Determination of the substrate specificity of turnip mosaic virus NIa 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-Gln decreased, with the scissile bond located after Gln. 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 decreased Ser, with Yaa being an aliphatic amino acid and the scissile bond being located between Gln and Ser. This result is consistent with the conserved cleavage sequence motif, and should provide insight into the molecular activity of the NIa protease.
Kang
Hara
H
Department of Life Science and National Research Laboratory, Kwangju Institute of Science and Technology, 1 Oryong-dong, Puk-gu, Kwangju 500-712, Korea1.
Lee
Yong Jae
YJ
Department of Life Science and National Research Laboratory, Kwangju Institute of Science and Technology, 1 Oryong-dong, Puk-gu, Kwangju 500-712, Korea1.
Goo
Jae Hwan
JH
Department of Life Science and National Research Laboratory, Kwangju Institute of Science and Technology, 1 Oryong-dong, Puk-gu, Kwangju 500-712, Korea1.
Park
Woo Jin
WJ
Department of Life Science and National Research Laboratory, Kwangju Institute of Science and Technology, 1 Oryong-dong, Puk-gu, Kwangju 500-712, Korea1.
D001665:Binding Sites; D016384:Consensus Sequence; D010450:Endopeptidases / Q000378:metabolism*; D005821:Genetic Techniques; D010935:Plant Diseases / Q000821:virology; D019176:Potyviridae / Q000201:enzymology*; D013379:Substrate Specificity; D014764:Viral Proteins / Q000378:metabolism*
D014764:Viral Proteins; D010450:Endopeptidases; C059992:nuclear inclusion protein a, mosaic viruses