Proteolytic processing at a novel cleavage site in the N-terminal region of the tomato ringspot nepovirus RNA-1-encoded polyprotein in vitro

Tomato ringspot nepovirus RNA-1-encoded polyprotein (P1) contains the domai ns for the putative NTP-binding protein, VPS, BC-like protease and a putati ve RNA-dependent RNA polymerase in its C-terminal region, The N-terminal re gion of P1, with a coding capacity for a protein (or a precursor) of 67 kDa , has not been characterized. Using partial cDNA clones, it is shown that t he 3C-like protease can process the N-terminal region of P1 at a novel clea vage site in vitro, allowing the release of two proteins, X1 (located at th e N terminus of pi) and X2 (located immediately upstream of the NTB domain) . P1 precursors in which the protease was inactive or absent were not cleav ed by exogenously added protease, suggesting that pi processing was predomi nantly in cis, Results from site-directed mutagenesis of putative cleavage sites suggest that dipeptides Q(423)/G and Q(620)/G are the X1-X2 and X2-NT B cleavage sites, respectively, The putative X1 protein contains a previous ly identified alanine-rich sequence which is present in nepoviruses but not in the related comoviruses. The putative X2 protein contains a region with similarity to the comovirus 32 kDa protease co-factor (the only mature pro tein released from the N terminus of comovirus pi polyproteins) and to the corresponding region of other nepovirus P1 polyproteins, These results rais e the possibility that the presence of two distinct protein domains in the N-terminal part of the P1 polyprotein may be a common feature of nepoviruse .