Genomic organization of RNA2 of Tomato ringspot virus: processing at a third cleavage site in the N-terminal region of the polyprotein in vitro

The proteinase of Tomato ringspot virus (genus Nepovirus) is responsible fo r proteolytic cleavage of the RNA2-encoded polyprotein (P2) at two cleavage sites, allowing definition of the domains for the movement protein (MP) an d coat protein. In this study, we have characterized a third cleavage site in the N-terminal region of P2 using an in vitro processing assay and parti al cDNA clones. Results from site-directed mutagenesis of putative cleavage sites suggest that cleavage occurs at dipeptide Q(301)/G. Cleavage at this site is predicted to result in the release of two proteins from the N-term inal region of P2: a 34 kDa protein located at the N terminus of P2 (assumi ng translation initiation at the first AUG codon) and a 71 kDa protein loca ted immediately upstream of the MP domain. In contrast, only one protein do main is present in the equivalent region of the P2 polyprotein of other cha racterized nepoviruses.