In vitro analysis of tobacco vein mottling virus NIa cistron: evidence for a virus-encoded protease.

Potential protease functions associated with the NIa nuclear inclusion protein of tobacco vein mottling virus (TVMV) were investigated. In the absence of treatments, in vitro translation of viral RNA produced various polypeptides representing each of the proposed TVMV cistrons--28K-HC-42K-CI-5.5K-NIa-NIb-CP. When viral RNA was first hybridized to DNA probes complementary to the NIa cistron, and then treated with RNase H prior to translation, a 48-kDa processing product, immunologically identified as the NIa protein, was abolished. In its place was observed a series of larger polypeptides, immunologically identified as fusion products of the cylindrical inclusion (CI) and NIa cistrons. The use of probes which permitted translation through as few as 15 nucleotide residues beyond the sequences encoding the proposed carboxyl terminus of NIa resulted in normal processing. None of the DNA probes affected an apparent cleavage between the helper component (HC) and 42K proteins. Cloned cDNA regions representing the NIa cistron and flanking sequences were inserted in transcription vectors. Translation of the in vitro transcript resulted in synthesis, not of a large fusion polyprotein, but, of a mature-sized NIa polypeptide. In vitro transcripts, lacking the 3'-most sequences that were expected to encode the protease active site of the NIa protein, were translated. These generated an apparent fusion polypeptide that reacted with antisera to both CI and NIa. The results indicate that the NIa gene product functions as a protease responsible for some but not all of the cleavage events which lead to the production of the mature forms of TVMV proteins.