Grapevine Fanleaf Virus and Tomato Ringspot Virus

Grapevine fanleaf virus (GFLV) and Tomato ringspot virus (ToRSV) are very closely related nepoviruses belonging to the subgroups a and c, respectively. Their bipartite genome encodes two polyproteins P1 and P2 that are cleaved into final maturation products by the RNAl-encoded proteinases (Hans and Sanfa^on 1995; Margis et al. 1994). Similarly to CPMV, the polyprotein P1 contains the domains for proteins likely to be involved in replication, including a putative helicase (Hel), the VPg, the proteinase (Pro), and the RNA-dependent RNA polymerase (Pol) (Ritzenthaler et al. 1991; Rott et al. 1995). For GFLV, five final products referred to as 1A, 1BHel, 1CVPg, 1DPro and 1Epo1 are generated by P1 processing, whereas ToRSV P1 is maturated into six proteins in vitro due to the presence of an additional cleavage site within protein 1A (Andret-Link et al. 2004; Wang et al. 1999; Wang and Sanfa^on 2000). Similarly, the processing of polyprotein P2 of GFLV generates three proteins termed 2A, 2B, 2C, whereas an additional N-terminally located protein is produced from the maturation of polyprotein P2 from ToRSV (Carrier et al. 2001; Margis et al. 1993). Protein 2B and 2C are involved in virus movement, transmission by nematode vectors and encapsidation (Andret-Link et al. 2004; Laporte et al. 2003; Ritzenthaler et al. 1995; Wang and Sanfa^on 2000).

Initial studies on the cytopathology of nepovirus-infected cells have revealed the presence of diffuse inclusions, often near the nuclei, which consist of complex membranous structures, some of which form vesicles (Francki et al. 1985). Further studies performed in our laboratory using infectious full-length transcripts of GFLV showed that RNA1 is able to self-replicate in protoplasts and therefore encodes all the functions needed for its own replication (Viry et al. 1993). However, analysis of a set of deletion mutants in the P2-coding sequence demonstrated that protein 2A is necessary but not sufficient for RNA2 replication (Gaire et al. 1999). Remarkably, 2A protein, when fused to GFP, distributed to punctate structures throughout the cytoplasm when expressed alone, and accumulated in a juxtanuclear area together with other RNA1-encoded proteins upon infection. This perinuclear area (Fig. 1a), also termed the viral compartment and initially described by Franski et al. (1985), could be defined as the site of viral RNA replication where newly synthesized RNA molecules, double-stranded replicative intermediates, and RNA1-encoded protein accumulate (Gaire et al. 1999; Ritzenthaler et al. 2002). It was hypothesised from these results that protein 2A enables RNA2 replication through its association with the replication complex assembled from RNA1-encoded proteins (Gaire et al. 1999).

Fig. 1 Effects of GFLV replication on the endomembrane system of tobacco BY-2 cells. a Ultrastructural analysis of the viral compartment made of numerous vesicular structures and proliferated membranes. b GFLV "rosette" isolated from infected BY-2 cells after anti-VPg affinity-purification and negative-staining. c Detection of VPg in GFLV-infected protoplasts (right cell infected, left cell healthy). The protein specifically accumulates in the viral compartment where replication occurs. d The ER is visualised in the same cell as in c. Note that the ER is highly condensed in the viral compartment. e Another example of modified ER in GFLV-infected protoplast. Golgi distribution in f an infected and g a healthy cell. Note the regularly-shaped Golgi in healthy cells compared to the modified ones observed in infected cells. Bars = 5 |im except in a (1 |im) and b (0.5 |im)

Fig. 1 Effects of GFLV replication on the endomembrane system of tobacco BY-2 cells. a Ultrastructural analysis of the viral compartment made of numerous vesicular structures and proliferated membranes. b GFLV "rosette" isolated from infected BY-2 cells after anti-VPg affinity-purification and negative-staining. c Detection of VPg in GFLV-infected protoplasts (right cell infected, left cell healthy). The protein specifically accumulates in the viral compartment where replication occurs. d The ER is visualised in the same cell as in c. Note that the ER is highly condensed in the viral compartment. e Another example of modified ER in GFLV-infected protoplast. Golgi distribution in f an infected and g a healthy cell. Note the regularly-shaped Golgi in healthy cells compared to the modified ones observed in infected cells. Bars = 5 |im except in a (1 |im) and b (0.5 |im)

How does the replication complex assemble, what type of intracellular membranes are used, and which are the viral and host proteins involved in the formation of the viral compartment, are questions that have been recently addressed for nepoviruses. By similarity with other picorna-like viruses, it was suggested that the 1BHel could act to anchor the replication complex to membranes. In view of this hypothesis, stretches of hydrophobic residues were identified within the 1BHel from both GFLV and ToRSV (Rott et al. 1995; Ritzenthaler, unpublished results). Thus, the 1BHel from ToRSV, also named nucleoside triphosphate-binding (NTB) protein, contains a hydrophobic region at its C terminus consisting of two adjacent stretches of hydrophobic amino acids separated by a few amino acids. Sanfa^on and colleagues recently showed that 1BHelVPg associates with canine microsomal membranes in the absence of other viral proteins in vitro and with ER membranes in planta (Wang et al. 2004). Analysis performed on truncated proteins fused to GFP confirmed the presence of a functional transmembrane domain within the 60 amino acids at the C terminus of protein 1B and also revealed the presence of a putative amphipathic helix within the N-terminal 80 amino acids of 1B (Zhang et al. 2005). In agreement with the replication of ToRSV on ER-derived membranes (Han and Sanfa^on 2003), both domains of the fusion proteins were sufficient to promote partial association with ER membranes, supporting the hypothesis that the HEL-VPg polyprotein acts as a membrane anchor for the replication complex of ToRSV (Zhang et al. 2005).

Similary to ToRSV, in silico analyses have revealed the presence of four stretches of 21 hydrophobic residues, predicted to correspond to transmembrane domains (TMD), situated near the N terminus (amino acids 518-538 and 547-567) and almost at the C terminus of protein 1BHel of GFLV 1B (amino acids 1168-1188 and 1191-1211). Transient expression of individual RNA1-encoded proteins as GFP fusions in vivo confirmed that only protein 1B was targeted to the ER and often led to the apparition of a highly modified ER network (R. Elamawi & C. Ritzenthaler, unpublished results). These 1BHel-induced modifications are very reminiscent of those observed in vivo upon infection with GFLV (Fig. 1c-e) (Gaire et al. 1999; Ritzenthaler et al. 2002). Whether the 1BHel alone will also allow the formation of rosettelike structures as shown during viral infection (Fig. 1b) still needs to be determined. Nevertheless, they are perfectly in agreement with the replication of the virus on ER membranes (Ritzenthaler et al. 2002). Remarkably, transient expression of protein 1BHel in tobacco BY-2 cells also prevented the export of the resident Golgi protein a-1,2 mannosidase I (R. Elamawi & C. Ritzenthaler, unpublished results). Together with the facts that GFLV replication (i) is inhibited by brefeldin A (Ritzenthaler et al. 2002) and (ii) may affect the number and structure of Golgi stacks within infected cells (compare the distribution of Golgi stacks in Figs. 1f and 1g), it is suggested that the virus could exploit the cellular COPII transport pathway for the assembly of the replication complex and the biogenesis of the perinuclear viral compartment. Current research in our laboratory is aimed at testing this hypothesis.

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