Determination of the substrate specificity of turnip mosaic virus Nla protease using a genetic method

The RNA genome of turnip mosaic potyvirus (TuMV) encodes a large polyprotein that is processed to mature proteins by virus-encoded proteases. The TuMV NIa protease is responsible for the cleavage of the polyprotein at seven different locations. These cleavage sites are defined by a conserved sequence motif Val-Xaa-His-Gin down arrow, with the scissile bond located after Gin. To determine the substrate specificity of the NIa protease, amino acid sequences cleaved by the NIa protease were obtained from randomized sequence libraries using a screening method referred to as GASP (genetic assay for site-specific proteolysis). Based on statistical analysis of the obtained sequences, a consensus substrate sequence was deduced: Yaa-Val-Arg-His-Gln down arrow Ser, with Yaa being an aliphatic amino acid and the scissile bond being located between Gin and Ser. This result is consistent with the conserved cleavage sequence motif, and should provide insight into the molecular activity of the Nla protease.