Proteolytic processing of tomato ringspot nepovirus 3C-like protease precursors: definition of the domains for the VPg, protease and putative RNA-dependent RNA polymerase

Tomato ringspot nepovirus (TomRSV) RNA-1 encodes a putative NTP-binding pro tein (NTB), a putative viral genome-linked protein (VPg), a putative RNA-de pendent RNA polymerase (Pol) and a serine-like protease (Pro), which have b een suggested to be involved in viral RNA replication. Proteolytic processi ng of protease precursors containing these proteins was studied in Escheric hia coil and in vitro, The TomRSV protease could cleave the precursor prote ins and release the predicted mature proteins or intermediate precursors, A lthough processing was detected at all three predicted cleavage sites (NTB- VPg, VPg-Pro and Pro-Pol), processing at the VPg-Pro cleavage site was inef ficient, resulting in accumulation of the VPg-Pro intermediate precursor in E. coil and in vitro. In addition, the presence of the VPg sequence in the precursor resulted in increased cleavage at the Pro-Pot cleavage site in E . coli and in vitro. Direct N-terminal sequencing of the genomic RNA-linked VPg, of the mature protease purified from E. coil extracts and of radiolab elled mature polymerase purified from in vitro translation products reveale d the sequences of the NTB-VPg, VPg-Pro and Pro-Pol dipeptide cleavage site s to be Q/S, Q/G and Q/S, respectively, in vitro processing at the NTB-VPg and Pro-Pot cleavage sites was not detected upon mutation or deletion of th e conserved glutamine at the -1 position of the cleavage site, These result s are discussed in light of the cleavage site specificity of the TomRSV pro tease.