg1-3D , VPg1 binds to the motif F of the finger
In the models raised from both the major and the minor domain and the a13 helix of the thumb domain, spanning
pathways, the VPg uridylylation reaction consists of two residues E166, I167, R168, K172, and R179 as well as
pol pol
steps: (i) VPg binds to 3D ; and (ii) 3D transfers UMP T407, A410, and I411, respectively [42 ]. The localization
to VPg [29,37]. Together with the fact that picornavirus of VPg1 at the catalytic tunnel and the interaction with
pol
3D s and VPgs share high primary sequence similarity the UMP molecule suggest that an in cis uridylylation
and structural homology [29], it seems reasonable that all mechanism is most likely favored by the FMDV VPg
pol
picornaviral VPgs would bind to the 3D active site. uridylylation process [42 ]. This structure presents a
However, several lines of evidence have revealed large precise picture of the VPg uridylylation catalytic reaction
discrepancies in this model. occurring.
Lyle et al. first reported that four surface residues near or Gruez et al. [43 ] further reported that in the crystal
pol pol
in motif E of PV 3D , that is, F377, R379, E382 and structure of CVB3 3D complexed with VPg and PPi,
pol
V391, were required for VPg or 3AB binding and affected VPg is located at the end palm region of 3D , encom-
VPg uridylylation [38] (Figure 2). A very recent result passing residues W369, T370, Y378–E383, V389, V392,
reported that amiloride inhibits VPg uridylylation and P394, and P222 (Figure 2). However, substitutions of
thus viral RNA replication by occupying this VPg binding such residues affect VPg binding but only slightly influ-
pol
site on PV 3D , supporting the model proposed by Lyle enced the uridylylation reaction [43 ]. This finding
et al. [39]. However, the mutations in the proposed 3AB- indicates a distinct in trans VPg uridylylation mechanism
pol
binding site of 3D were less active for VPg priming than in CVB3 and that this VPg-binding position can either act
pol
RNA elongation [38], suggesting that 3D -bound VPg as a substrate in an intermolecular uridylylation complex
could be extended by a second RdRp molecule [40]. or stabilize the VPg uridylylation complex [43 ]. Very
Later studies investigating the structure of human rhino- interestingly, the VPg binding sites structurally identified
pol pol
virus 16 (HRV16) 3D identified a distinct large cleft on in CVB3 3D partially overlap with those found in the
pol
the front face of HRV16 3D and led to the develop- PV system and are at least somewhat consistent with the
ment of a ‘front-loading’ model for VPg uridylylation [41]. ‘front-loading’ model proposed from HRV16 [41].
pol
The structures of 3D -VPg complexes from the foot- Two recent biochemical and structural studies have
and-mouth disease virus (FMDV), coxsackie virus B3 revealed a further distinct VPg binding site in EV71,
pol
(CVB3) and enterovirus-71 (EV71) have provided direct located on the palm domain of the EV71 3D
evidence for multiple distinct locations of VPg binding [28 ,29]. EV71 VPg forms a V-shape conformation and
pol
sites in 3D s and indicate variation in the mechanisms of is anchored at the bottom of the palm domain of EV71
Figure 2
FMDV VPg1
CVB3 VPg
90° 90°
EV71 VPg
Current Opinion in Virology
pol pol
Comparison of identified VPg binding sites in picornavirus 3D s. Because all reported structures of picornavirus 3D s share high structural
pol
similarities, we used the structure of the EV71 3D (PDB code: 3N6L) [55] as a representative model in this figure and covered it with a white surface.
The bound VPgs with FMDV, CVB3 and EV71 are shown as green, cyan and purple sticks, respectively. The residues for VPg (or 3AB) binding in PV
pol
[38], FMDV [42 ], CVB3 [43 ] and EV71 [28 ,29] 3D are colored as orange, red, pale green and blue, respectively, in the surface representation.
www.sciencedirect.com Current Opinion in Virology 2014, 9:24–30
28 Virus replication in animals and plants
pol pro
3D [28 ] (Figure 2). Substitutions of the interacting interaction of each 3C molecule with a single-stranded
pol
residues in EV71 3D , that is, L319, D320, and Y335, portion of the stem [40]. However, binding to the stem-
with alanine significantly disrupted VPg uridylylation by loop d of oriL only requires monomer binding to an intact
pol
weakening VPg binding to 3D but did not affect the double-stranded stem [53], suggesting that some 3C resi-
pol
RNA elongation activity of 3D [28 ]. In vitro trans- dues are most likely used for binding to all RNAs and some
complementation of VPg uridylylation could be achieved 3C residues are likely only used for specific RNA inter-
by mixing VPg-binding-defective and catalytic-defective actions. A recent review suggested that the combination of
pol
mutant 3D proteins, indicating again a trans mechan- protein–protein and protein–RNA interactions occurring at
ism for EV71 VPg uridylylation [28 ]. oriL, oriR and oriI facilitate the formation of a single,
functional VPg uridylylation complex [40].
Further key evidence for the formation of the
picornaviral VPg uridylylation complex
pol Conclusion and perspective
The interactions between VPg and 3D only reflect a
VPg uridylylation plays a crucial role in the initiation of
partial aspect of the formation of the VPg uridylylation
picornavirus replication. Previous results have revealed
complex. Actually, in the opinion of the authors, the sites
distinct interactions within the VPg uridylylation com-
for VPg binding or uridylylation identified by both func-
plex of picornaviruses. In our opinion, these variations are
tional and structural studies provide only individual snap-
not likely to be caused by the difference in viruses, as the
shots of particular stages of the overall picornaviral VPg pol
VPg and 3D of picornaviruses share very high primary
uridylylation process. To dissect the formation, dynamic
sequence and structural similarities. We speculate that it
and working mechanism of the entire VPg uridylylation pol
is likely that the variable VPg–3D structures represent
complex, it is quite necessary to investigate the inter-
the binding position of VPg at different stages during VPg
actions among other P3 proteins and RNA. Fortunately,
uridylylation or virus replication.
extensive work has been performed to describe these
interactions, including 3C–3C [37,44], 3CD–3CD [37,45],
Based on reported biochemical and generic evidence, a
3D–3D [46 ], 3CD–3D [37], 3C–3D [37,44] and 3C or
number of structural studies would be of great help to
3CD with distinct CREs [45].
dissect the precise mechanism of the picornaviral VPg
uridylylation complex. First, PV is a representative model
After a head-to-tail 3D–3D interaction mode was first
pol to study VPg uridylylation, but the structure of PV VPg–
observed in the crystal structure of PV 3D [47], a great pol
pol 3D is still lacking, despite the extensive biological
breakthrough was achieved to identify that PV 3D
results that have been generated. The structure of PV
forms a lattice through interface I and implicated a unique pol
VPg–3D would be helpful to interpret those findings
2D mechanism for RNA synthesis, in which RNA is
in an atomic level. Second, a systematic comparison
elongated by moving from one active site to another pol
amongst VPg–3D complexes of all picornaviruses
within the lattice instead of having the RdRp tracking
would provide further information to understand the
along the RNA [46 ,48,49]. A recent crystal structure of pol
variation of VPg–3D interactions. However, to the best
the PV elongation complex supports this model [50 ].
of our knowledge, there remain several difficult hurdles to
Furthermore, we have come to understand that the back
pol overcome to perform this research. For example, VPgs for
of the thumb of the PV 3D interacts with the top of the
pro some genera in the Picornaviridae family have yet to be
3C dimer and that this interaction is essential for VPg pol
defined, and the interaction between VPg and 3D for
uridylylation in vitro and genome replication in cells [44].
some picornaviruses are too weak for structural biology
Next, the crystal structure of the PV 3CD fusion protein
studies. Moreover, a structure of 3CD-VPg complex
provided important insights, in which extensive 3C–3C,
would make it clear how 3CD fusion protein functions
3C–3D and 3D–3D interactions were observed [37].
within the VPg uridylylation complex and clarify the
However, the bottom of the palm domain accounted
controversial data from previous studies. Eventually,
for the interaction of 3C in a VPg uridylylation complex
the elucidation of the entire uridylylation complex
model proposed based on the structure of PV 3CD
pol VPg-3CD-CRE (or different CRE) will undoubtedly
(Figure 8B in Ref. [37]), but anchored VPg in EV71 3D
provide detailed insights into and finalize the precise
[28 ].
mechanism of the picornaviral VPg uridylylation process.
We believe that these efforts will help us to integrate all
Viral RNA is another key component within the VPg
the individual ‘snapshots’ into an entire ‘movie’ that will
uridylylation complex. VPg uridylylation is dependent on
allow us to understand the dynamics and mechanism of
the stem-loop structure of CRE, and 3C is understood to
the picornaviral VPg uridylylation complex.
be the major determinant that binds all CREs in the VPg
uridylylation complex [22,51 ], although 3AB has also Acknowledgments
been shown to participate in CRE recognition [52]. The
We apologize to all colleagues whose contributions we could not adequately
formation of the VPg uridylylation complex requires a
discuss due to space constraints. This work was supported by the National
pro
3C dimer and melting of the oriI element for the Natural Science Foundation of China (Grant nos. 81322023, 31370733,
Current Opinion in Virology 2014, 9:24–30 www.sciencedirect.com
Picornavirus VPg uridylylation complex Sun, Guo and Lou 29
31170678, 31100208, and 31000332), the National Basic Research Program International Committee for the Taxonomy of Viruses. Edited by
of China 973 program (Grant nos. 2013CB911100 and 2014CB542800), and Van Regen-mortel MHV, Fauquet CM, Bishop DHL, Calisher
Tsinghua University Initiative Scientific Research Program (2009THZ01). CH.et al.: 2000:657-673.
18. McMinn PC: An overview of the evolution of enterovirus 71 and
its clinical and public health significance
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