The CTC series of cobalt chelates display in vitro and in vivo activity against herpes simplex virus types 1 and 2 (HSV-1 and HSV-2). The experiments described here identify the stage in the virus life cycle where CTC-96 acts and demonstrate that the drug inhibits infection of susceptible cells. CTC-96 at 50 g/ml has no effect on adsorption of virions to Vero cell monolayers. Penetration assays reveal that CTC-96 inhibits entry of the virus independent of gC and cellular entry receptors. This observation was supported by the failure to detect the accumulation of virus-specified proteins and ␣ mRNA transcripts when CTC-96 is present at the onset of infection. Moreover, virion-associated ␣TIF does not accumulate in the nucleus of cells infected in the presence of CTC-96. CTC-96 targets the initial fusion event between the virus and the cell and also inhibits cell-to-cell spread and syncytium formation. Furthermore, CTC-96 inhibits plaque formation by varicellazoster virus and vesicular stomatitis virus as efficiently as by HSV-1. Collectively, these experiments suggest that CTC-96 is a broad-spectrum inhibitor of infection by enveloped viruses and that it inhibits HSV-1 infection at the point of membrane fusion independent of the type of virus and cellular receptors present.Infection by the alphaherpesviruses herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) results in a variety of viral diseases including oral and genital epithelial lesions, encephalitis, and ocular keratitis (15,16,20,96,103). Among these, herpetic ocular infection is the leading infectious cause of blindness in developed countries (48,55,56,96). Herpesvirus infections are characterized by their ability to establish latency and reactivate from the latent state (80). In immunocompetent and immunocompromised patients herpesvirus infections are among the most frequent causes of viral disease (78,93,104). Both primary and recrudescent infections in immunocompromised patients are life threatening (78,93,104). Thus, there exists considerable interest in developing treatments for preventing infection and reducing the pathogenesis of primary and recurrent infections by HSV.Several nucleoside analogs are approved for use in the treatment of herpesvirus infections (e.g., acyclovir, penciclovir, valaciclovir, and famciclovir), and derivatives of these are being developed and/or are undergoing clinical trials (2, 5, 17). These drugs are activated by the HSV thymidine kinase, and thus their primary target is virus DNA synthesis (26, 32). Not surprisingly, drug-resistant strains are appearing with increasing frequency (13,14,17,28,29,54,70,85). Resistance arises from mutations in the TK gene (18,27) or mutations in the gene encoding DNA polymerase (13,14,47,70,85). Therefore, new drugs need to be developed that target other aspects of the virus life cycle in order to find more effective treatments against the existing drug-resistant strains as well as all the known herpesviruses.The CTC series of cobalt-containing compounds possess anti-inflammatory (105) ...

The herpes simplex virus type 1 (HSV-1) immediate-early (IE) protein ICP0 has been implicated in the regulation of viral gene expression and the reactivation of latent HSV-1. Evidence demonstrates that ICP0 is an activator of viral gene expression yet does not distinguish between a direct or indirect role in this process. To further our understanding of the function of ICP0 in the context of the virus life cycle, site-directed mutagenesis of the consensus C 3 HC 4 zinc finger domain was performed, and the effects of these mutations on the growth and replication of HSV-1 were assessed. We demonstrate that alteration of any of the consensus C 3 HC 4 cysteine or histidine residues within this domain abolishes ICP0-mediated transactivation, alters the intranuclear localization of ICP0, and significantly increases its stability. These mutations result in severe defects in the growth and DNA replication of recombinant herpesviruses and in their ability to initiate lytic infections at low multiplicities of infection. These viruses, at low multiplicities of infection, synthesize wild-type levels of the IE proteins ICP0 and ICP4 at early times postinfection yet exhibit significant decreases in the synthesis of the essential IE protein ICP27. These findings reveal a role for ICP0 in the expression of ICP27 and suggest that the multiplicity-dependent growth of ␣0 mutant viruses results partially from reduced levels of ICP27.

The ordered expression of herpes simplex virus type 1 (HSV-1) genes, during the course of a productive infection, requires the action of the virus immediate-early regulatory proteins. Using a protein interaction assay, we demonstrate specific in vitro protein-protein interactions between ICP4 and ICP27, two immediateearly proteins of HSV-1 that are essential for virus replication. We map multiple points of contact between these proteins. Furthermore, by coimmunoprecipitation experiments, we demonstrate the following. (i) ICP4-ICP27 complexes are present in extracts from HSV-1 infected cells. (ii) ICP27 binds preferentially to less modified forms of ICP4, a protein that is extensively modified posttranslationally. We also demonstrate, by performing electrophoretic mobility shift assays and supershifts with monoclonal antibodies to ICP4 or ICP27, that both proteins are present in a DNA-protein complex with a noncanonical ICP4 binding site present in the HSV thymidine kinase (TK) gene. ICP4, in extracts from cells infected with ICP27-deficient viruses, is impaired in its ability to form complexes with the TK site but not with the canonical site from the ␣4 gene. However, ICP4 is able to form complexes with the TK probe, in the absence of ICP27, when overproduced in mammalian cells or expressed in bacteria. These data suggest that the inability of ICP4 from infected cell extracts to bind the TK probe in the absence of ICP27 does not reflect a requirement for the physical presence of ICP27 in the complex. Rather, they imply that ICP27 is likely to modulate the DNA binding activity of ICP4 by affecting its posttranslational modification status. Therefore, we propose that ICP27, in addition to its established role as a posttranscriptional regulator of virus gene expression, may also modulate transcription either through direct or indirect interactions with HSV regulatory regions, or through its ability to modulate the DNA binding activity of ICP4.

During a productive infection by herpes simplex virus type 1 (HSV-1), ICP4, the major regulatory protein encoded by the ␣4 gene, binds to its transcription initiation site and represses the accumulation of ␣4 RNA. Evidence suggests that the degree of repression by ICP4 is a function of the absolute distance of an ICP4 binding site 3 from a TATA box. However, repression of HSV-1 gene expression by ICP4 through binding sites located 5 of TATA boxes, as in the case of the ␣0 gene, has not been adequately addressed. To this end, recombinant ␣0 promoters with various arrays of ICP4 binding sites flanking the ␣0 TATA box were constructed and recombined into the HSV-1 genome. Our results demonstrate the following. (i) Destruction of the endogenous ␣0 ICP4 binding site, located 5 of the TATA box, results in derepression of ␣0 protein and RNA accumulation in infected Vero cells. (ii) The degree of ␣0 derepression is equivalent to that reported for the ␣4 gene following destruction of the ICP4 binding site at the ␣4 mRNA cap site in HSV-1. (iii) Introduction of an ICP4 binding site at the ␣0 mRNA cap site represses the accumulation of ␣0 RNA greater than threefold relative to the wild type. (iv) Changes in the abundance of ␣0 protein and RNA in infected cells do not affect replication or growth of HSV-1 in tissue culture. Our findings are consistent with the conclusion that ␣0 transcription is repressed by ICP4. These results demonstrate that repression by ICP4 can occur through binding sites located 5 of virus gene TATA boxes in HSV-1. Thus, models addressing repression of HSV-1 gene expression by ICP4 should incorporate the role of binding sites located 5, as well as 3, of virus gene TATA boxes.

The transcriptional program of herpes simplex virus is regulated by the concerted action of three immediate-early (α) proteins, ICP4, ICP27, and ICP0. The experiments described in this study examine the role of the acidic amino terminus (amino acids 1 to 103) of ICP0 in gene activation. When tethered to a DNA binding domain, this sequence activates transcription in the yeast Saccharomyces cerevisiae. Deletion of these amino acids affects the ability of ICP0 to activate α-gene promoter reporters in transient expression assays, while it has little or no effect on a β- and a γ-gene reporter in the same assay. Viruses that express the deleted form of ICP0 (ICP0-NX) have a small-plaque phenotype on both Vero cells and the complementing cell line L7. Transient expression and immunofluorescence analyses demonstrate that ICP0-NX is a dominant negative form of ICP0. Immunoprecipitation of ICP0 from cells coinfected with viruses expressing ICP0-NX and ICP0 revealed that ICP0 oligomerizes in infected cells. These data, in conjunction with the finding that ICP0-N/X is dominant negative, provide both biochemical and genetic evidence that ICP0 functions as a multimer in infected cells.