In vitro expression of a full-length DNA copy of cowpea mosaic virus B RNA: identification of the B RNA encoded 24-kd protein as a viral protease

Abstract

Double-stranded cDNA was synthesized from B component RNA of cowpea mosaic virus and cloned into appropriate vectors. Using four clones, together representing the entire B RNA sequence, a full-length DNA copy was constructed and subsequently positioned downstream of a phage SP6 or T7 promoter. RNA molecules transcribed from this full-size DNA copy using SP6 or T7 RNA polymerase were efficiently translated in rabbit reticulocyte lysates into a 200-kd polypeptide similar to RNA isolated from viral B components. Moreover this polypeptide was rapidly cleaved into 32-kd and 170-kd polypeptides, exactly like the 200-kd polypeptide encoded by viral B RNA. In vitro transcription and translation of a DNA copy in which an 87-bp-long deletion in the coding sequence for the 24-kd polypeptide was introduced revealed that the 24-kd polypeptide bears the proteolytic activity involved in the primary cleavage of the B RNA-encoded polyprotein.