Genetic evidence for multiple nuclear functions of the herpes simplex virus ICP8 DNA-binding protein

Abstract

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 ICP8-40-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-,1-D-galactopyranoside. Mutated ICP8 gene plasmids contransfected 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. MATERIALS AND METHODS Cells and viruses. Vero cells were grown and maintained as described previously (23). The growth medium for the Neor cell lines S2 and B10 (see below) included 200 ,ug of the antibiotic G418 per ml during the first passage of cells after thawing or 500 pg of G418 per ml every five passages. The herpes simplex virus type 1 wild-type strain KOS1.1 was propagated and assayed as described previously (23, 25). Mutant viruses were grown in ICP8-expressing S2 and 5258

Replication-defective virus vaccine-induced protection of mice from genital herpes simplex virus 2 requires CD4 T cells

“…A partially purified preparation of the ICP8 − , replication-defective HSV-2 strain 5BlacZ (Da Costa, Bourne, Stanberry, & Knipe, 1997) was prepared for immunizations by high speed centrifugation of clarified supernatants of infected, ICP8-complementing S2 cells (Gao & Knipe, 1989) as previously described (Morrison, Da Costa, & Knipe, 1998). Stocks of HSV-2 strain G-6, a plaque-purified isolate of strain G, were grown in Vero cells and prepared as previously described (Morrison & Knipe, 1996).…”

Release of the catalytic domain N(o) from the herpes simplex virus type 1 protease is required for viral growth

“…For Western blot (immunoblot) analysis of infected cell lysates, cell monolayer cultures were infected with KOS1.1 or A247S virus at a multiplicity of infection of 10 and were harvested as indicated in the text. Procedures for SDS-PAGE and Western blot analysis were described previously (9,44). The anti-ICP35 MAb MCA406 (1:1,000 dilution; Serotec) was used to detect ICP35 and the protease-related products Pra, Prb, Na, and Nb.…”

Immunization with a replication-deficient mutant of herpes simplex virus type 1 (HSV-1) induces a CD8+ cytotoxic T-lymphocyte response and confers a level of protection comparable to that of wild-type HSV-1

“…Infection with d301 is characterized by a distinct pattern of viral gene expression. During the course of the abortive infection, ␣, ␤, and ␥1 genes are expressed, but ␥2 genes are not (22). Because d301 does not replicate, the input virus alone must express levels of immunogenic HSV proteins sufficient to evoke a CTL response.…”

“…The d301 virus contains a large deletion in the gene encoding ICP8, the HSV major DNA binding protein, which results in the inability of this virus to replicate its DNA. During the course of infection with d301, the pattern of gene expression is characterized by the expression of the ␣, ␤, and ␥1 genes but not ␥2 genes (22). Whereas infection with SC16⌬gH results in the production of wild-type levels of noninfectious progeny virus (19), d301 is unable to produce any progeny virus.…”