Mutagenesis of Amino Acids at Two Tomato Ringspot Nepovirus Cleavage Sites: Effect on Proteolytic Processingin cisandin transby the 3C-like Protease

Abstract

Tomato ringspot nepovirus (ToRSV) encodes two polyproteins that are processed by a 3C-like protease at specific cleavage sites. Analysis of ToRSV cleavage sites identified previously and in this study revealed that cleavage occurs at conserved Q/(G or S) dipeptides. In addition, a Cys or Val is found 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 protease and putative RNA-dependent RNA polymerase, which is processedin cis, and the cleavage site at the N-terminus of the movement protein, which is cleavedin trans. The effect of the mutations on proteolytic processing at these sites was tested usingin vitrotranslation systems. Substitution of conserved amino acids at the −2, −1, and +1 positions resulted in a significant reduction in proteolytic processing at both cleavage sites. The effects of individual substitutions were stronger on the cleavage site processedin transthan on the one processedin cis. The cleavage site specificity of the ToRSV protease is discussed in comparison to that of related proteases.