In this study, we have used immunoprecipitation and mass spectrometry to identify over 50 cellular and viral proteins that are associated with the herpes simplex virus 1 (HSV-1) ICP8 single-stranded DNA-binding protein. Many of the coprecipitating cellular proteins are known members of large cellular complexes involved in (i) DNA replication or damage repair, including RPA and MSH6; (ii) nonhomologous and homologous recombination, including the catalytic subunit of the DNA-dependent protein kinase, Ku86, and Rad50; and (iii) chromatin remodeling, including BRG1, BRM, hSNF2H, BAF155, mSin3a, and histone deacetylase 2. It appears that DNA mediates the association of certain proteins with ICP8, while more direct protein-protein interactions mediate the association with other proteins. A number of these proteins accumulate in viral replication compartments in the infected cell nucleus, indicating that these proteins may have a role in viral replication. WRN, which functions in cellular recombination pathways via its helicase and exonuclease activities, is not absolutely required for viral replication, as viral yields are only very slightly, if at all, decreased in WRN-deficient human primary fibroblasts compared to control cells. In Ku70-deficient murine embryonic fibroblasts, viral yields are increased by almost 50-fold, suggesting that the cellular nonhomologous endjoining pathway inhibits HSV replication. We hypothesize that some of the proteins coprecipitating with ICP8 are involved in HSV replication and may give new insight into viral replication mechanisms.Herpes simplex virus 1 (HSV-1) is a large, double-stranded DNA virus that replicates in the host cell nucleus. HSV encodes over 80 gene products that contribute to viral replication in either cultured cells or animal hosts (76). Due to the limited size of the HSV-1 genome, the virus cannot code for every function required for its propagation; thus, HSV-1 must rely upon factors supplied by the host cell for replication. For example, HSV exclusively uses the host cell RNA polymerase II for the transcription of viral genes (4, 16). The exact number and identity of the cellular factors required for HSV replication is unknown, but the identification of such factors is an active area of research as it may shed light on mechanisms of viral replication, the cellular process, or the factor itself. It is this concept that induced us to identify cellular proteins that associate with HSV-1 ICP8.The HSV-1 single-stranded DNA-binding protein, ICP8, is a 128-kDa multifunctional zinc metalloprotein (31, 37) encoded by the U L 29 gene (61). ICP8, in concert with the other HSV DNA replication proteins, including the helicase-primase complex (U L 5, U L 8, U L 52), the origin-binding protein (U L 9), and the polymerase holoenzyme (U L 30/U L 42), is required for viral DNA synthesis (11,12). While the seven HSV DNA replication proteins are known, it is currently unclear as to what host proteins are involved in viral DNA replication. In addition to its role in DNA synthesis, IC...

Herpes simplex virus (HSV) uses intranuclear compartmentalization to concentrate the viral and cellular factors required for the progression of the viral life cycle. Processes as varied as viral DNA replication, late gene expression, and capsid assembly take place within discrete structures within the nucleus called replication compartments. Replication compartments are hypothesized to mature from a few distinct structures, called prereplicative sites, that form adjacent to cellular nuclear matrix-associated ND10 sites. During productive infection, the HSV single-stranded DNA-binding protein ICP8 localizes to replication compartments. To further the understanding of replication compartment maturation, we have constructed and characterized a recombinant HSV-1 strain that expresses an ICP8 molecule with green fluorescent protein (GFP) fused to its C terminus. In transfected Vero cells that were infected with HSV, the ICP8-GFP protein localized to prereplicative sites in the presence of the viral DNA synthesis inhibitor phosphonoacetic acid (PAA) or to replication compartments in the absence of PAA. A recombinant HSV-1 strain expressing the ICP8-GFP virus replicated in Vero cells, but the yield was increased by 150-fold in an ICP8-complementing cell line. Using the ICP8-GFP protein as a marker for replication compartments, we show here that these structures start as punctate structures early in infection and grow into large, globular structures that eventually fill the nucleus. Large replication compartments were formed by small structures that either moved through the nucleus to merge with adjacent compartments or remained relatively stationary within the nucleus and grew by accretion and fused with neighboring structures.

The herpes simplex virus (HSV) immediate early ICP27 protein plays an essential role in stimulating viral early and late gene expression. ICP27 appears to be multifunctional in that it has been reported to stimulate viral late gene transcription, polyadenylation site usage, and RNA export. We report here on proteomic studies involving immunoprecipitation of ICP27 and mass spectrometric identification of co-precipitated proteins. These studies show an association of ICP27 with the cellular translation initiation factors poly A binding protein (PABP), eukaryotic initiation factor 3 (eIF3), and eukaryotic initiation factor 4G (eIF4G) in infected cells. Immunoprecipitation-western blot studies confirmed these associations. Finally, purified MBP-tagged ICP27 (MBP-27) can interact with eIF3 subunits p47 and p116 in vitro. These results suggest that ICP27 may also play a role in stimulating translation of certain viral and host mRNAs and/or in inhibiting host mRNA translation.

Herpes simplex virus (HSV) commonly causes human infections in the orofacial region (HSV-1) and in the genital region (HSV-2). Productive viral infection in mucosal epithelial cells may result in clinical symptoms and is followed by a latent infection within sensory neurons. During productive infection a large number of viral gene products are expressed while during latent infection few or no viral proteins are expressed. Reactivation from latency results in recurrent infections and disease at or near the primary site of infection. Understanding the details of the two stages of the HSV life cycle is a particular focus of current research on HSV. The virus interacts with and modifies numerous host cell functions in both epithelial and neuronal cells, and studies of HSV have enhanced our knowledge of many fundamental processes in eukaryotic cells. Ongoing research continues to uncover novel effects of HSV on cells, and a complete understanding of HSV infection during both productive and latent infection should allow the design of new antiviral agents and vaccines and increased knowledge of basic cell and molecular biology. This review article is designed to provide an introduction to HSV biology and key aspects of the infection cycle.

Small molecules can provide valuable tools to investigate virus biology. We developed a chemical screening approach to identify small molecule inhibitors of poorly understood, pre-early gene expression steps in herpes simplex virus infection, using green fluorescent protein fused to an early protein. Our assay identified ouabain, a cardiac glycoside. Ouabain reversibly decreased viral yield by 100-fold without affecting cellular metabolic activity in an overnight assay. The antiviral potencies of other cardiac glycosides correlated with their potencies against the known target of these compounds, the cellular sodium potassium ATPase. Ouabain had a reduced effect if added 8 h post-infection. It did not inhibit viral attachment or entry, but did reduce the expression of viral immediate-early and early genes by at least 5-fold. Collectively, these results implicate a cellular target that was hitherto not considered important for a stage of HSV replication prior to viral gene expression.