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- [email protected]) 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].