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Virology. 1999 Jan 20;253(2):288-98.

Stable ubiquitination of the ICP0R protein of herpes simplex virus type 1 during productive infection.

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Infectious Diseases Section, Parke-Davis Pharmaceutical Research, Division of Warner-Lambert Company, 2800 Plymouth Road, Ann Arbor, Michigan, 48105, USA. WEBERP@AA.WL.COM


ICP0R is the polypeptide product of an alternatively spliced transcript of the gene encoding the transactivator protein ICP0 of herpes simplex virus type 1 (HSV-1). Although it has been shown to act as a transrepressor of gene expression in transfection assays, overexpression of the ICP0R protein in the recombinant virus HSV-KST was previously found to have no detectable effect on virus replication, so that the role it plays in HSV-1 infection remains unclear. Analysis of HSV-KST-infected cell lysates by Western blotting revealed the presence of not only the 41-kDa ICP0R polypeptide but also a 64-kDa processed form of the protein. This processing event was the result of ubiquination of the ICP0R protein, as demonstrated by the reactivity of the 64-kDa species with antibody specific for the influenza virus hemagglutinin (HA) protein epitope in experiments where the gene encoding ICP0R was coexpressed with a gene encoding HA-tagged ubiquitin. Surprisingly, the 64-kDa form of ICP0R was found to be remarkably stable and persisted in infected cells for many hours after processing, despite the fact that ubiquitination normally functions as a means of tagging proteins for rapid degradation. Analyses of mutant polypeptides containing arginine substitutions at each of the lysine residues of ICP0R, which represent potential ubiquitin conjugation sites, revealed that a single lysine residue at codon 248 was both necessary and sufficient for the appearance of the 64-kDa processed form. However, a number of ICP0R mutants that retained the ubiquitination site at lysine 248 but contained disruptions of sequences at distant sites also lacked detectable 64-kDa protein, indicating that the integrity of the overall structure of ICP0R was an additional determinant for ubiquitination.

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