Genetic evidence for multiple nuclear functions of the herpes simplex virus ICP8 DNA-binding protein.
We have isolated several mutant herpes simplex viruses, specifically mutated in the infected cell protein 8 (ICP8) gene, to define the functional domains of ICP8, the major viral DNA-binding protein. To facilitate the isolation of these mutants, we first isolated a mutant virus, HD-2, with the lacZ gene fused to the ICP8 gene so that an ICP8-beta-galactosidase fusion protein was expressed. This virus formed blue plaques on ICP8-expressing cell lines in the presence of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Mutated ICP8 gene plasmids cotransfected with HD-2 DNA yielded recombinant viruses with the mutant ICP8 gene incorporated into the viral genome. These recombinants were identified by formation of white plaques. Four classes of mutants were defined: (i) some expressed ICP8 that could bind to DNA but could not localize to the cell nucleus; (ii) some expressed ICP8 that did not bind to DNA but localized to the nucleus; (iii) some expressed ICP8 that neither bound to DNA nor localized to the nucleus; and (iv) one expressed ICP8 that localized to the cell nucleus and bound to DNA in vitro, but the mutant virus did not replicate its DNA. These classes of mutants provide genetic evidence that DNA binding and nuclear localization are distinct functions of ICP8 and that ICP8 has nuclear functions other than binding to DNA. Furthermore, the portion of ICP8 needed for a nuclear function(s) distinct from DNA binding is the part of ICP8 showing sequence similarity to that of the cellular protein cyclin or proliferating cell nuclear antigen.
Gao
M
M
Department of Microbiology and Molecular Genetics, Harvard Medical School, Boston, Massachusetts 02115.
Knipe
D M
DM
D000818:Animals; D001483:Base Sequence; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics*; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D005814:Genes, Viral; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D011485:Protein Binding; D018139:Simplexvirus / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D014709:Vero Cells; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D015678:Viral Structural Proteins / Q000235:genetics
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C045462:ICP8 protein, Simplexvirus; D014764:Viral Proteins; D015678:Viral Structural Proteins