Specificity and Properties of the Genome-linked Proteins of Nepoviruses

Abstract

SUMMARYThe infectivity of the RNA of six nepoviruses was decreased or abolished by proteinase K treatment, whereas that of the RNA of cowpea mosaic virus (comovirus group) or tomato bushy stunt virus was unaffected. The extent of the decrease in infectivity was characteristic for each nepovirus and was independent of the plant species used as virus source or as assay host. The infectivity of raspberry ringspot virus (RRV) RNA was less affected than that of the other nepoviruses but treatment with Pronase decreased infectivity more than treatment with proteinase K. Proteinase K treatment also abolished the infectivity for tobacco mesophyll protoplasts of RNA of tobacco ringspot virus (TRSV) and tomato black ring virus (TBRV).Tests on virus RNA, labelled with ~25I by the chloramine T method, provided evidence that three nepoviruses and Echtes Ackerbohnenmosaik-Virus (EAMV; comovirus group) have genome-linked proteins (VPg). Pronase treatment rendered about half (RNA of strawberry latent ringspot virus; SLRV), or nearly all (RNA of the other nepoviruses and EAMV), of the '25I soluble in 70 % ethanol. Treatment of nepovirus RNA with ribonuclease P 1 yielded a product with an estimated mol. wt. of 4000 __+ 900. Mobilities in polyacrylamide gels of VPg from the RNA of different viruses differed slightly (SLRV > TBRV > TRSV > RRV). TRSV VPg yielded one ~25I-labelled tryptic peptide whereas the genome-linked proteins of RRV and TBRV both yielded two major products, of which one was resistant to further digestion and the other was converted, apparently via intermediates, to a second more stable product. No difference was detected between the tryptic peptides obtained from VPg of different strains of RRV, or of TBRV, or between those obtained from RNA-1, RNA-2 or RNA-3 (satellite RNA) of TBRV. Nepovirus VPg is therefore virus-specific. It seems to be coded on RNA-1 and probably has multiple functions.

Defective Multiplication of a Pseudorecombinant of Tomato Black Ring Virus in Tobacco Protoplasts

“…Perhaps RNA-2 was replicated but either the synthesis and breakdown balanced at a level equivalent to that originally taken up by the protoplasts, or the RNA which was synthesized, and which functioned as a messenger, was not infective. The infectivity of TBRV RNA is almost completely abolished by treatment with protease (Harrison & Barker, 1978), probably because the genome-linked protein is destroyed (Mayo et al, 1982). Perhaps the RNA-2 synthesized in protoplasts inoculated with RNA-I(A) + RNA-2(G) was not infective because it lacked a genome-linked protein.…”

“…Two isolates of TBRV were used: strain A, of the Scottish (= beet ringspot) serotype, and strain G of the German (= potato bouquet) serotype. Virus was propagated in Nieotiana clevelandii plants and purified from infected leaves by trituration in phosphate buffer, clarification with 8-5~ (v/v) butan-1-ol, precipitation with 10~ polyethylene glycol (PEG) 6000 + 0.17 M-NaC1, and differential centrifugation as described by Mayo et al (1982).…”

Translation of Tobacco Ringspot Virus RNA in Reticulocyte Lysate: Proteolytic Processing of the Primary Translation Products

“…White Burley. Leaves were harvested 7 days after inocukltion and virus was purified as described by Mayo et al (1982). Virus which was to be used for the production of antiserum was further purified by centrifugation (3.5 h at 45000 g, 4 °C) in a density gradient of 5 to 20~ sucrose m 0./t7 M-sodium phosphate pH 7.…”

Nucleotide sequence of the coat protein genes of strawberry latent ringspot virus: lack of homology to the nepoviruses and comoviruses

“…The genome of SLRV consists of two RNA species of 9.0 and 5-15 kb (Everett et al, 1994) that are translated as polyproteins (Hellen et al, 1991). The RNAs are polyadenylated at their 3' termini (Mayo et al, 1979), and contain a protein covalently bound to their 5' termini (Mayo et al, 1982). Some isolates of SLRV have The nucleotide sequence reported in this paper will appear in the EMBL, GenBank and DDBJ nucleotide sequence databases under the accession number X75165.…”

“…Some isolates of SLRV have The nucleotide sequence reported in this paper will appear in the EMBL, GenBank and DDBJ nucleotide sequence databases under the accession number X75165. a 1"2 kb satellite RNA species (Mayo et al, 1974(Mayo et al, , 1982Gallitelli et al, 1982).…”