Proteolytic processing of oligopeptides containing the target sequences bythe recombinant tobacco vein mottling virus NIa proteinase

Tobacco vein mottling virus (TVMV) belongs to the potyviridae that consists of about 200 plant viruses. Potyviruses have RNA genomes of approximately 10,000 bases from which a single polyprotein is expressed fl om each virus upon infection, The NIa proteinase is known to process the polyprotein at s even distinct junctions between proteins. Kinetic constants were determined for the reactions of the recombinant TVMV NIa protease (27 kDa) with synth etic oligopeptides containing the sequences for the cleavage sites. For opt imum activity, the substrate needs to have six amino acids (P6-P1) in the a mino region and four (P1'-P4') in the carboxy region, including four conser ved amino acids (V-R-F-Q) in P4-P1 positions. Mutation of any of four conse rved amino acids to Gly made the substrate inert to the enzyme. Among the s ubstrates, the oligopeptides containing the sequences for junctions, P3-6K1 , NIa (VPg-Pro), and NIa-NIb were not processed by the NIa protease, Those junctions have Glu at P3, Glu at P1, and Thr at P2, The implications of hig h substrate specificity and size dependence in polyprotein processing and v iral replication are discussed.