Characterization of the protease and other products of amino-terminus-proximal cleavage of the herpes simplex virus 1 UL26 protein.
The herpes simplex virus 1 UL26 open reading frame encodes a protease which cleaves a small carboxyl-terminal peptide of itself and its substrate encoded by an overlapping, 3'-coterminal transcriptional unit, designated UL26.5. The translational product of UL26.5 is infected-cell protein 35c,d (ICP35c,d) (F. Liu and B. Roizman, J. Virol. 65:206-212, 1991; F. Liu and B. Roizman, J. Virol. 65:5149-5156, 1991). The protease activity maps at the amino terminus of UL26 translation product designated Pra. Cleavage of Pra to remove the carboxyl-terminal 25 amino acids converts the protein to Prb (F. Liu and B. Roizman, Proc. Natl. Acad. Sci. USA 89:2076-2080, 1992). Other studies reported a second, amino-terminus-proximal cleavage in UL26 gene products made in Escherichia coli (I. C. Deckman, M. Hagen, and P. J. McCann III, J. Virol 66:7362-7367, 1992; C. L. DiIanni, D. A. Drier, I. C. Deckman, P.J. McCann III, F. Liu, B. Roizman, R. J. Colonno, and M. G. Cordingley, J. Biol. Chem., 368:2048-2051, 1993). We report the following results. (i) The amino-terminus-proximal cleavage of UL26 protein in eukaryotic cells generates two polypeptides, an apparent M(r)-25,000 amino-terminal polypeptide designated Prn and a carboxyl-terminal polypeptide which corresponds in electrophoretic mobility to ICP35a. Cleavage of the carboxyl-terminal 25 amino acids by the UL26 protease converted ICP35a to ICP35b. (ii) Replacement of Ala-247-Ser-248 with Arg-Pro precluded the amino-terminus-proximal cleavage. (iii) Prn, the amino-terminal product of the cleavage reaction at amino acid 247 functions as a protease. (iv) Additional amino acid substitutions in the putative domain of the protease yielded results consistent with the hypothesis that UL26 encodes a serine protease. (v) The domain of the UL26 protein whose modification confers the formation of double bands for all products (Pra, Prb, ICP35a, ICP35b, ICP35c,d, and ICP35e,f) except Prn maps in the domain shared by UL26 and UL26.5, between codons 307 and 417.
Liu
F
F
Marjorie B. Kovler Viral Oncology Laboratories, University of Chicago, Illinois 60637.
Roizman
B
B
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002478:Cells, Cultured; D002874:Chromosome Mapping; D006224:Cricetinae; D003545:Cysteine; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D010450:Endopeptidases / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D016679:Genome, Viral; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010446:Peptide Fragments / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics; D011499:Protein Processing, Post-Translational; D018139:Simplexvirus / Q000201:enzymology*; D013379:Substrate Specificity; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis
D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; C054582:ICP35 protein, Human herpesvirus 1; D010446:Peptide Fragments; D014764:Viral Proteins; C079287:scaffold protein, Herpes simplex virus-1; D010450:Endopeptidases; D003545:Cysteine