Proteolytic cleavage of the herpes simplex virus type-1 single-strand DNA-binding protein, ICP8, produces active fragments

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Infected cell protein 8 (ICP8) is a 128 kDa multi-functional protein that is one of seven viral gene products required for origin-dependent DNA replication. The major function of ICP8 is that of a single-strand DNA-binding protein. ICP8 also interacts with the herpes simplex virus type-1 (HSV-1) origin-binding protein (UL9 protein), DNA polymerase, and helicase-primase. To isolate functional domains of ICP8, purified protein was subjected to limited trypsin digestion. Anion-exchange chromatography resolved trypsinized ICP8 into two species. The major species consisted of a complex of proteolytic polypeptides in the range of 20-65 kDa. This complex was resistant to reducing agents suggesting that it was stabilized by non-covalent interactions. It retained the ability to bind single-stranded DNA and to stimulate the DNA helicase and DNA polymerase activities of the HSV-1 UL9 protein and DNA polymerase. The minor species contained proteolytic polypeptides in the range of 30-34 kDa. It also retained single-strand DNA-binding activity and the ability to stimulate and interact with the UL9 protein. However, it was unable to stimulate DNA polymerase activity. N-terminal amino acid sequencing showed that this fraction contained at least four distinct polypeptides, starting at residues 7,300,569, and 794. These findings show that: 1. ICP8, even when covalently cut, is stabilized by strong non-covalent interactions that maintain the function and structure of the protein. 2. Limited proteolysis of ICP8 generates 30-34 kDa domains that retain single-strand and UL9 protein binding activity. Supported by NIH grant AI38335.

Original languageEnglish (US)
Pages (from-to)A1194
JournalFASEB Journal
Issue number9
StatePublished - 1997

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics


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