Immunodetection of the proteins encoded by grapevine chrome mosaic nepovirus RNA2

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...

Section: Subcellular Localization Of the P38 Proteinmentioning

confidence: 44%

Section: Total Proteinsmentioning

confidence: 99%

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

Ritzenthaler

Pinck

1995

“…The role served by this protein remains unknown but the amino acid triplet ' LPL' which Koonin et al (1991) implicated with virion movement within plants occurred at amino acid positions 63 to 65 in the SLRSV-H 26-6K coding sequence and a movement function for the protein coded in that region by SLRSV-H would accord with the experiences or suggestions of others [viz. CPMV (Wellink et al, 1987;Wellink & Van Kammen, 1989), TBRV (Hibrand et al, 1992;Demangeat et al, 1992) or other nepoviruses (Meyer et al, 1986;Brault et al, 1989;Rott et al, 1991)]. Nevertheless, comparisons between the 26.6K polypeptide of SLRSV-H and the 58/48K polypeptide of CPMV, the 30K protein of tobacco mosaic virus (Atabekov & Dorokhov, 1984;Meshi et al, 1987) or the region upstream of the coat protein coding sequences in ArMV, GCMV, GFLV, RRV, TBRV or TomRSV showed no unexpected affinities.…”

Section: Tbrv-smentioning

confidence: 99%

Sequence analysis and location of capsid proteins within RNA 2 of strawberry latent ringspot virus

Kreiah¹,

Strunk

Cooper³

1994

The nucleotide sequence of the RNA 2 of a strawberry isolate (H) of strawberry latent ringspot virus (SLRSV) comprised 3824 nucleotides and contained one long open reading frame with a theoretical coding capacity of 890 amino acids equivalent to a protein of 98.8K. The Nterminal amino acid sequences ofvirion-derived proteins were determined by Edman degradation allowing the capsid coding regions to be located and serine/glycine cleavage sites to be identified within the polyprotein. The amino acid sequence in the capsid coding region of an isolate of SLRSV from flowering cherry in New Zealand was 97 % identical to that of SLRSV-H. Except in the 3' and 5' terminal non-coding sequences, computer-based alignment and comparison algorithms did not reveal any substantial homologies between RNA 2 of SLRSV-H and the equivalent genomic segments in the nepoviruses arabis mosaic, cherry leaf roll, grapevine fanleaf, raspberry ringspot, grapevine hungarian chrome mosaic, tomato blackring, tomato ringspot, tobacco ringspot, or in the comoviruses cowpea mosaic and red clover mottle. Despite the similarities in overall genome organization, data from RNA 2 remain insufficient for unambiguous positioning of SLRSV in relation to species/genera in the Comoviridae.

“…If this is the case, it would be facilitated by coordinate expression of the movement and coat proteins. The steady-state levels of the putative movement proteins from two other nepovirus have been reported to be transient in infected plants and it was thus concluded that the proteins were unstable (Demangeat et al, 1992;Hibrand et al, 1992). In contrast, the grapevine fanleaf nepovirus movement protein, which is present at late stages of infection of protoplasts and whole plants, is found mainly in the cytoplasmic fraction and was therefore suggested to be stable (Ritzenthaler et al, 1995).…”

mentioning

confidence: 99%

Expression of the tomato ringspot nepovirus movement and coat proteins in protoplasts

Sanfaçon

Wieczorek²,

Hans³

1995

Tomato ringspot nepovirus (TomRSV) produces a 45 kDa movement protein and a 58 kDa coat protein in infected plants. Accumulation of the movement protein in relation to that of the coat protein was studied in infected protoplasts using a monoclonal antibody against the movement protein and polyclonal antibodies against the coat protein. Unlike most other viral movement proteins, the TomRSV movement protein was present at late stages of infection. Pulse-chase labelling experiments revealed that the release of the movement protein from the precursor polyprotein was coordinated with that of the coat protein. However, the movement protein was less stable than the coat protein in the extractable fraction of the protoplasts. The expression pattern of the TomRSV movement protein is discussed in the light of the proposed mechanism of cell-to-cell movement of virus-like particles through tubular structures composed of the movement protein.