The Experts below are selected from a list of 222 Experts worldwide ranked by ideXlab platform
N. Yoshikawa - One of the best experts on this subject based on the ideXlab platform.
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spherical virus: a new virus classified into the family
2013Co-Authors: N. Yoshikawa, T Takahashi, T Ito, K Yoshida, H KoganezawaAbstract:A virus with isometric virus particles (ca. 25 nm) was isolated from an apple tree and named Apple latent spherical virus (ALSV). Virus particles purified from infected Chenopodium quinoa formed two bands with densities of 1�41 and 1�43 g/cm 3 in CsCl equilibrium density-gradient centrifugation, indicating that the virus is composed of two components. The virus had two ssRNA species (RNA1 and RNA2) and three capsid proteins (Vp25, Vp24 and Vp20). The complete nucleotide sequences of RNA1 and RNA2 were determined to be 6815 nt and 3384 nt excluding the 3� poly(A) tail, respectively. RNA1 contains two partially overlapping ORFs encoding polypeptides of molecular mass 23 kDa (‘23K’; ORF1) and 235 kDa (‘235K’; ORF2); RNA2 has a single ORF encoding a polypeptide of 108 kDa (‘108K’). The 235K protein has, in order, consensus motifs of the protease cofactor, the NTP-binding helicase, the cysteine protease and the RNA polymerase, in good agreement with the gene arrangement of viruses in the Comoviridae. The 108K protein contains an LPL movement protein (MP) motif near the N terminus. Direct sequencing of the N-terminal amino acids of the three capsid proteins showed that Vp25, Vp20 and Vp24 are located in this order in the C-terminal region of the 108K protein. The cleavage sites of the 108K polyprotein were Q/G (MP/Vp25 and Vp25/Vp20) and E/G (Vp20/Vp24). Phylogenetic analysis of the ALSV RNA polymerase domain showed that ALSV falls into a cluster different from the nepo-, como- and fabavirus lineages
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Cheravirus and Sadwavirus: two unassigned genera of plant positive-sense single-stranded RNA viruses formerly considered atypical members of the genus Nepovirus (family Comoviridae)
Archives of Virology, 2007Co-Authors: O. Gall, Alexander Karasev, Joan Wellink, K Lehto, M. Ikegami, T. Iwanami, T. Jones, T. Wetzel, N. YoshikawaAbstract:The genus Nepovirus (family Comoviridae ) was known both for a good level of homogeneity and for the presence of atypical members. In particular, the atypical members of the genus differed by the number of capsid protein (CP) subunits. While typical nepoviruses have a single CP subunit with three structural domains, atypical nepoviruses have either three small CP subunits, probably corresponding to the three individual domains, or a large and a small subunit, probably containing two and one structural domains, respectively. These differences are corroborated by hierarchical clustering based on sequences derived from both genomic RNAs. Therefore, these atypical viruses are now classified in two distinct genera, Cheravirus (three CP subunits; type species Cherry rasp leaf virus ) and Sadwavirus (two CP subunits; type species Satsuma dwarf virus ).
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Nucleotide sequence and genome organization of apple latent spherical virus: a new virus classified into the family Comoviridae.
The Journal of general virology, 2000Co-Authors: N. Yoshikawa, T Takahashi, T Ito, K Yoshida, H KoganezawaAbstract:A virus with isometric virus particles (ca. 25 nm) was isolated from an apple tree and named Apple latent spherical virus (ALSV). Virus particles purified from infected Chenopodium quinoa formed two bands with densities of 1.41 and 1.43 g/cm(3) in CsCl equilibrium density-gradient centrifugation, indicating that the virus is composed of two components. The virus had two ssRNA species (RNA1 and RNA2) and three capsid proteins (Vp25, Vp24 and Vp20). The complete nucleotide sequences of RNA1 and RNA2 were determined to be 6815 nt and 3384 nt excluding the 3' poly(A) tail, respectively. RNA1 contains two partially overlapping ORFs encoding polypeptides of molecular mass 23 kDa ('23K'; ORF1) and 235 kDa ('235K'; ORF2); RNA2 has a single ORF encoding a polypeptide of 108 kDa ('108K'). The 235K protein has, in order, consensus motifs of the protease cofactor, the NTP-binding helicase, the cysteine protease and the RNA polymerase, in good agreement with the gene arrangement of viruses in the Comoviridae: The 108K protein contains an LPL movement protein (MP) motif near the N terminus. Direct sequencing of the N-terminal amino acids of the three capsid proteins showed that Vp25, Vp20 and Vp24 are located in this order in the C-terminal region of the 108K protein. The cleavage sites of the 108K polyprotein were Q/G (MP/Vp25 and Vp25/Vp20) and E/G (Vp20/Vp24). Phylogenetic analysis of the ALSV RNA polymerase domain showed that ALSV falls into a cluster different from the nepo-, como- and fabavirus lineages.
Helene Sanfacon - One of the best experts on this subject based on the ideXlab platform.
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secoviridae a family of plant picorna like viruses with monopartite or bipartite genomes
eLS, 2015Co-Authors: Helene SanfaconAbstract:Members of the family Secoviridae, referred to as secovirids, are plant viruses that share features with animal and human viruses of the family Picornaviridae and other insect or marine viruses of the order Picornavirales. These common features include a conserved structure of the icosahedral virus particles, the expression of viral proteins by proteolytic cleavage of large polyproteins and viral replication proteins with conserved sequence motifs, including the viral RNA-dependent RNA polymerase and protease. Secovirids also share the distinguishing feature of encoding specialised proteins that enable their movement in plant and counteract plant defence mechanisms, such as RNA silencing. The family Secoviridae includes eight genera. Members of the genera Comovirus, Fabavirus, Nepovirus, Cheravirus, Sadwavirus and Torradovirus have a bipartite positive-strand RNA genome, whereas members of the genera Sequivirus and Waikavirus have a monopartite genome. Key Concepts Many plant viruses are related to animal and human viruses of the family Picornaviridae and to other picorna-like viruses infecting algae and arthropods. A recent update in the taxonomy of plant picorna-like viruses has led to the creation of the family Secoviridae which amalgamates the previous families Comoviridae and Sequiviridae as well as the genera Cheravirus, Sequivirus and Torradovirus. Secovirids share common characteristics including having both similar virus particle structures and genomic organizations and requiring specialised proteins to facilitate their movement within the host plant or counteract plant defence mechanism. Secovirids produce their proteins in the form of large polyproteins that are cleaved at specific sites by a viral protease. Replication of the viral RNA occurs in large protein complexes in association with intracellular membranes from the host. Plant cells infected with secovirids generally display tubular structures that are composed of the viral movement protein, contain virus-like particles and traverse the cell wall. These tubular structures are involved in the movement of the virus from cell to cell. Secovirids can be transmitted through seeds and pollen or with the help of nematode or arthropod vectors and their spread in the field is largely dependent on their mode of transmission. Some secovirids have been successfully exploited as plant vectors, allowing epitope presentation for vaccine production, expression of proteins in plants and silencing of endogenous plant genes. Keywords: picornavirales; protease; virus taxonomy; plant–virus interactions; cell-to-cell movement; virus replication
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Insertion of large amino acid repeats and point mutations contribute to a high degree of sequence diversity in the X4 protein of tomato ringspot virus (genus Nepovirus)
Archives of Virology, 2009Co-Authors: Bita Jafarpour, Helene SanfaconAbstract:Tomato ringspot virus (ToRSV) is a serious pathogen of small fruits and fruit trees in North America [14]. Several ToRSV isolates have been described that differ in natural host range and intensity of symptoms in herbaceous hosts [3, 17]. Tomato ringspot virus is a species of the genus Nepovirus [15], which has recently been reassigned to the proposed new family ‘‘Secoviridae’’ (subfamily ‘‘Comovirinae’’) within the order Picornavirales [16]. Nepoviruses have a bipartite positive-strand RNA genome. Each RNA encodes a polyprotein that is cleaved by the viral proteinase (Pro) at specific cleavage sites. RNA1 encodes replication proteins, while RNA2 codes for the coat protein (CP), movement protein (MP) and other protein(s) of less defined function. Using in vitro processing assays, three proteinase cleavage sites have been identified in the RNA2encoded polyprotein of ToRSV (a subgroup C nepovirus), allowing the definition of four protein domains (X3, X4, MP and CP) [4, 5]. In contrast, only three protein domains are present in the polyprotein of subgroup A and B nepoviruses (2a, MP and CP; see Fig. 1a). The ToRSV X3 protein shares conserved sequence motifs with the 2a protein of subgroup A and B nepoviruses [12]. The 2a protein of grapevine fanleaf virus has been shown to play a role in the replication of RNA2 [7]. The ToRSV X4 protein does not have significant sequence identity with proteins available in the database and does not have a functional equivalent in the genome of nepoviruses of subgroups A and B (Fig. 1a). The only other nepovirus of subgroup C for which the entire nucleotide sequence is available is blackcurrant reversion virus (BRV). The extent of sequence identity between ToRSV and BRV is very low in the deduced amino acid (a.a.) sequence of the X4 protein (8%) but higher in the deduced a.a. sequence of the CP (29%), VPg (viral protein linked to the genome), Pro and polymerase (Pol) (36%). In this study, we examined the extent of sequence diversity in the X4 protein among closely related ToRSV isolates. We show that there is a high degree of sequence diversity in the X4 protein, which is due in part to the insertion of multiple copies of two types of large amino acid repeats.
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secoviridae the amalgamation of the families sequiviridae and Comoviridae
eLS, 2009Co-Authors: Helene SanfaconAbstract:Several plant viruses share features with animal and human viruses of the family Picornaviridae, including a conserved structure of both the virus particle and the viral genome, expressing viral proteins by proteolytic cleavage of large polyproteins and encoding replication proteins with conserved sequence motifs. Members of the family Comoviridae were originally described as the only plant picorna-like viruses. Other plant picorna-like viruses were later discovered and classified in the family Sequiviridae. Sequiviridae and Comoviridae are related to each other in phylogenetic studies and share the common property of encoding specialized proteins to enable their movement in the plant. Recently, it was proposed to regroup plant picorna-like viruses into a single family termed ‘secoviridae’. The proposed family amalgamates the families Comoviridae and Sequiviridae, and incorporates other plant picorna-like viruses currently classified in the genera Sadwavirus and Cheravirus, and the proposed genus ‘Torradovirus’. Key concepts: Many plant viruses are related to the animal and human picornaviridae and to other picorna-like viruses infecting algae and arthropods. A recent update in the taxonomy of plant picorna-like viruses has lead to the creation of the family ‘secoviridae’ which amalgamates the families Comoviridae and Sequiviridae as well as the existing genera Cheravirus, Sequivirus and the proposed genus ‘torradovirus’. Secoviridae share many common characteristics including having both similar virus particle structures and genomic organizations, and requiring a specialized protein to facilitate their movement within the host plant. Secoviridae produce their proteins in the form of large polyproteins that are cleaved at specific sites by a viral proteinase. Replication of the viral RNA occurs in large protein complexes in association with intracellular membranes from the host. Plant cells infected with secoviridae generally display tubular structures that are composed of the viral movement protein, contain virus-like particles and traverse the cell wall. These tubular structures are probably involved in the movement of the virus from cell to cell. Secoviridae can be transmitted through seeds and pollen or with the help of nematode or arthropod vectors and their spread in the field is largely dependent on their mode of transmission. Keywords: picornavirales; proteinase; virus taxonomy; plant–virus interactions; cell-to-cell movement; virus replication
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Characterization of Membrane Association Domains within the Tomato Ringspot Nepovirus X2 Protein, an Endoplasmic Reticulum-Targeted Polytopic Membrane Protein
Journal of virology, 2006Co-Authors: Guangzhi Zhang, Helene SanfaconAbstract:Replication of nepoviruses (family Comoviridae) occurs in association with endoplasmic reticulum (ER)-derived membranes. We have previously shown that the putative nucleoside triphosphate-binding protein (NTB) of Tomato ringspot nepovirus is an integral membrane protein with two ER-targeting sequences and have suggested that it anchors the viral replication complex (VRC) to the membranes. A second highly hydrophobic protein domain (X2) is located immediately upstream of the NTB domain in the RNA1-encoded polyprotein. X2 shares conserved sequence motifs with the comovirus 32-kDa protein, an ER-targeted protein implicated in VRC assembly. In this study, we examined the ability of X2 to associate with intracellular membranes. The X2 protein was fused to the green fluorescent protein and expressed in Nicotiana benthamiana by agroinfiltration. Confocal microscopy and membrane flotation experiments suggested that X2 is targeted to ER membranes. Mutagenesis studies revealed that X2 contains multiple ER-targeting domains, including two C-terminal transmembrane helices and a less-well-defined domain further upstream. To investigate the topology of the protein in the membrane, in vitro glycosylation assays were conducted using X2 derivatives that contained N-glycosylation sites introduced at the N or C termini of the protein. The results led us to propose a topological model for X2 in which the protein traverses the membrane three times, with the N terminus oriented in the lumen and the C terminus exposed to the cytoplasmic face. Taken together, our results indicate that X2 is an ER-targeted polytopic membrane protein and raises the possibility that it acts as a second membrane anchor for the VRC.
Marc Fuchs - One of the best experts on this subject based on the ideXlab platform.
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multiple interspecies recombination events within rna2 of grapevine fanleaf virus and arabis mosaic virus
Archives of Virology, 2008Co-Authors: Emmanuelle Vigne, Marc Fuchs, Aurelie MarmonierAbstract:Sequence alignments and SISCAN analyses inferred multiple interspecies recombination events within RNA2 of strains GHu of Grapevine fanleaf virus (GFLV) and Ta of Arabis mosaic virus (ArMV), two closely related subgroup A nepoviruses in the family Comoviridae. Interspecies recombination events were identified in the 5′ untranslated region, the putative homing protein and movement protein genes but not in the coat protein gene and 3′ untranslated region. These findings suggest a dynamic relationship between GFLV and ArMV, and a differential selection pressure on RNA2-encoded proteins with constraints in terms of function and co-adaptation that limit interspecies recombination to certain gene segments.
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grapevine fanleaf virus still a major threat to the grapevine industry
Journal of Plant Pathology, 2004Co-Authors: P Andretlink, Christophe Ritzenthaler, Gerard Demangeat, Emmanuelle Vigne, C Stussigaraud, Laure Valat, C Laporte, V Laval, P Pfeiffer, Marc FuchsAbstract:SUMMARY Grapevine fanleaf virus (GFLV) is responsible for fanleaf degeneration, which is one of the most severe virus diseases of grapevines worldwide. GFLV causes substantial crop losses, reduces fruit quality and shortens the longevity of grapevines in the vineyard. GFLV is transmitted specifically from grapevine to grapevine by the ectoparasitic nematode Xiphinema index, and belongs to the genus Nepovirus in the family Comoviridae. Since the discovery of the nematode vector in the late 1950’s and the identification of GFLV as the agent responsible for fanleaf degeneration in the early 1960’s, a wealth of information has been accumulated on its transmission, biological properties and serological characteristics, as well as on the structure and expression of the GFLV genome. Although dissemination of the virus through propagation material has been drastically reduced over the past two decades by implementing rigorous certification schemes and establishing quarantine facilities, effective strategies are still needed to control GFLV in naturally infected vineyards. Recently, significant progress has been made on the elucidation of the function(s) of most GFLV proteins, in particular those involved in critical steps of the virus multiplication cycle, including RNA replication, cell-to-cell movement, and transmission by X. index. New insights have also been gained into the population structure and genomic variability among isolates from naturally infected vineyards, which have opened new avenues for designing alternative strategies to control this destructive virus. This review article offers a comprehensive overview of the most significant advances made over the past 15 years on GFLV and discusses novel control strategies for one of the major threats to the grapevine industry worldwide.
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population structure and genetic variability within isolates of grapevine fanleaf virus from a naturally infected vineyard in france evidence for mixed infection and recombination
Journal of General Virology, 2004Co-Authors: Emmanuelle Vigne, Marc Bergdoll, Sebastien Guyader, Marc FuchsAbstract:The nematode-borne Grapevine fanleaf virus, from the genus Nepovirus in the family Comoviridae, causes severe degeneration of grapevines in most vineyards worldwide. We characterized 347 isolates from transgenic and conventional grapevines from two vineyard sites in the Champagne region of France for their molecular variant composition. The population structure and genetic diversity were examined in the coat protein gene by IC-RT-PCR-RFLP analysis with EcoRI and StyI, and nucleotide sequencing, respectively. RFLP data suggested that 55 % (191 of 347) of the isolates had a population structure consisting of one predominant variant. Sequencing data of 51 isolates representing the different restrictotypes confirmed the existence of mixed infection with a frequency of 33 % (17 of 51) and showed two major predominant haplotypes representing 71 % (60 of 85) of the sequence variants. Comparative nucleotide diversity among population subsets implied a lack of genetic differentiation according to host (transgenic vs conventional) or field site for most restrictotypes (17 of 18 and 13 of 18) and for haplotypes in most phylogenetic groups (seven of eight and six of eight), respectively. Interestingly, five of the 85 haplotypes sequenced had an intermediate divergence (0·036–0·066) between the lower (0·005–0·028) and upper range (0·083–0·138) of nucleotide variability, suggesting the occurrence of homologous RNA recombination. Sequence alignments clearly indicated a mosaic structure for four of these five variants, for which recombination sites were identified and parental lineages proposed. This is the first in-depth characterization of the population structure and genetic diversity in a nepovirus.
Joan Wellink - One of the best experts on this subject based on the ideXlab platform.
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Secoviridae: a proposed family of plant viruses within the order Picornavirales that combines the families Sequiviridae and Comoviridae, the unassigned genera Cheravirus and Sadwavirus, and the proposed genus Torradovirus
Archives of Virology, 2009Co-Authors: Joan Wellink, Olivier Gall, Alexander Karasev, René Vlugt, Thierry WetzelAbstract:The order Picornavirales includes several plant viruses that are currently classified into the families Comoviridae (genera Comovirus , Fabavirus and Nepovirus ) and Sequiviridae (genera Sequivirus and Waikavirus ) and into the unassigned genera Cheravirus and Sadwavirus . These viruses share properties in common with other picornavirales (particle structure, positive-strand RNA genome with a polyprotein expression strategy, a common replication block including type III helicase, a 3C-like cysteine proteinase and type I RNA-dependent RNA polymerase). However, they also share unique properties that distinguish them from other picornavirales. They infect plants and use specialized proteins or protein domains to move through their host. In phylogenetic analysis based on their replication proteins, these viruses form a separate distinct lineage within the picornavirales branch. To recognize these common properties at the taxonomic level, we propose to create a new family termed “Secoviridae” to include the genera Comovirus , Fabavirus , Nepovirus , Cheravirus , Sadwavirus , Sequivirus and Waikavirus . Two newly discovered plant viruses share common properties with members of the proposed family Secoviridae but have distinct specific genomic organizations. In phylogenetic reconstructions, they form a separate sub-branch within the Secoviridae lineage. We propose to create a new genus termed Torradovirus (type species, Tomato torrado virus) and to assign this genus to the proposed family Secoviridae.
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Cheravirus and Sadwavirus: two unassigned genera of plant positive-sense single-stranded RNA viruses formerly considered atypical members of the genus Nepovirus (family Comoviridae)
Archives of Virology, 2007Co-Authors: O. Gall, Alexander Karasev, Joan Wellink, K Lehto, M. Ikegami, T. Iwanami, T. Jones, T. Wetzel, N. YoshikawaAbstract:The genus Nepovirus (family Comoviridae ) was known both for a good level of homogeneity and for the presence of atypical members. In particular, the atypical members of the genus differed by the number of capsid protein (CP) subunits. While typical nepoviruses have a single CP subunit with three structural domains, atypical nepoviruses have either three small CP subunits, probably corresponding to the three individual domains, or a large and a small subunit, probably containing two and one structural domains, respectively. These differences are corroborated by hierarchical clustering based on sequences derived from both genomic RNAs. Therefore, these atypical viruses are now classified in two distinct genera, Cheravirus (three CP subunits; type species Cherry rasp leaf virus ) and Sadwavirus (two CP subunits; type species Satsuma dwarf virus ).
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Comoviridae: plant picorna-like viruses
2000Co-Authors: Le Gall, Joan WellinkAbstract:The Comoviridae are plant viruses that share many structural and functional features with animal and human viruses of the family Picornaviridae.
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The cowpea mosaic virus RNA 1-encoded 112 kDa protein may function as a VPg precursor in
1995Co-Authors: Er A. Peters, Joan Wellink, Jean-michel T Mesnard, Ingeborg M. Kooter, Jan Verver, Ab Van KammenAbstract:Processing of the 112 kDa ( ' 112K') protein encoded by cowpea mosaic virus RNA 1 was examined in cowpea mesophyll protoplasts using a transient expression system. Cleavage of the 112K protein occurred via two alternative pathways either into VPg and l l0K (24K+ 87K) or into 26K (VPg +24K) and 87K proteins. The 26K protein can be further cleaved into VPg and 24K proteins. The results support a model in which the 112K protein functions as the precursor of VPg during initiation of replication. Cowpea mosaic virus (CPMV), the type member of the family Comoviridae, is a positive-strand RNA plant virus with a bipartite genome. Both RNA 1 and RNA 2 have a small protein, VPg (viral protein genome linked), at their 5 ' end, are polyadenylated at the 3 " end and ar
C. Stussi-garaud - One of the best experts on this subject based on the ideXlab platform.
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Grapevine Fanleaf Virus Replication Occurs on Endoplasmic Reticulum-Derived Membranes
Journal of Virology, 2002Co-Authors: Christophe Ritzenthaler, Claude Laporte, Patrice Dunoyer, Fabien Gaire, S. Duval, A. Piéquet, A. M. Loudes, Odette Rohfritsch, Corinne Schmitt, C. Stussi-garaudAbstract:More than 30 years ago, it was suggested from electron microscopic and/or biochemical analyses that replication of positive-strand RNA viruses, whether in plants or in animals, takes place in association with intracellular membranes (for reviews, see references 13 and 14). The type of membranes involved in replication depends on the virus considered: tymoviruses, for instance, induce vesicular structures by invagination of chloroplast membranes in which both viral RNA and nonstructural proteins were detected (24, 25), whereas infection by tombusviruses leads to the formation of multivesicular bodies derived from mitochondria or peroxisomes (9, 56, 57). Several plant viruses belonging to different groups, such as tobamoviruses (30), bromoviruses (50), potyviruses (59), and comoviruses (11), similarly appear to strongly modify the endomembrane compartments. For Cowpea mosaic virus (CPMV), virus-induced accumulation of vesicles derived from the endomembrane system was described as early as 1974 by de Zoeten et al. (15), and in several instances fibrilar material tentatively identified as double-stranded RNA was shown to accumulate in such vesicles (20, 28). Viral proteins involved in replication were immunodetected on these vesicles in CPMV-infected cells (66). Similar information is available with animal viruses and has been described extensively in the literature, more particularly for poliovirus, the type member of picornaviridae. Evidence for the involvement of virus-induced vesicles in poliovirus replication was obtained already in the sixties (10), and the molecular mechanisms underlying the formation of virus-induced vesicles, their origin, and their role in viral replication were recently unraveled (19, 58, 62). The fact that distinct types of membranes are involved in the replication of different viruses implies the establishment of specific interactions between such host membranes and virus-encoded proteins. In some cases, the transmembrane domains responsible for their anchoring on specific membranes could be identified (18, 35, 56) allowing the formation of dedicated structures where coupled translation and synthesis of both minus- and plus-strand RNA take place (8). Such complexes probably ensure protection of the viral RNA being synthesized from degradation by cellular RNases. In the case of poliovirus, it was shown that specific viral proteins were responsible for vesicle formation (6, 7, 62) and that formation of the poliovirus replication complex is a process that requires coupled viral translation, vesicle production, and viral RNA synthesis (19). Grapevine fanleaf nepovirus (GFLV) has a bipartite RNA genome of positive polarity and, like all members of the Comoviridae, carries a small protein, VPg, covalently linked to the 5′ end of the viral genomic RNAs, which are both polyadenylated at their 3′ end. Each RNA encodes a single polyprotein, and this family of plant viruses shares therefore many common features with Picornaviridae. RNA1 encodes polyprotein P1 (253 kDa), which is processed by an embedded proteinase activity into five proteins required for replication, namely, 1A (of unknown function), 1B (probably the helicase), 1C (VPg), 1D (proteinase), and 1E (polymerase) (38, 40). These proteins are the only proteins required for RNA1 replication (65), and they function in trans to ensure RNA2 replication. RNA2 encodes polyprotein P2 (122 kDa), which is processed in trans by 1D into three proteins (39). Protein 2A is necessary for RNA2 replication, together with RNA1-encoded proteins (23). It is associated with membranous structures and is recruited by the RNA1-encoded replication machinery. We hypothesized that the 2A moiety of polyprotein P2 could mediate the transport of the nascent P2-RNA2 complexes from their initial location in the cytosol to the perinuclear replication sites where RNA2 replication and P2 cleavage take place (23). Therefore, protein 2A could play the role of a “homing protein.” Protein 2B is the movement protein (MP) forming tubules through which viral particles are delivered to uninfected adjacent cells (54). Finally, protein 2C is the coat protein (CP). Using epifluorescence microscopy, we have previously described the formation of a perinuclear complex where viral RNA was synthesized and viral proteins accumulated (23), but this could not be further analyzed due to technical limitations. In the present study, the structural preservation of cells and the intensity of immunolabelings were markedly enhanced, and image resolution was further improved by using high-resolution confocal microscopy. Moreover, the use of transgenic T-BY2 cell lines expressing green fluorescent protein (GFP) targeted to the endoplasmic reticulum (ER) and the Golgi apparatus (GA) provided a direct means to follow the virus-induced modifications of the endomembrane system and allowed us to conclude that GFLV replicates in association with specific membranes mainly derived from ER.