Mutagenesis of amino acids at two tomato ringspot nepovirus cleavage sites: Effect on proteolytic processing in cis and in trans by the 3C-like protease

Tomato ringspot nepovirus (ToRSV) encodes two polyproteins that are process ed by a 3C-like protease at specific cleavage sites. Analysis of ToRSV clea vage sites identified previously and in this study revealed that cleavage o ccurs at conserved Q/(G or S) dipeptides. In addition, a Cys or Val is foun d in the -2 position. Amino acid substitutions were introduced in the -6 to +1 positions of two ToRSV cleavage sites: the cleavage site between the pr otease and putative RNA-dependent RNA polymerase, which is processed in cis , and the cleavage site at the N-terminus of the movement protein, which is cleaved in trans. The effect of the mutations on proteolytic processing at these sites was tested using in vitro translation systems. Substitution of conserved amino acids at the -2, -1, and +1 positions resulted in a signif icant reduction in proteolytic processing at both cleavage sites. The effec ts of individual substitutions were stronger on the cleavage site processed in trans than on the one processed in cis. The cleavage site specificity o f the ToRSV protease is discussed in comparison to that of related protease s. (C) 1999 Academic Press.