Gene expression by a hypovirulence-associated virus of the chestnut blight fungus involves two papain-like protease activities. Essential residues and cleavage site requirements for p48 autoproteolysis.
Proteolytic processing plays a fundamental role in gene expression of a recently characterized viral-like double-stranded RNA associated with biological control of the chestnut blight fungus. Polypeptide p29, a papain-like protease, was shown to autocatalytically release itself from the NH2 terminus of the polyprotein specified by the first of two encoded open reading frames, ORF A (Choi, G. H., Shapira, R., and Nuss, D. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1167-1171; Choi, G. H., Pawlyk, D. M., and Nuss, D. L. (1991) Virology 183, 747-752). The characterization of a second autocatalytic protease, p48, which is encoded by ORF B, is the subject of this report. Deletion analysis revealed that the catalytic domain resides within the carboxyl-terminal region, while site-specific mutational analysis identified Cys-341 and His-388 as residues essential for autoproteolysis. Autoproteolytic processing by p48 was also demonstrated when expressed in Escherichia coli and microsequence analysis of the recovered COOH-terminal cleavage product indicated that cleavage occurred between Gly-418 and Ala-419. The requirements for a functional cleavage site, including confirmation of the cleavage dipeptide, were defined by amino acid substitution analysis. Similarities between p29 and p48 suggest that the respective coding domains could have arisen as a result of a gene duplication event.
Department of Molecular Oncology and Virology, Roche Institute of Molecular Biology, Roche Research Center, Nutley, New Jersey 07110.
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D000595:Amino Acid Sequence; D001203:Ascomycota / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism*; D015870:Gene Expression; D005814:Genes, Viral; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D010206:Papain / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012328:RNA Viruses / Q000235:genetics* / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics; D016415:Sequence Alignment; D014158:Transcription, Genetic
D012330:RNA, Double-Stranded; D012367:RNA, Viral; D010450:Endopeptidases; D010206:Papain