We have cloned the region of tomato ringspot nepovirus (TomRSV) RNA-1 coding for the putative TomRSV 3C-related protease (amino acids 1213 to 1508) in a transcription vector and in a transient expression vector. Using cell-free transcription and translation systems and plant protoplasts, we have demonstrated that proteins produced from these clones possess a proteolytic activity in on the cleavage site between the TomRSV movement and coat proteins. By amino acid homology of the TomRSV 3C-related protease with other nepo- and comovirus proteases, His, Glu (or Asp) and Cys have been predicted to constitute the catalytic triad. Site-directed mutagenesis of His to Asp abolished the TomRSV protease activity, and . The cleavage site between the TomRSV movement and coat proteins has been determined to be Q/G, by direct protein sequencing. Previously, His located in the substrate binding pocket of the TomRSV 3C-related protease has been suggested to be involved in the cleavage site specificity. We show that an inactive TomRSV 3C-related protease is obtained after substitution of His with Leu. These results are discussed in light of the possible relation of the TomRSV 3C-related protease to 3C-related proteases of nepo-, como- and potyviruses.


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