Thermolabile in vivo DNA-binding activity associated with a protein encoded by mutants of herpes simplex virus type 1.
The major DNA-binding protein encoded by several temperature-sensitive mutants of herpes simplex virus type 1 was thermolabile for binding to intracellular viral DNA. The ability of DNase I to release this protein from isolated nuclei was used as a measure of the amount of protein bound to viral DNA. This assay was based upon our previous observation that the fraction of herpesviral DNA-binding protein which can be eluted from nuclei with DNase I represents proteins associated with progeny viral DNA (D. M. Knipe and A. E. Spang, J. Virol. 43:314-324, 1982). In this study, we found that several temperature-sensitive mutants encoded proteins which rapidly chased from a DNase I-sensitive to a DNase I-resistant nuclear form upon shift to the nonpermissive temperature. We interpret this change in DNase I sensitivity to represent the denaturation of the DNA-binding site at the nonpermissive temperature and the association with the nuclear framework via a second site on the protein. The DNA-binding activity measured by the DNase I sensitivity assay represents an important function of the protein in viral replication because three of five mutants tested were thermolabile for this activity. A fourth mutant encoded a protein which did not associate with the nucleus at the nonpermissive temperature and therefore would not be available for DNA binding in the nucleus. We also present supportive evidence for the binding of the wild-type protein to intracellular viral DNA by showing that a monoclonal antibody coprecipitated virus-specific DNA sequences with the major DNA-binding protein.
D002467:Cell Nucleus / Q000378:metabolism; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D004268:DNA-Binding Proteins; D003850:Deoxyribonuclease I; D004706:Endodeoxyribonucleases / Q000494:pharmacology; D005814:Genes, Viral; D008954:Models, Biological; D009154:Mutation; D018139:Simplexvirus / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; D004706:Endodeoxyribonucleases; D003850:Deoxyribonuclease I; D004265:DNA Helicases