Mutational analysis of the tobacco etch potyviral 35-kDa proteinase: identification of essential residues and requirements for autoproteolysis.
The tobacco etch potyvirus (TEV) polyprotein is processed by three virus-encoded proteinases, termed Nla, HC-Pro, and the 35-kDa proteinase. The 35-kDa proteinase is derived from the amino-terminal region of the polyprotein. Analysis of polyproteins containing beta-glucuronidase fused to the expected carboxy terminus of the 35-kDa proteinase confirmed the previously identified Tyr304-Ser305 dipeptide as the cleavage site between the 35-kDa proteinase and HC-Pro. The 35-kDa proteinase of TEV was unable to catalyze proteolysis when synthetic substrate polyproteins were supplied in a bimolecular or trans reaction, suggesting that processing occurs by an autolytic mechanism. The results of a mutational analysis within the 35-kDa proteolytic domain indicated that His214, Asp223, Ser256, and Asp288 were required for optimal autoproteolytic activity. Replacement of Ser256 with either Thr or Cys resulted in low but detectable proteinase activity, as did substitution of Asp223 and Asp288 with Glu. These results are consistent with the hypothesis that the 35-kDa proteinase resembles cellular serine-type proteinases, with Ser256 functioning as the nucleophilic residue within the active site. Cleavage mediated by the 35-kDa proteinase has been shown previously to occur after polyprotein synthesis in wheat germ extracts and transgenic plants, but not in rabbit reticulocyte lysate. We were able to demonstrate that processing in vitro may require a heat-labile factor present in wheat germ extracts.
Verchot
J
J
Department of Biology, Texas A&M University, College Station 77843.
Herndon
K L
KL
Carrington
J C
JC
D000595:Amino Acid Sequence; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010942:Plant Viruses / Q000201:enzymology* / Q000235:genetics; D010947:Plants, Toxic; D014026:Nicotiana / Q000382:microbiology*
D010450:Endopeptidases