Replication of positive-strand RNA viruses in plants: contact points between plant and virus components

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1 1529 REVIEW / SYNTHÈSE Replication of positive-strand RNA viruses in plants: contact points between plant and virus components Hélène Sanfaçon Abstract: Positive-strand RNA viruses constitute the largest group of plant viruses and have an important impact on world agriculture. These viruses have small genomes that encode a limited number of proteins and depend on their hosts to complete the various steps of their replication cycle. In this review, the contact points between positive-strand RNA plant viruses and their hosts, which are necessary for the translation and replication of the viral genomes, are discussed. Special emphasis is placed on the description of viral replication complexes that are associated with specific membranous compartments derived from plant intracellular membranes and contain viral RNAs and proteins as well as a variety of host proteins. These complexes are assembled via an intricate network of protein protein, protein membrane, and protein RNA interactions. The role of host factors in regulating the assembly, stability, and activity of viral replication complexes are also discussed. Key words: replication complexes, translation, positive-strand RNA virus, plant virus, plant virus interactions, intracellular membranes, RNA-dependent RNA polymerase. Résumé : Les virus ARN à brin positif constituent le plus grand groupe de virus des plantes et ont un impact important en agriculture, partout au monde. Ces virus ont de petits génomes qui codent pour un nombre limité de protéines et dépendent de leurs hôtes pour compléter diverses étapes de leur cycle de réplication. Dans cette revue, l auteur discute des points de contact entre les virus végétaux ARN à brin positif et leurs hôtes, lesquels sont nécessaires pour la traduction et la réplication des génomes viraux. On met l emphase sur la description des complexes de réplication virale, qui sont associésàdes compartiments membraneux spécifiques dérivés de membranes végétales intracellulaires et contiennent les protéines et les ARN viraux, ainsi qu une variété de protéines de l hôte. Ces complexes sont assemblés via un réseau complexe d interactions protéine-protéine, protéine-membrane et protéine-arn. On discute également le rôle des facteurs de l hôte dans la régulation de l assemblage, de la stabilité et de l activité des complexes de réplication virale. Mots clés :complexes de réplication, traduction, virus ARN à brin positif, virus des plantes, interactions plante virus, membranes intracellulaires, polymérase ARN dépendant de l ARN. [Traduit par la Rédaction] Introduction Positive-strand ((+)-strand) RNA viruses constitute a very large group of viruses that infect plants, animals, insects, and fungi. In plants, the vast majority of viruses characterized to date are (+)-strand RNA viruses. Even though (+)- strand RNA viruses are successful pathogens, they have small genomes, between 4 and 15 kilobases (kb), that en- Received 12 July Published on the NRC Research Press Web site at on 10 February Abbreviations: (+)-strand, positive-strand or plus-strand; ( )-strand, negative-strand or minus-strand; AMV, Alfalfa mosaic alfamovirus; BMV, Brome mosaic bromovirus; BYDV, Barley yellow dwarf luteovirus; CIRV, Carnation Italian ringspot tombusvirus; CMV, Cucumber mosaic cucumovirus; CNV, Cucumber necrosis tombusvirus; CPMV, Cowpea mosaic comovirus; CymRSV, Cymbidium ringspot tombusvirus; eif, eukaryotic translation initiation factor; ER, endoplasmic reticulum; GFLV, Grapevine fanleaf nepovirus; IRES, internal ribosome entry site; kb, kilobase; m 7 GTP, methyl-guanidine triphosphate or cap structure; mrna, messenger RNA; PABP, polya binding protein; PVA, potato potyvirus A; RCNMV, Red clover necrotic mosaic dianthovirus; RdRp, RNA-dependent RNA polymerase; RNA, ribonucleic acid; sgrna, subgenomic RNA; TBSV, Tomato bushy stunt tombusvirus; TEV, Tobacco etch potyvirus; TMV, Tobacco mosaic tobamovirus; ToRSV, Tomato ringspot nepovirus; trna, transfer RNA; TuMV, Turnip mosaic potyvirus; TYMV, Turnip yellow mosaic tymovirus; UTR, untranslated region; VPg, viral protein linked to the genome; VRC, viral replication complex. H. Sanfaçon. Agriculture and Agri-Food Canada, Pacific Agri-Food Research Centre, 4200 Highway 97, Summerland, BC V0H 1Z0, Canada ( SanfaconH@agr.gc.ca). Can. J. Bot. 83: (2005) doi: /b05-121

2 1530 Can. J. Bot. Vol. 83, 2005 code a limited set of genes. As a result, they are dependent on their hosts to perform the various steps of their replication cycle (Maule et al. 2002; Whitham and Wang 2004). The molecular characterization of plant-virus interactions leading to the establishment of viral disease in susceptible plants or to the elimination or restriction of the virus in resistant plants has drawn considerable attention in the last few years (Carrington and Whitham 1998). In susceptible hosts, interactions between viral and plant cellular components are necessary for translation of the virus genome, replication of the viral RNA, and spread of the virus throughout the plant. In turn, the hijacking and modification of host factors by plant viruses often have dramatic effects on the physiology of the host, ultimately resulting in symptom development, including mosaic, necrosis, wilting, and in some cases death of the plant. In this review, I will describe the multiple contact points between viruses and susceptible hosts, which are instrumental for the translation and replication of the viral genome. The discussion will be restricted to plant viruses and the role of plant factors in regulating the efficiency of the various steps of viral RNA synthesis. The readers are referred to complementary recent reviews discussing the involvement of host factors in RNA replication of other eukaryotic (+)-strand RNA viruses (Bushell and Sarnow 2002; van der Heijden and Bol 2002; Ahlquist et al. 2003; Schneider and Mohr 2003; Salonen et al. 2005). Fig. 1. Summary of the various steps necessary for (+)-strand RNA virus genome replication. To simplify the figure, the viral genome (shown at the top of the figure) is represented as a single molecule of (+)-strand RNA thus representing a monopartite virus. Similar steps are involved in the replication of multipartite viruses that have their genome split into two or more RNA molecules (as discussed in the text). The schematic representation of viral replication complexes (VRCs) is shown by an invagination of the plant intracellular membrane containing the viral membrane anchor protein (empty circles), the viral RNA-dependent RNA polymerase (RdRp) (black circles), host proteins (grey circles), and viral RNA (thick line). 5' 3' (+) strand RNA Replication of (+)-strand RNA viruses: a stepby-step guide of the interplay between hosts and viruses All (+)-strand RNA viruses must accomplish the same basic steps to replicate their genomes (Fig. 1) (Buck 1996). The first step is the synthesis of the viral replication proteins. The single-strand RNA genome is of positive polarity, which means that it can act as a messenger RNA (mrna) immediately after uncoating of the virus particles. As they do not encode translation factors or ribosomes, viruses must use the host translational machinery to complete this step. Because successful virus infection requires high levels of protein synthesis, viral RNAs possess various elements that interact directly or indirectly with the host translational machinery and allow them to outcompete cellular mrnas for the scarce translational resources of the host (Gale et al. 2000; Bushell and Sarnow 2002; Schneider and Mohr 2003). The next step is the assembly of the viral replication complex (VRC), which is associated with intracellular membranes and contains viral and host proteins and the template viral RNA or RNAs, in the case of multipartite viruses in which the genome is divided in two or more molecules of RNA. Because viral RNA serves both as the mrna for translation and the template for replication, translation and replication are often tightly linked. Consequently, regulatory mechanisms must be in place to allow the switch from the translation mode to the replication mode and avoid collisions of the ribosomes, traveling from the 5 to the 3 end of the viral RNA, and the viral RNA-dependent RNA polymerase (RdRp), traveling from the 3 to the 5 end of the viral RNA (Agol et al. 1999; Ahlquist et al. 2003). The association of VRCs with intracellular membranes is common to all (+)-strand RNA viruses characterized to date 3' 5' (-) strand RNA 5' 3' (+) strand progeny RNA (Salonen et al. 2005). This association results in the formation of membrane vesicles or spherules that constitute subcellular compartments, in which replication proteins and the viral RNAs are sequestered and isolated from the cytoplasmic content of the cell. Thus, the formation of this compartment may serve a protecting role against degradation of the viral RNAs. In addition, membranes have also been shown to play an active role in viral replication, and modification of the lipid composition of intracellular membranes results in decreased viral replication (Lee et al. 2001). Integral membrane proteins, usually viral proteins, are targeted to specific intracellular membranes and act as membrane anchors for the complexes. Other viral and host proteins are recruited to the VRCs often through protein protein interactions with the membrane anchor. Host factors (such as chaperones and kinases) may regulate the folding and interactions of viral proteins (Noueiry and Ahlquist 2003). In some viruses, viral replication proteins are brought into

3 Sanfaçon 1531 the VRCs as polyprotein precursors that include the domain for the membrane anchor (Bedard and Semler 2004). These precursors often have differential activities from the corresponding mature proteins. Thus, regulated processing of the precursors by viral proteinases offers additional opportunities to control the timing of the various replication steps. The viral RNAs are brought into the VRCs through protein-rna interactions with one or several replication proteins. The final step in viral RNA replication is the actual synthesis of progeny RNA. A complementary minus-strand (( ) -strand) RNA is synthesized by the viral RdRp using the (+)-strand RNA as a template. Cis-acting elements, located at the 3 end of the (+)-strand RNA, consist of RNA secondary structures, such as stem-loops and pseudoknots. These are recognized by the RdRp and act as promoters for the synthesis of the ( )-strand RNA. A viral helicase is often, but not always, responsible for unwinding doublestrand RNA regions formed during the synthesis of the ( )-strand RNA. This allows the newly formed ( )-strand RNA to serve as a template for the synthesis of the progeny (+)-strand RNA (Kadare and Haenni 1997). The progeny (+)-strand RNA is synthesized in 10- to 100-fold excess over the ( )-strand RNA, suggesting that mechanisms are in place to differentially regulate the activity of the RdRp during ( )- and (+)-strand synthesis. For example, the composition of the VRCs and (or) the requirement for cis-acting sequences may differ during ( )-strand and (+)-strand synthesis (Buck 1996). In animal picornaviruses, a uridylated form of a viral protein (VPg, for virus protein genome-linked) is used as a primer for the synthesis of the (+)-strand progeny (Bedard and Semler 2004). In a related plant potyvirus, uridylation of the VPg by the viral polymerase was demonstrated, suggesting that VPg may also play a role in priming viral synthesis, although this remains to be confirmed experimentally (Puustinen and Makinen 2004). In other viruses, RNA synthesis occurs without the need for a protein primer. When a cap structure is present at the 5 end of the viral RNA, a viral-encoded methyltransferase is responsible for capping the RNA after its synthesis. In addition to producing the genomic RNA, some viruses also synthesize subgenomic RNAs (sgrna). sgrnas are 3 -coterminal with the genomic RNA and allow the translation of the coding region for various proteins, including the movement and coat proteins. These coding regions are translationally silent on the polycistronic genomic RNA. sgrnas are produced by recognition of internal subgenomic promoters within the ( )- strand RNA or alternatively by premature termination during ( )-strand synthesis followed by synthesis of the (+)- strand sgrna using the truncated ( )-strand template (Miller and Koev 2000; White 2002). In the first and most common mechanism of sgrna synthesis, the full-length ( )-strand RNA is a template for both (+)-strand synthesis and sgrna transcription. These two processes mutually interfere with each other, probably because they compete for common factors (Grdzelishvili et al. 2005). Because sgrnas are synthesized during the final steps of viral RNA replication, the proteins encoded by these RNAs are not produced until later in infection, when encapsidation and cell-to-cell movement of viral RNAs are initiated. Translation initiation of viral RNA As mentioned above (+)-strand RNA viruses must sequester the host translational machinery to synthesize their proteins (Bushell and Sarnow 2002; Schneider and Mohr 2003). In animal viruses, this often results in a host translational shutdown (Bushell and Sarnow 2002; Schneider and Mohr 2003). In plant viruses, transcriptional host gene shutdown in response to virus infection has been observed (Aranda and Maule 1998). However, experimental evidence is still lacking to confirm that translational shutdown also occurs in plants. In this section, I will briefly review the role of key translation factors in the translation of cellular mrnas and discuss some examples of recruitment of these factors by plant viruses. Translation initiation of cellular mrnas Cellular mrnas contain a methyl-guanidine triphosphate (m 7 GTP) structure, or cap at their 5 end, and a polya tail at their 3 end. Both elements are necessary to recruit host translation factors and form the initial translation complex (Gallie 1998; Gallie 2002b; Sonenberg and Dever 2003). The cap structure is recognized by eukaryotic translation initiation factor 4E (eif4e), while another translation factor, the polya binding protein (PABP) binds to the polya tail. A third translation factor, eif4g interacts with both eif4e and PABP. This results in the creation of a protein bridge that brings the 5 and 3 ends of mrnas in close proximity. This circularization of mrnas is essential for their translation. The complex of eif4e and eif4g is often referred to as the eif4f translation factor. The initial translation complex also includes other translation factors, eif4a and eif4b, which interact with the scaffold eif4g protein. eif4a is a helicase that stimulates translation by unwinding secondary structures present at the 5 end of the mrna. eif4b also enhances translation, although its mode of action is less well characterized. The eif4g scaffold protein also interacts with eif3, a large factor composed of at least 11 subunits. The eif3 translation factor is part of the ternary complex that contains the 40S ribosomal subunit, the aminoacylated met-transfer RNA (trna), eif1a (or 1a), and eif2. The translation complex then scans mrnas to locate the initiation codon. Two additional factors, eif5 and eif5b, promote the joining of the 60S ribosome subunit to form the active 80S ribosome. Many translation factors consist of multiple isoforms that are encoded by large gene families (Browning 2004; Hernandez and Vazquez-Pianzola 2005). Each isoform plays specific roles in mrna translation and sometimes in other cellular processes. In some cases, two isoforms of the same translation factor can functionally replace each other. For example, multiple isoforms of eif4e are present in Arabidopsis, including eif4e, eif(iso)4e, and a novel cap-binding protein (Ruud et al. 1998; Browning 2004). A mutant line with a transposon insertion in the eif(iso)4e gene is phenotypically indistinguishable from wild-type plants (Duprat et al. 2002). The lack of ei- F(iso)4E mrna and protein in this line is apparently compensated by increased expression of eif4e. While viruses are dependent on the host translational machinery to synthesize their proteins, many viral RNAs differ from cellular mrnas in that they lack the 5 cap structure,

4 1532 Can. J. Bot. Vol. 83, 2005 the 3 end polya tail, or both. Viruses use various mechanisms to recruit cellular translation factors, promote the circularization of their RNAs, and assemble active translation complexes (Gallie 1996). As will be discussed below, these include: (i) the presence of RNA structures such as pseudoknots, internal ribosome entry sites, or trna-like structures that interact directly with translation factors; (ii) the formation of alternative protein bridges that include viral proteins; and, (iii) base pairing of RNA structures present at the 5 and 3 ends of the RNA. Translation of viral RNA that lack a 5 cap In picorna-like viruses, a viral protein, the VPg is covalently linked to the 5 end of the RNA, thereby replacing the cap structure. The 5 untranslated region (UTR) of the genomic RNA of several picorna-like viruses contains internal ribosome entry sites (IRES) that are composed of a complex group of stem-loop structures. IRES were initially discovered in the genomic RNA of animal picornaviruses (Jang et al. 1990; Hellen and Sarnow 2001) and have also been found in the genome of several picorna-like plant viruses, including two potyviruses (Carrington and Freed 1990; Basso et al. 1994) and possibly one comovirus (Thomas et al. 1991; Verver et al. 1991), although the function of this last IRES has been contested (Belsham and Lomonossoff 1991). Translational enhancement promoted by the IRES of one potyvirus is dependent on the presence of eif4g, suggesting that it acts by recruiting this factor thereby bypassing the need for the cap-eif4e interaction (Gallie 2001). However, the presence of IRES may not be a universal feature of this virus group. In fact, at least one potyvirus does not contain an IRES (Simon-Buela et al. 1997). Another possible mechanism to enhance translation of viral RNAs lacking a cap structure involves the VPg protein. VPg has been suggested to play a role in recruiting eif4e, thereby functionally replacing the cap structure in the creation of a protein bridge between the 5 and 3 ends of the RNAs (Wittmann et al. 1997). In support of this model, the Turnip mosaic potyvirus (TuMV) and Tobacco etch potyvirus (TEV) VPgs were shown to interact with eif(iso)4e and eif4e, respectively (Wittmann et al. 1997; Leonard et al. 2000; Schaad et al. 2000; Leonard et al. 2004). The TuMV VPg could compete with a cap analogue for interaction with eif(iso)4e in vitro (Leonard et al. 2000; Plante et al. 2004). In planta, eif(iso)4g and PABP are associated with the TuMV VPg-eIF(iso)4E complex, suggesting that the interaction of VPg with eif(iso)4e promotes the recruitment of other translation factors (Leonard et al. 2004; Plante et al. 2004). However, conclusive evidence that the VPg-eIF4E interaction enhances viral translation and (or) inhibits host translation in vivo is still lacking. The importance of this interaction in the replication cycle of potyviruses has been confirmed using a number of methods, including (i) analysis of potyvirus variants carrying mutations in the VPg, (ii) characterization of recessive resistance genes that correspond to eif4e isoforms, and (iii) targeted mutations of eif4e isoforms in various plants (Leonard et al. 2000; Duprat et al. 2002; Lellis et al. 2002; Ruffel et al. 2002; Nicaise et al. 2003; Gao et al. 2004; Moury et al. 2004; Kang et al. 2005; Sato et al. 2005). However, VPg is a multifunctional protein, and its interaction with eif4e isoforms may also be involved in other important steps of the replication cycle of potyviruses, including replication and (or) cell-tocell movement (Urcuqui-Inchima et al. 2001; Gao et al. 2004). An interaction between a nepovirus proteinase and eif(iso)4e was demonstrated in vitro (Leonard et al. 2002). However, this interaction has not yet been confirmed in infected plants, and a potential role for eif4e isoforms in nepovirus replication cycle is only tentative at this point. Translation of viral RNAs that lack a 3 polya tail Many viral RNAs have trna-like structures and (or) pseudoknots at their 3 ends, which can functionally replace the polya tail in enhancing translation (Gallie and Walbot 1990; Leathers et al. 1993). The 3 trna-like structures are often but not always aminoacylated by host aminoacyl-transferases and interact with eif1a (Bastin and Hall 1976; Joshi et al. 1986; Hall et al. 1987; van Belkum et al. 1987; Goodwin et al. 1997; Dreher and Goodwin 1998; Dreher et al. 1999). Similarly, the 3 pseudoknot structure of Tobacco mosaic tobamovirus (TMV) interacts with eif1a (Zeenko et al. 2002). As discussed above, eif1a is implicated in translation initiation of cellular mrnas and is likely to play a similar role in stimulating the translation of these viral RNAs. In addition, binding of eif1a to the aminoacylated 3 trnalike structure of Turnip yellow mosaic tymovirus (TYMV) also results in a reduction of ( )-strand synthesis by the RdRp. This suggests that the switch from translation to replication is down-regulated by eif1a (Matsuda et al. 2004; Matsuda and Dreher 2004). The 3 UTR of Alfalfa mosaic alfamovirus (AMV) on its own does not have significant translational enhancement activity (Gallie and Kobayashi 1994). However, it acts as a potent translational enhancer in the presence of the viral coat protein (CP) (Bol 2005; Krab et al. 2005). Addition of a polya tail at the 3 end of AMV RNAs eliminates the need to add CP to enhance their translation. This suggests that the AMV CP can replace PABP in the formation of a protein bridge between the 5 and 3 end of the viral RNA (Neeleman et al. 2001). In support of this model, CP was recently shown to interact with eif4g and eif(iso)4g (Krab et al. 2005). It has also been suggested that the interaction of CP with the 3 UTR of viral RNAs may be involved in the switch from translation to replication, although the exact mechanism of this regulation remains to be determined (Olsthoorn et al. 1999; Bol 2005; Guogas et al. 2005; Petrillo et al. 2005). In the TMV genomic RNA, an additional translational enhancer, the omega sequence, is present in the 5 UTR of the RNA. The omega sequence consists of a series of repeated motifs that are recognized by HSP101, a heat-shock-induced chaperone (Gallie and Walbot 1992; Wells et al. 1998). The omega sequence also recruits eif4g, possibly with the help of HSP101 (Gallie 2002a). While the interactions mentioned above are thought to enhance general translation of viral proteins, mutations of a yeast translation initiation factor DED1 revealed that this factor is required for the translation of one but not all Brome mosaic bromovirus (BMV) RNAs (Noueiry et al. 2000). The translational dependence for DED1 of BMV RNA2 participates in down-regulating the translation of the RdRp, which is encoded by this RNA (Noueiry et al. 2000). DED1 enco-

5 Table 1. Membrane association of viral replication complexes (VRC) in various plant RNA viruses. Virus Genus (family) Membranes Probable membrane anchor protein(s) RdRp (mechanism of assembly in VRC) No. of RNA molecules (mechanism of assembly in VRC) Alpha-like viruses CymRSV Tombus (tombus) Peroxysome p33 and p92 p92 (not known) 1 CIRV Tombus (tombus) Mitochondria p36 and p93 p93 (not known) 1 TBSV Tombus (tombus) Not known p33 p92 (associate with membrane, interaction 1 (interaction with p33 and p92) with p33) RCNMV Diantho (tombus) ER p27 p88 (probably interaction with p27) 1 TMV Tobamo ER Tom1 host protein Heterodimer 126 kda - readthrough 183 kda (interaction with Tom1) 1 (interaction with 126 and 183 kda) BMV Bromo (bromo) ER 1a (MT+Hel) 2a (interaction with 1a) 3 (RNA1:co-translational, RNA2 3: interaction with 1a) AMV Alfamo (bromo) Vacuole P1 (MT+Hel) P2 (interaction with P1) 3 CMV Cucumo (bromo) Vacuole P1 (MT+Hel) 2a (interaction with 1a) 3 TYMV Tymo (tymo) Chloroplast 140 kda (MT+Pro+Hel) 66 kda (interaction with 140 kda) 1 Picorna-like viruses TEV Poty (poty) ER 6 kda (polyprotein containing 6 kda) NIb (interaction with 6K-NIa) 1 (interaction with NIa) CPMV Como (como) ER 32 kda, 60 kda (Hel-VPg) Pol (interaction with polyprotein 2 (RNA1: co-translational RNA2: containing 60 kda) ToRSV Nepo (como) ER X2, NTB-VPg (Hel-VPg) VPg-Pro-Pol (possible protein protein interaction) possible role of 68 kda protein) 2 (not known) Note: AMV, Alfalfa mosaic alfamovirus; BMV, Brome mosaic bromovirus; BYDV, Barley yellow dwarf luteovirus; CIRV, Carnation Italian ringspot tombusvirus; CMV, Cucumber mosaic cucumovirus; CNV, Cucumber necrosis tombusvirus; CPMV, Cowpea mosaic comovirus; CymRSV, Cymbidium ringspot tombusvirus; ER, endoplasmic reticulum; GFLV, Grapevine fanleaf nepovirus; Hel, helicase; MT, methyltransferase; Pro, proteinase; PVA, Potato potyvirus A; RCNMV, Red clover necrotic mosaic dianthovirus; RdRp, RNA-dependent RNA polymerase; TBSV, Tomato bushy stunt tombusvirus; TEV, Tobacco etch potyvirus; TMV, Tobacco mosaic tobamovirus; ToRSV, Tomato ringspot nepovirus; TuMV, Turnip mosaic potyvirus; TYMV, Turnip yellow mosaic tymovirus; VPg, viral protein linked to the genome. Sanfaçon 1533

6 1534 Can. J. Bot. Vol. 83, 2005 des a RNA helicase involved in translation initiation of yeast mrnas, and is related to eif4a. Finally, translation of BMV genomic RNAs, but not subgenomic RNAs, requires deadenylation-dependent decapping factors (LSM1/ LSM7) (Noueiry et al. 2003). In plant cells, these factors play a crucial role in cellular mrna turnover, trna processing, ribosome biogenesis, and telomere maintenance, but are dispensable for the translation of cellular mrnas (Noueiry and Ahlquist 2003). Possibly, these factors act as chaperones to facilitate RNA-RNA interactions. Mutation of LSM1 does not prevent association of viral RNA with polysomes, but rather affected the translation elongation rate. Translation of viral RNAs that lack both a 5 cap and a 3 polya tail The genomic RNAs of Barley yellow dwarf luteovirus (BYDV) and members of the tombusviridae family lack both a 5 cap and a 3 polya tail. To compensate for this, they contain translational enhancers in their 3 UTRs (Wang and Miller 1995; Meulewaeter et al. 1998; Wu and White 1999; Qu and Morris 2000; Shen and Miller 2004). In BYDV and Tomato bushy stunt tombusvirus (TBSV), the 3 translational enhancers can functionally replace the missing cap structure and consist of a series of stems and loops (Wang et al. 1997; Allen et al. 1999; Wu and White 1999). One of these loops is involved in base-pairing with sequences in the 5 UTR. This promotes circularization of the genome and mimicks the cap-polya tail protein bridge formed during translation of cellular mrnas (Guo et al. 2000; van Lipzig et al. 2002; Fabian and White 2004). In the case of BYDV, complementation assays determined that the 3 element is sufficient for recruiting translation factors and ribosomes, while the 5 UTR plays a role in base pairing with the 3 element (Wang et al. 1997). Assembly of viral replication complexes in association with host intracellular membranes All (+)-strand RNA viruses replicate in close association with intracellular membranes (Salonen et al. 2005). However, the nature of these membranes varies from one virus family to another and even in some cases, from one virus to another. In plants, VRCs have been found in association with the endoplasmic reticulum (ER), the chloroplast outer membrane, the vacuole membrane, the peroxysomal membrane, and the mitochondrial membrane. Immunogold labelling of viral replication proteins revealed that they are localized in modified membrane structures, including membrane invaginations (spherules) and double membrane vesicles or multivesicles. Interestingly, although each virus induces specific membrane alterations with which VRCs are associated, recent results suggest that (i) alternate membrane rearrangements can support virus replication and (ii) VRCs can be retargeted to alternative membranes (Miller et al. 2003; Schwartz et al. 2004). Thus, the requirement for membrane association is somewhat flexible. As briefly discussed above, one or several viral or host proteins serve as a membrane anchor for the VRCs. These proteins are usually integral membrane proteins that bring other replication proteins to the VRCs by protein protein interactions or as polyprotein precursors. The viral membrane anchor proteins also have the ability to modify the membrane structure. In animal viruses, some of these proteins are viroporins that create pores in intracellular membranes and alter membrane permeability (Agirre et al. 2002; Gonzalez and Carrasco 2003). Although the ability of plant virus membrane proteins to act as viroporins has not yet been demonstrated experimentally, one nepovirus protein is a potential candidate (see below). The molecular basis for the targeting of membrane-anchor proteins to specific membranes, and for the recruitment of other viral and host components in the VRCs is still poorly understood. In the next few sections, I will review specific examples from various virus families (summarized in Table 1). These sections are not meant to be an exhaustive list of all known plant virus VRCs, but rather to discuss a few representative viruses that have been characterized in molecular detail. Tombusviruses: a small hydrophobic replication protein recruits viral RdRp and RNA to peroxysome and mitochondria membranes Members of the Tombusvirus genus have a relatively simple genome consisting of a single molecule of RNA that encodes two replication proteins (Fig. 2a) (White and Nagy 2004). Three additional proteins, including the viral coat protein, are also encoded by this RNA but are translated from two subgenomic RNAs. The first replication protein is a small protein that contains membrane-anchoring domains (Rubino and Russo 1998; Turner et al. 2004). The second replication protein is a larger protein that is produced by readthrough of a translational stop codon located at the C- terminus of the small protein. This protein contains the membrane-anchoring domains present in the small protein as well as a unique RdRp domain. The readthrough mechanism is inefficient, and the ratio of the small N-terminal protein to the full-length RdRp is approximately 20:1, at least in the case of TBSV (Scholthof et al. 1995). The small replication protein is an integral membrane protein that associates with membranes independently and induces drastic modification of the membrane morphology (Rubino et al. 2000, 2001; Weber-Lotfi et al. 2002; Navarro et al. 2004; Panavas et al. 2005). The nature of the membranes affected varies from one virus to another. Cymbidium ringspot tombusvirus (CymRSV) and Cucumber necrosis tombusvirus (CNV) p33 proteins associate with peroxisomal membranes, while Carnation Italian ringspot tombusvirus (CIRV) p36 protein is targeted to mitochondrial membranes. Targeting of CymRSV p33 to peroxysomal membranes and of CIRV p36 to mitochondrial membranes have striking similarities. In both cases, two transmembrane helices interspaced by a small hydrophilic loop are the primary determinants of membrane association (Weber-Lotfi et al. 2002; Navarro et al. 2004). The N- and C-termini of the two proteins are exposed to the cytosol, while the small hydrophilic loop is translocated into the membrane lumen. Expression of CymRSV p33, or CIRV p36, independently of other viral proteins results in membrane proliferation. However, expression of these proteins is not sufficient for the formation of infection-specific spherules. This suggests that other protein domains, possibly the RdRp domain, are involved in the induction of these structures. The specificity of association

7 Sanfaçon 1535 Fig. 2. Assembly of tombusvirus viral replication complexes (VRCs). (a) Genomic organization of Tomato bushy stunt virus. The monopartite genomic RNA is shown at the top of the figure with the boxes representing the different open reading frames. Stars, shaded diamonds, and black ovals represent predicted transmembrane helices, RNA-binding motifs, and RNA-dependent RNA polymerase (RdRp) domains, respectively. A protein protein interaction is shown by the open arrows. (b) Model for the assembly of tombusvirus VRCs (see text for details). Intracellular membranes are represented by the double thick hatched lines with the luminal side of the membranes towards the top of the figure and the cytosolic side towards the bottom. The viral RNA is represented by the thick black line. (a) Stop codon CP Fig. 3. Assembly of tobamovirus viral replication complexes (VRCs). (a) Genomic organization of Tobacco mosaic virus. The monopartite genomic RNA is shown at the top of the figure with the boxes representing the different open reading frames. Spiked ovals, shaded diamonds, shaded triangles, and black ovals represent predicted methyl-transferase domains, RNA-binding motifs, helicase domains, and RNA-dependent RNA polymerase (RdRp) domains, respectively. A protein protein interaction is shown by the open arrows. (b) Model for the assembly of tobamovirus VRCs (see text for details). (a) Stop codon 126 kda MP CP p kda (b) p33 p92 (b) 126 kda Tom1 Tom1 Tom2 Tom1 MP p33 p92 (1) (2) (3) p92 with mitochondrial, or peroxysomal, membranes is conferred by sequences within or adjacent to the transmembrane domains. In the case of CymRSV p33, a cluster of three positively charged amino acids upstream of the transmembrane domains is involved in peroxysome targeting (Navarro et al. 2004). Deletion of a similar sequence from the CNV p33 protein does not prevent its association with the peroxysome, suggesting that other sequences are also involved in peroxysome targeting (Panavas et al. 2005). The sequences directing mitochondria targeting of CIRV p36 protein are still poorly defined, but do not include a putative mitochondrial targeting signal at the N-terminus of the protein (Weber-Lotfi et al. 2002). The two replication proteins of TBSV interact with each other, and viral replication can be achieved when these proteins are expressed from two separate RNAs (Oster et al. 1998; Rajendran and Nagy 2004; Panavas et al. 2005). Two small domains at the C-terminus of p33 are responsible for p33 self-interaction and for its interaction with the larger p92 protein. Deletion of the membrane-targeting sequences from the CNV p92 protein does not prevent its association with peroxysomes when it is expressed in the presence of p33. In contrast, deletion of the p33:p33/p92 interaction domain prevents p92 peroxysome targeting (Panavas et al. 2005). These results suggest that formation of the p33-p92 (1) (2) (3) 183 kda complex occurs prior to peroxysome targeting, and that membrane-targeting sequences are predominantly active in the context of the p33 protein. TBSV p33 and p92 also contain several RNA-binding domains, one of which is in the overlapping region and is essential for viral replication (Panaviene et al. 2003; Panaviene and Nagy 2003; Rajendran and Nagy 2003). Two other RNA-binding domains are in the unique domain of p92. The region of the viral RNA interacting with the replication proteins has been delineated to a sequence within the RdRp coding region (Monkewich et al. 2005; Pogany et al. 2005). A current model (summarized in Fig. 2b) suggests that the template RNA interacts with p33 and p92 during translation and is then recruited to the membrane-bound VRCs. This may allow the switch from the translation to the replication mode. Co-expression of template viral RNA, in addition to the replication proteins, resulted in stimulated replicase activity. This suggests that the viral RNA plays an active role in VRC assembly and (or) stability (Panaviene et al. 2004). Tobamoviruses: host integral membrane proteins recruit VRCs to the ER The TMV genome consists of a single molecule of RNA that encodes four proteins (Buck 1999; Beachy and Heinlein 2000) (Fig. 3a). One of these, the 126 kda replication protein, contains the domain for a methyltransferase and a helicase. Another replication protein, the 183 kda protein, is synthesized after readthough of a translational stop codon located at the C-terminus of the 126 kda protein. The

8 1536 Can. J. Bot. Vol. 83, kda protein contains the domains present in the 126 kda as well as a unique RdRp domain. The movement and coat proteins are translated from subgenomic RNAs and are dispensable for viral RNA replication but play an accessory role in VRC assembly (see below) (Meshi et al. 1987; Beachy and Heinlein 2000; Asurmendi et al. 2004). The 126 and 183 kda proteins self-interact and interact with each other. They can be supplied from individual RNA molecules, and heterodimers of the 126 and 183 kda proteins are detected in purified VRCs (Ogawa et al. 1991; Osman and Buck 1996; Watanabe et al. 1999; Goregaoker et al. 2001). Mutants that produce only the 183 kda protein are able to replicate, but at a much lower level than the wildtype virus, indicating that hetero-oligomer formation is important (Lewandowski and Dawson 2000). The interface between the two interacting proteins includes the helicase domain and the region immediately upstream (Goregaoker et al. 2001). Oligomerization is required for the activity of recombinant helicase (Goregaoker and Culver 2003). A region of the 126 kda protein located between the methyltransferase and the helicase domains interacts with the trna-like structure at the 3 end of the viral RNA in a co-translational manner (Lewandowski and Dawson 2000; Osman and Buck 2003). This suggests that the 126 kda protein recruits the viral RNA to the VRCs immediately following translation. TMV infection results in drastic alterations in the ER structure (Reichel and Beachy 1998). Viral replication occurs in association with membranes derived from the ER, and possibly also from the vacuole membrane (Heinlein et al. 1998; Mas and Beachy 1999; Hagiwara et al. 2003). The 126 and 183 kda proteins associate with the ER independently of other viral proteins (dos Reis Figueira et al. 2002). However, a large proportion of these proteins remains soluble in the context of a viral infection, suggesting that membrane attachment is not always efficient (Hagiwara et al. 2003). The 126 and 183 kda proteins do not contain hydrophobic domains that could serve as transmembrane domains. Thus, ER association of these proteins could occur either through a direct interaction of a putative amphipathic helix with the membranes or through protein protein interactions with host membrane proteins (dos Reis Figueira et al. 2002). Analysis of a recessive Arabidopsis mutant with reduced ability to support TMV replication allowed the identification of two related integral membrane proteins, Tom1 and Tom3 (Ishikawa et al. 1993; Yamanaka et al. 2000, 2002). Tom1 and Tom3 can functionally replace each other, and both proteins have the ability to interact with the helicase domain of the TMV 126 and 183 kda proteins. In TMV-infected plants, Tom1 is associated with intracellular membranes that have TMV-specific replication activity, suggesting that it is an integral part of the VRCs (Hagiwara et al. 2003). An additional Arabidopsis mutant (tom2) that is also deficient in TMV replication has been identified (Ohshima et al. 1998). Two host factors (Tom2A and Tom2B) interact differently with specific strains of TMV (Tsujimoto et al. 2003). One of these factors, Tom2A, is a transmembrane protein that does not interact directly with TMV replication proteins, but interacts with itself and with the Tom1 protein. In subcellular fractionation experiments, the Tom2A protein is present in some but not all membrane fractions having RdRp activity. This suggests that it plays Fig. 4. Assembly of bromovirus viral replication complexes (VRCs). (a) Genomic organization of Brome mosaic virus. The tripartite genomic RNA is shown at the top of the figure with the boxes representing the different open reading frames. Spiked ovals, stars, shaded triangles, and black ovals represent predicted methyltransferase domains, membrane-association domains, helicase domains, and RNA-dependent RNA polymerase (RdRp) domains, respectively. Intramolecular and intermolecular protein protein interactions are shown by the open arrows. (b) Model for the assembly of bromovirus VRCs (see text for details). To simplify the figure, only one of the three viral RNAs is shown (thick black line). (a) (b) RNA-1 1a 1a RNA-2 1a 2a (1) (2) (3) (4) RNA-3 an accessory role in replication, possibly by allowing the formation of Tom1-Tom2 multimers in the membranes and enhancing the interaction of Tom1 with the TMV 126 and 183 kda proteins (Tsujimoto et al. 2003). Several lines of evidence suggest that the TMV movement protein (MP) plays an accessory role in VRC assembly (Beachy and Heinlein 2000). Although it is not strictly required for replication and it does not interact directly with the replication proteins (Meshi et al. 1987; Mas and Beachy 1999; Hirashima and Watanabe 2003), MP is found in association with the VRCs (Heinlein et al. 1998; Mas and Beachy 1999; Asurmendi et al. 2004; Kawakami et al. 2004). Early in infection, the VRCs are localized to small vesicles associated with the ER, often at a perinuclear location. At middle stages of infection, large irregular bodies associated with the ER, which contain viral RNA, MP, and replication proteins, are formed. At late stages of infection, cytopathic structures are present as aggregates near the plasma membrane, which then slowly migrate towards the plasmodesmata. Recent evidence suggests that the entire VRC is translocated through the plasmodesmata to adjacent cells, allowing immediate initiation of the replication cycle in the new cell (Kawakami et al. 2004). MP is probably responsible for the formation of the large irregular bodies and for their translocation to the plasma membrane and through the plasmodesmata. However, the absence of MP does not prevent the formation of the initial complexes at the perinuclear location (Mas and Beachy 1999). MP is an integral membrane protein that associates with and modifies the structure of the ER when it is expressed individually (Kahn 2a CP 2a

9 Sanfaçon 1537 et al. 1998; Reichel and Beachy 1998). Migration of VRCs to the plasma membrane and movement to adjacent cells is prevented by treatment with inhibitors of actin filaments, suggesting that VRCs move in association with these filaments (Kawakami et al. 2004). A model for the assembly of tobamovirus VRCs outlining the interactions between viral replication proteins and RNAs and between viral and host proteins is summarized in Fig. 3b. Bromoviridae: the 1a replication protein recruits the viral RdRp and RNAs to intracellular membranes Members of the bromoviridae family are tripartite viruses and include the bromovirus, cucumovirus, and alfalmovirus genus (Noueiry and Ahlquist 2003; Palukaitis and Garcia- Arenal 2003; Bol 2005). RNA1 and RNA2 each encode a replication protein: 1a, containing the domains for the methyltransferase and helicase, and 2a, the RdRp (Fig. 4a). RNA3 encodes the movement and coat proteins. Replication occurs in membranous spherules that contain 1a and 2a and are associated with the ER in the case of bromoviruses, or with vacuolar membranes in the case of alfamoviruses and cucumoviruses (Restrepo-Hartwig and Ahlquist 1996; Van Der Heijden et al. 2001; Cillo et al. 2002). Within the BMV VRCs, the ratio of 1a to 2a is 25:1 (Schwartz et al. 2002). 1a is the membrane-anchor for the VRCs as it can associate with the ER independently and can redirect 2a, which is otherwise cytoplasmic, to ER membranes (Restrepo-Hartwig and Ahlquist 1999; Chen and Ahlquist 2000). 1a does not contain extensive hydrophobic regions. Yet, the membrane association of 1a is resistant to high salt treatment and high ph suggesting that it is tightly associated with the membranes (den Boon et al. 2001). Regions of 1a involved in membrane association have been identified, but the nature of the protein-membrane interaction remains to be elucidated. 1a expressed alone stimulates membrane spherule formation and membrane lipid accumulation (Schwartz et al. 2002; Lee and Ahlquist 2003). BMV does not replicate in the OLE1 yeast mutant. This mutant has a defect of D9 fatty acid desaturase that results in reduced levels of unsaturated fatty acid (Lee et al. 2001; Lee and Ahlquist 2003). In this mutant, ( )-strand synthesis is severely inhibited, even though 1a associates with membranes, induces spherule formation, and recruits other components of the VRC. This result suggests that the membrane spherules do not only serve a protective role for the nascent RNA, but that they play a direct role in RNA replication, which is influenced by the lipid composition of the membrane. Protein 1a interacts with itself, and with 2a, and these interactions are essential for virus replication (Kao and Ahlquist 1992; Kao et al. 1992; Dinant et al. 1993; O Reilly et al. 1995, 1997, 1998). The N-terminal half of 1a is involved in self-interaction, while the C-terminal half of 1a interacts with the N-terminal region of 2a. In addition, an intramolecular interaction between the N-terminal and C-terminal regions of 1a has been identified (O Reilly et al. 1998). This interaction may be temporary and help delay the interaction of 2a with 1a until proper oligomers of 1a have been formed (Fig. 4b). The interacting domains of 1a and 2a are conserved among members of the bromoviridae family, suggesting that there are similarities in the assembly and architecture of the VRCs (O Reilly et al. 1998). The three bromovirus RNAs are recruited to the VRCs by protein-rna interactions. RNA1 probably remains associated with 1a following translation. 1a also interacts directly or indirectly with a conserved motif present at the 5 end of RNA2 and in the central region of RNA3 (Sullivan and Ahlquist 1999; Chen et al. 2001). This motif is similar to the box B sequence of cellular trnas, which is recognized by the host RNA polymerase III. This region of the viral RNAs is also modified by the host machinery to resemble the structure of the TCC stem-loop of host trnas (Baumstark and Ahlquist 2001). Thus, host factors may be involved in the interaction between 1a and RNAs 2 and 3. 1adependent recruitment of viral RNAs into the membrane spherules protects these RNAs from degradation by exogenous RNases (Schwartz et al. 2002). An alternative pathway for the recruitment of RNA2 to the VRCs has been recently suggested, in which 1a interacts with the nascent 2a protein during translation, thereby recruiting the translating mrna to the VRCs (Chen et al. 2003). Tymoviruses: the 140 kda protein recruits viral RNA and RdRp to chloroplast membranes The TYMV genome consists of a single molecule of RNA that contains three open reading frames (Martelli et al. 2002). Two replication proteins are released from a 206 kda polyprotein precursor through the action of the viral proteinase (Bransom et al. 1996). The first protein is a 140 kda protein that contains the domains for the methyltransferase, proteinase, and helicase. The second protein is a 66 kda protein that contains the domain for the RdRp. Replication proteins are associated with membrane invaginations of the chloroplast envelope and co-fractionate with the replication activity in a highly purified chloroplast membrane fraction (Prod homme et al. 2001). The 140 kda protein is the membrane anchor protein, as evidenced by its ability to target the chloroplast envelope when expressed independently of other viral proteins, and redirect the RdRp to the chloroplast envelope through protein protein interactions (Prod homme et al. 2003; Jakubiec et al. 2004). The proteinase domain within the 140 kda protein is responsible for the interaction with the RdRp and also contains chloroplasttargeting sequences (Jakubiec et al. 2004). TYMV RNA is preferentially replicated in cis, suggesting that the replication proteins interact with the viral RNA during, or immediately following, translation (Weiland and Dreher 1993; Dreher and Weiland 1994). Because the requirement for cis replication is stronger for the 140 kda protein, this protein probably contains the RNA-binding domain (Weiland and Dreher 1993; Dreher and Weiland 1994). The RNA-binding and chloroplast-targeting domains within the 140 kda protein remain to be examined in further detail. Plant picorna-like viruses: hydrophobic proteins recruit other viral replication proteins to ER membranes either as polyprotein precursors or via protein protein interactions Plant picorna-like viruses induce severe modifications of ER membranes, and their VRCs are associated with ER-derived membranes (Schaad et al. 1997; Carette et al. 2000; Carette et al. 2002a; Ritzenthaler et al. 2002; Han and Sanfacon 2003). Replication proteins are found in association

10 1538 Can. J. Bot. Vol. 83, 2005 Fig. 5. Assembly of potyvirus viral replication complexes (VRCs). (a) Genomic organization of Tobacco etch virus. The monopartite genomic RNA is shown at the top of the figure with the boxes representing the different open reading frames. Although various cleavage products (including intermediate precursors and mature proteins) are produced by alternative processing pathways, only a few relevant cleavage products are shown in the figure. Scissors, shaded triangles, stars, black rectangle, shaded diamonds, and black ovals represent the predicted domains for proteinases, helicase, membrane-association sequences, viral protein linked to the genome (VPg), and RNA-dependent RNA polymerase (RdRp), respectively. A protein protein interaction domain is shown by the open arrow. (b) Model for the assembly of potyvirus VRCs (see text for details). (a) VPg (b) P1 HC-Pro P3 VPg CI 6k Pro NIb CI-6k CI-6k 6k-NIa (1) (2) NIa 6k-NIa with membranous vesicles induced during viral infection (Wellink et al. 1988; Restrepo-Hartwig and Carrington 1994; Ritzenthaler et al. 2002). Replication of comoviruses and nepoviruses requires de novo synthesis of phospholipids, as demonstrated by its sensitivity to cerulenin, an inhibitor of lipid biosynthesis (Carette et al. 2000; Ritzenthaler et al. 2002). This property is not shared by TMV and Peanut clump pecluvirus, two unrelated viruses that replicate in association with ER membranes (Carette et al. 2000; Dunoyer et al. 2002; Ritzenthaler et al. 2002). Replication of Grapevine fanleaf nepovirus (GFLV) is inhibited by Brefeldin A, an inhibitor of vesicle trafficking between the ER and the Golgi apparatus. This suggests that the virus may be using the host anterograde transport pathway to recruit membranes for viral replication (Ritzenthaler et al. 2002). This is similar to the model suggested for the formation of replication-competent vesicles in animal cells during poliovirus infection. Cellular COP proteins are involved in the formation of poliovirus-induced vesicles (Rust et al. 2001; Gazina et al. 2002), but this remains to be tested for the plant picornalike viruses. Potyviruses The genome of members of the potyvirus genus consists of a single RNA encoding a large polyprotein, from which mature proteins and intermediate precursors are released NIb CP NIb through the action of three viral-encoded proteinases (Riechmann et al. 1992; Urcuqui-Inchima et al. 2001) (Fig. 5). The putative membrane-anchor for the TEV VRCs is a small 6 kda protein, also called the 6K protein. The 6 kda protein is largely responsible for the membrane modifications observed in infected plants and contains a highly hydrophobic domain that directs its association with intracellular membranes (Restrepo-Hartwig and Carrington 1994; Schaad et al. 1997). Various intermediate polyprotein precursors containing the domain for the 6 kda protein, including CI-6K and 6K-NIa, which are produced by alternative processing pathways of a larger polyprotein precursor, have been detected in plants. CI is a helicase and has the ability to oligomerize through its N-terminal domain (Eagles et al. 1994; Fernandez et al. 1995, 1997; Lopez et al. 2001). NIa contains the domains for VPg and a proteinase, and accumulates in the nucleus, unless it is retargeted to the membrane-bound VRCs by the domain for the 6 kda protein present on the 6K-NIa polyprotein (Garcia et al. 1989; Carrington et al. 1991; Laliberte et al. 1992; Restrepo-Hartwig and Carrington 1992; Carrington et al. 1993). The RdRp (NIb protein) is brought into the VRCs through protein protein interactions with the VPg or proteinase domains of NIa (Hong et al. 1995; Li and Carrington 1995; Li et al. 1997; Fellers et al. 1998; Daros et al. 1999). In the case of Tobacco vein mottling potyvirus, the activity of NIb is stimulated by its interaction with the VPg (Fellers et al. 1998). A large network of interactions between these and other accessory viral replication proteins, such as P1 and P3, has been documented for several members of the potyviridae family (Verchot and Carrington 1995; Merits et al. 1999; Choi et al. 2000; Guo et al. 2001). These interactions may be important for the stability or assembly of the VRCs, although this remains to be shown experimentally. The viral RNA is probably recruited to VRCs by the NIa proteinase domain (Daros and Carrington 1997), although other viral proteins including P1 and CI also interact with the viral RNA in vitro (Brantley and Hunt 1993; Soumounou and Laliberte 1994; Fernandez et al. 1995; Fernandez and Garcia 1996; Merits et al. 1998). Comoviruses and nepoviruses The genome organization of members of the comoviridae family, including the comovirus and nepovirus genus, is similar to that of potyviruses, but with one major difference: the genome is divided into two RNAs (Fig. 6a). RNA1 codes for the replication proteins and can replicate independently, while RNA2 codes for structural and movement proteins and is dependent on RNA1 for its replication (Sanfacon 1995; Goldbach and Wellink 1996; Mayo and Robinson 1996). Each RNA codes for a large polyprotein that is processed by a single viral proteinase. Two proteins have been identified as putative membrane anchors for the VRCs. These proteins associate with and modify ER membranes independently of other viral proteins (Carette et al. 2002b; Zhang et al. 2005). The Cowpea mosaic comovirus (CPMV) 60 kda protein and the corresponding nepovirus NTB-VPg protein contain the domains for the putative helicase and the VPg. The Tomato ringspot nepovirus (ToRSV) NTB-VPg protein is an integral membrane protein associated with ER-bound VRCs in infected plant cells (Han and