TY - JOUR
T1 - Mutational analysis of the tobacco etch potyviral 35-kDa proteinase
T2 - Identification of essential residues and requirements for autoproteolysis
AU - Verchot, Jeanmarie
AU - Herndon, Kerri L.
AU - Carrington, James C.
N1 - Funding Information:
We thank Larry HarrisHaIler for synthesis of oligonucleotides and for helpful suggestions with sequencing through difficult regions; Rohit Duggal and Clayton Huntley for assistance with scanning densitometry; and Valerian Dolja, Maria Restrepo-Hartwig, and Xiao Hua Li for valuable comments on the manuscript. This work was supported by grants from the U.S. Department of Agriculture (91-37303-6435) and the National Institute of Allergy and Infectious Disease (Al-27842).
PY - 1992/9
Y1 - 1992/9
N2 - 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 β-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.
AB - 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 β-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.
UR - https://www.scopus.com/pages/publications/0026794659
U2 - 10.1016/0042-6822(92)91216-H
DO - 10.1016/0042-6822(92)91216-H
M3 - Article
C2 - 1529535
AN - SCOPUS:0026794659
SN - 0042-6822
VL - 190
SP - 298
EP - 306
JO - Virology
JF - Virology
IS - 1
ER -