Replication of RNA-1 of Tomato Black Ring Virus Independently of RNA-2

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: 99%

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

Ritzenthaler

Pinck

1995

“…Preparation of 3H-labeUed cDNA copies, conditions for hybrid formation and conditions for assay using S1 nuclease were as described by Robinson et al (1980). Estimates of Rot~ (initial RNA concentration × time, for 50% hybridization) were obtained by fitting the equation for pseudo-first order kinetics using the R'EVOL programme of Koeppe & H amann (1980) on a Tektronix 4051 microcomputer.…”

Section: Introductionmentioning

confidence: 99%

RNA Species of Tobacco Rattle Virus Strains and Their Nucleotide Sequence Relationships

Robinson¹

1983

Self Cite

SUMMARYThe nucleotide sequences of the genome RNA species (RNA-1 and RNA-2) of three strains of tobacco rattle virus OR, SYM and CAM, were compared by hybridization with complementary DNA copies. Strain OR was found to contain two RNA-2 species, which differ by about 5 × 104 in mol. wt. (150 nucleotides), but about 90~ of whose nucleotide sequences are homologous. The larger of the two contained the genetic information for yellow symptoms in infected Nicotiana spp., whereas the smaller, which probably arose from it by mutation, did not. The RNA-2 species of strains OR, SYM and CAM had no detectable nucleotide sequences in common. RNA-1 of strain OR and of strain SYM were almost identical in sequence, but had no detectable sequence in common with RNA-1 of strain CAM. Only in strain CAM was any nucleotide sequence homology between RNA-1 and RNA-2 detected, amounting to about 450 residues.

“…Preparation of 3H-labelled cDNA, conditions for hybrid formation and conditions for assay using S1 nuclease were as described by Robinson et al (1980).…”

Section: Methodsmentioning

confidence: 99%

Origin and Messenger Activity of Two Small RNA Species Found in Particles of Tobacco Rattle Virus Strain SYM

Robinson¹,

Mayo²,

Fritsch

et al. 1983

Self Cite

SUMMARYNucleoprotein preparations of tobacco rattle virus (TRV) strain SYM contain particles of four modal lengths. Particles of length 188 nm and 101 nm contain RNA-1 and RNA-2 respectively. Those of length 57 nm and 48 nm contain RNA molecules that are not required for infectivity but include specific sequences derived from both RNA-I and RNA-2. No products attributable to translation of RNA-2 were obtained in reticulocyte lysates, whereas RNA-2 of other TRV strains serves as messenger RNA for virus coat protein, In strain SYM, one of the smaller RNA species, RNA-3 (mol. wt. 0.6 x 106, 1750 nucleotides), was translated in vitro to give coat protein, and is apparently a subgenomic messenger RNA derived from RNA-2. The other small RNA species, RNA-4 (mol. wt. 0-54 × 106, 1550 nucleotides), was translated into a polypeptide of apparent mol. wt. 29000, unrelated to coat protein. It is suggested that RNA-4 is a subgenomic messenger RNA derived from RNA-1.