Grapevine fanleaf nepovirus P38 putative movement protein is not transiently expressed and is a stable final maturation product in vivo

Abstract

The putative 38 kDa movement protein (P38) gene, located on the RNA2 of grapevine fanleaf nepovirus (GFLV), was cloned in Escherichia coli and expressed as a fusion protein fused to six histidines (6HisP38). This protein was purified and used to produce a specific polyclonal antiserum from which immunoglobulins were isolated by immunoaffinity against the recombinant 6HisP38. Western immunoblot analyses on GFLV RNA2 in vitro maturation products showed that the antibodies were specific for P38 protein. This protein was detected as early as 18 h post-inoculation in GFLVinfected Chenopodium quinoa protoplasts and accumulated to very high levels. Tissue-prints and time course experiments on infected C. quinoa plants confirmed that P38 is present at a high level late in infection and is a final maturation product of the GFLV RNA2 polyprotein in vivo. P94 and P66 intermediates of maturation and polyprotein P2 were also detected in vivo but in very low concentrations. No significant difference was observed in the relative amounts of P66 and P94 detected in vivo, contrary to what occurs in vitro. Subcellular fractionation studies showed that P38, although mainly cytosolic, is also found in association with cell wall and membranes. Thought to be the GFLV movement protein, P38 would thus behave in an 'atypical' manner. IntroductionGrapevine fanleaf virus (GFLV) belongs to the genus Nepovirus in the family Comoviridae (Ward, 1993). Its genetic information is divided over two plus-stranded RNA molecules: RNA1 (7342 nt) (Ritzenthaler et al., 1991) encodes a 253 kDa polyprotein (P1) and RNA2 (3774nt) (Serghini et al., 1990) encodes a 122kDa polyprotein (P2). In vitro both polyproteins are proteolytically processed by the RNAl-encoded 24 kDa chymotrypsin-like cysteine proteinase (Margis & Pinck, 1992;Margis et al., 1991). Polyprotein P1 is cleaved at various sites: Cys415/Ala 416 (Margis et al., 1994), Cys1217/Ser 121s, Gly124X/Glu ~242 , and Arg1461/Gly 1402 (Ritzenthaler et al., 1991) to release, from the N terminus to the C terminus, a 46 kDa protein, an 88 kDa putative helicase, the VPg, the 24 kDa proteinase and a 94 kDa putative viral RNA polymerase. Upon in vitro translation, polyprotein P2 is converted into three final products: a 28 kDa N-terminal protein (P28), a 38 kDa protein (P38) and the C-terminal 56 kDa coat protein (CP) (Fig. 1 a). The cleavages occur at the Cys257/Ala 258 and Arg6°5/Gly 6°6 sites between P28/P38 and P38/CP respectively Serghini et al., 1990 Biologically active full-length transcripts of RNA2 (clone pACN) and RNAI (clone pMV) have been obtained (Viry et al., 1993) which allowed us to demonstrate that GFLV RNA1 can autoreplicate in protoplasts and thus encodes the viral replication functions. In contrast RNA2, which requires for its replication the functions encoded by RNA1, is needed for virus spread in plants and thus encodes functions necessary for viral movement (Viry et al., 1993). By analogy with related comoviruses, it has been proposed that the protein adjacent to the capsid protein cont...

Involvement of the Secretory Pathway and the Cytoskeleton in Intracellular Targeting and Tubule Assembly of Grapevine fanleaf virus Movement Protein in Tobacco BY-2 Cells

“…Additional bands of lower molecular mass, probably corresponding to GFP:MP degradation products or translational intermediates, also were detected after induction. These results confirm the previously described high specificity of the MP antibodies toward GFP:MP (Ritzenthaler et al, 1995a) and also indicate that tight control of GFP:MP expression can be achieved in transgenic BY-2 cells using the glucocorticoid induction system.…”

“…This accumulation was slightly faster than that observed in GFLV-infected protoplasts, in which MP became detectable only after 18 h and tubules were seen only rarely before 30 h after infection (Ritzenthaler et al, 1995a(Ritzenthaler et al, , 1995b). We do not know exactly how the kinetics of the appearance of tubules in these experiments is related to the rate of cell-to-cell movement of GFLV in a natural infection.…”

Grapevine Fanleaf Virus Replication Occurs on Endoplasmic Reticulum-Derived Membranes

“…For immunofluorescence microscopy, polyclonal anti-2B antibodies (named anti-MP) raised in rabbits as described in (53) were used at a 1:1,000 dilution; mouse monoclonal antibodies directed against 2C (named anti-CP) or against BrdU (Sigma) were used at a 1:500 dilution, and guinea pig polyclonal antidouble-stranded RNA (dsRNA) antibodies (36) were used at a 1:500 dilution.…”

A Stretch of 11 Amino Acids in the βB-βC Loop of the Coat Protein of Grapevine Fanleaf Virus Is Essential for Transmission by the Nematode Xiphinema index

“…Since recombinants AG1 and G1 are infectious in planta, region R1 is likely not involved in the specific 2C CP -2B MP or virion-2B MP interactions required for tubule-guided GFLV movement through plasmodesmata (27,6).…”