| A region of ORF 1 from Grapevine rupestris stem pitting-associated virus was expressed in Escherichia coli, baculovirus, and in vitro transcription/translation expression systems to test its putative proteolytic activity. The region expressed included the predicted protein domains for an ovarian tumour-like protease, papain-like protease, helicase, and RNA-dependant RNA polymerase. A protein equivalent to the theoretical size of the cloned region was expressed in the E. coli and baculovirus systems. The protein produced in these systems was largely insoluble and likely inactive. The activity or cleavage sites of the putative proteases could not be determined from either system by Western analysis. Knock-outs of the predicted active sites of either or both protease domains showed no difference in expression profile to the wild-type constructs. Upon review of results obtained from the expression systems, the putative proteases appear to be inactive when expressed in E. coli, or insect cells infected by a recombinant baculovirus. |
