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Cleavage site specificity of the tomato ringspot nepovirus protease Carrier, Karma James


Tomato ringspot nepovirus (TomRSV) encodes two polyproteins, which are processed by a 3C-like protease at specific cleavage sites. In this study two new cleavage sites have been identified: the cleavage site between the protease and putative RNA-dependent RNA polymerase (Pro-Pol), which is processed in cis and the cleavage site at the N-terminus of the movement protein (X-MP), which is cleaved in trans. A new trans-cleavage site was also localized in the N - terminal region of the polyprotein encoded by RNA-2 (P2) using partial cDNA clones and in vitro translations systems. These results suggest that at least two proteins are released from the region of P2 upstream of the movement proteia In contrast, only one protein is released from this region of P2 in characterized nepoviruses of subgroup A/B. Comparison of the sequence of the X-MP and Pro-Pol cleavage sites to that of other TomRSV cleavage sites allowed us to propose the following cleavage site consensus sequence for the TomRSV protease: (Cys,Val)- Gln/(Ser,Gly). This consensus is similar to cleavage sites recognized by proteases from picornaviruses, potyviruses and comoviruses but not to those from nepoviruses of subgroups A or B. Amino acid substitutions were introduced in the -6 to +1 positions of the Pro-Pol and XMP cleavage sites. Substitution of conserved amino acids at the -2, -1 and +1 positions resulted in a significant reduction of proteolytic processing in vitro in both cleavage sites suggesting that these amino acids play a key role in the recognition of the cleavage sites by the protease. The effects of individual substitutions were stronger on the cleavage site processed in trans than on the one processed in cis. It has been predicted that the specificity of the TomRSV protease for cleavage sites similar to those of picornaviruses, potyviruses and comoviruses is due to the presence of a conserved His residue in the substrate-binding pocket. However, substituting this His residue in the TomRSV protease with a Leu found at a similar position in the proteases of nepovirus subgroups A and B did not allow recognition of cleavage sites in which amino acids typical of cleavage sites from those nepoviruses were introduced.

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