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↓, 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↓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.
HawkinsC. J.,
WangS. L.,
HayB. A.1999; A cloning method to identify caspases and their regulators in yeast: identification of Drosophila IAP1 as an inhibitor of the Drosophila caspase DCP-1. Proceedings of the National Academy of Sciences, USA 96:2885–2890
KimD. H.,
HwangD. C.,
KangB. H.,
LewJ.,
HanJ.,
SongB. D.,
ChoiK. Y.1996; Effects of internal cleavages and mutations in the C-terminal region of NIa protease of turnip mosaic potyvirus on the catalytic activity. Virology 226:183–190
KimS. Y.,
ParkK. W.,
LeeY. J.,
BackS. H.,
GooJ. H.,
ParkO. K.,
JangS. K.,
ParkW. J.2000; In vivo determination of substrate specificity of hepatitis C virus NS3 protease: genetic assay for site-specific proteolysis. Analytical Biochemistry 284:42–48
LeeY. J.,
KangH.,
RhoS. H.,
EomS. H.,
ParkW. J.2001; Assessment of substrate specificity of hepatitis G virus NS3 protease by a genetic method. Biochemical and Biophysical Research Communications 286:171–175
ShattuckV. I.,
BrolleyB.,
StobbsL. W.,
LougheedE. C.1989; The effect of turnip mosaic virus infection on the mineral content and storability of field-grown rutabaga. Communications in Soil Science and Plant Analysis 20:581–595
SteinerH.,
PesoldB.,
HaassC.1999; An in vivo assay for the identification of target proteases which cleave membrane-associated substrates. FEBS Letters 463:245–249