PURIFICATION OF TURNIP MOSAIC POTYVIRUS VIRAL PROTEIN GENOME-LINKED PROTEINASE EXPRESSED IN ESCHERICHIA-COLI AND DEVELOPMENT OF A QUANTITATIVE ASSAY FOR PROTEOLYTIC ACTIVITY

The 49-kDa, nuclear inclusion a-like, viral protein genome-linked prot einase (VPg-Pro) of turnip mosaic potyvirus (TuMV) was expressed in Es cherichia coli. The protein was produced in a soluble form at high lev els and was active, as demonstrated by intermolecular cleavage of the polymerase capsid protein (Pol-CP) substrate. The VPg-Pro was purified by metal-chelation and ion-exchange chromatographies. Two forms of VP g-Pro, which differed in molecular masses, were obtained during isolat ion; their identities were confirmed by immunoblot analysis and N-term inal amino acid sequencing. Data indicated that cleavage took place at a site near the C-terminus of VPg-Pro and was the result of the prote olytic activity of the viral protein. The purified proteinase retained enzymic activity on its natural substrate (Pol-CP) and was also capab le of hydrolysing the synthetic peptide acyl-Ala-Ala-Val-Tyr-His-Gln-A la-Ala-NH2, derived from the consensus cleavage site for the TuMV poly protein. Analysis by mass spectrometry of the two fragments resulting from this reaction indicated that cleavage took place between the Gin and Ala residues, as expected. A fluorogenic derivative of this peptid e was hydrolysed by VPg-Pro, affording a convenient quantitative assay for intermolecular proteolytic activity, and was used to determine th e pH-activity profile. The availability of large quantities of pure pr oteinase and of a rapid and sensitive assay will permit detailed kinet ic and structural studies which are essential to obtain a better under standing of the mode of action of this and related viral proteinases, such as the 3C proteinase of picornaviruses.