Two herpes simplex virus mutants containing mutated residues within the conserved Exo III motif of the polymerase gene were previously shown to be defective in 3′–5′ exonuclease activity and exhibited extremely high mutation frequencies. In this study, we have shown that these mutants also exhibited higher resistance to phosphonoacetic acid and sensitivity to aphidicolin and all nucleoside analogs tested, including acyclovir and gancicolvir, compared to wild-type virus. Marker transfer experiments and sequencing analyses demonstrated that these altered phenotypes were the result of mutations within the Exo III motif. The data indicate that, aside from leading to exonuclease deficiency, mutations in the Exo III motif may also affect interaction of nucleoside triphosphates with the catalytic sites of polymerase activity.

A novel nucleoside analogue, (±)-(1α, 2β, 3α)-9-[2-hydroxy -3-(hydroxymethyl)cyclobutyl]guanine [(±)-HHCG] was synthesized and has antiviral activity against herpes simplex virus (HSV) types 1 and 2, human cytomegalovirus (HCMV) and varicella-zoster virus (VZV) in plaque reduction assays. The antiviral activity of (±)-HHCG against HSV-2 shows a 10-40-fold dependence on the presence of a virally encoded thymidine kinase. (±)-HHCG is a substrate for HSV-1 thymidine kinase with a phosphorylation rate of 28 μm h −1 compared to 15 μm h −1 for acyclovir under identical conditions. Enzymatically prepared HHCG-triphosphate is a competitive inhibitor of dGTP incorporation into DNA by HSV-1 DNA polymerase with an inhibition constant corresponding to 0.0077 μm. Hybridization studies using an HSV-1-specific DNA probe indicated that DNA synthesis is reduced in HSV-1-infected WI-38 cells treated with (±)-HHCG, with an ED 50 comparable to that of acyclovir. These results suggest that the antiviral activity of (±)-HHCG is due to preferential inhibition of viral DNA synthesis.

(±)-(1α,2β,3α)-9-[2,3-bis(hydroxymethyl)cyclobutyl]guanine [(±)-BHCG or SQ 33 054] is a newly synthesized nucleoside analog with potent and selective antiviral activity against members of the herpesvirus group, including human cytomegalovirus. The activity against a thymidine kinase deficient HSV-2 mutant was 25-fold poorer than against the parent virus, suggesting that phosphorylation is an important prerequisite for antiviral activity against HSV-2. (±)-BHCG is readily phosphorylated by purified HSV-1 thymidine kinase, and BHCG triphosphate synthesized enzymatically is a selective inhibitor of HSV-1 DNA polymerase. (±)-BHCG did not inhibit host cell growth at concentrations at least 1000-fold higher than HSV-2 inhibitory concentrations. Subcutaneous administration of (±)-BHCG was protective against HSV-1 systemic infections in mice. BHCG is an exciting antiviral agent and represents a new class of nucleoside analogs.

The analysis of the deduced amino acid sequence of the herpes simplex virus type 1 (HSV-1) DNA polymerase reported here suggests that the polymerase structure consists of domains carrying separate biological functions. The HSV-1 enzyme is known to possess 5'-3'-exonuclease (RNase H), 3'-5'-exonuclease, and DNA polymerase catalytic activities. Sequence analysis suggests an arrangement of these activities into distinct domains resembling the organization of Escherichia coli polymerase I. In order to more precisely define the structure and C-terminal limits of a putative catalytic domain responsible for the DNA polymerization activity of the HSV-1 enzyme, we have undertaken in vitro mutagenesis and computer modeling studies of the HSV-1 DNA polymerase gene. Sequence analysis predicts that the major DNA polymerization domain of the HSV-1 enzyme will be contained between residues 690 and 1100, and we present a three-dimensional model of this region, on the basis of the X-ray crystallographic structure of the E. coli polymerase I. Consistent with these structural and modeling studies, deletion analysis by in vitro mutagenesis of the HSV-1 DNA polymerase gene expressed in Saccharomyces cerevisiae has confirmed that certain amino acids from the C terminus (residues 1073 to 1144 and 1177 to 1235) can be deleted without destroying HSV-1 DNA polymerase catalytic activity and that the extreme N-terminal 227 residues are also not required for this activity.

(+-)-(1 alpha,2 beta,3 alpha)-9-[2,3-bis(hydroxymethyl)cyclobutyl] guanine [(+-)-BHCG or SQ 33,054] is a newly synthesized nucleoside analog with potent and selective antiviral activity against members of the herpesvirus group, including human cytomegalovirus. The activity against a thymidine kinase deficient HSV-2 mutant was 25-fold poorer than against the parent virus, suggesting that phosphorylation is an important prerequisite for antiviral activity against HSV-2. (+-)-BHCG is readily phosphorylated by purified HSV-1 thymidine kinase, and BHCG triphosphate synthesized enzymatically is a selective inhibitor of HSV-1 DNA polymerase. (+-)-BHCG did not inhibit host cell growth at concentrations at least 1000-fold higher than HSV-2 inhibitory concentrations. Subcutaneous administration of (+-)-BHCG was protective against HSV-1 systemic infections in mice. BHCG is an exciting antiviral agent and represents a new class of nucleoside analogs.

The herpes simplex virus type 1 ICP4 and ICP0 polypeptides are immediate-early proteins that positively and negatively regulate expression of other viral genes in trans. ICP4 has recently been shown to bind DNA bearing the consensus sequence 5'-ATCGTCNNNN(T/C)CG(A/G)C-3', present upstream of a number of viral genes. To test the hypothesis that this DNA-binding activity is involved in ICP4-mediated gene regulation, site-specific mutagenesis was employed to mutate the version of this sequence in the promoter of the ICP0 gene. The mutation eliminated detectable binding of ICP4 to the promoter as measured in vitro by a gel electrophoresis band shift assay. The ability of the mutated ICP0 promoter to direct synthesis of a reporter gene was also investigated in a transient transfection assay. Whereas ICP4 was found to transactivate the wild-type ICP0 promoter two- to threefold, the mutated promoter was transactivated seven- to ninefold. In assays containing the ICP0 transactivator gene, ICP4 down regulated the wild-type promoter far more efficiently than the mutated promoter. Finally, both the wild-type and mutated ICP0 promoters exhibited a similar response to ICP4 in transfections that included a vector expressing the viral transactivator protein VP16. These experiments suggest that the sequence-specific DNA-binding activity of ICP4 is an essential element of its role as a negative regulator of gene expression.

A mutation (asparagine 815 to serine 815) was introduced into the herpes simplex virus type 1 (HSV-1) DNA polymerase (pol). The HSV-1 pol enzyme in lysates of Saccharomyces cerevisiae cells expressing the mutant protein showed increased resistance to acyclovir triphosphate and increased sensitivity to phosphonoacetate but was not substantially altered with respect to sensitivity to phosphonoformate or aphidicolin. These results directly demonstrate that both resistance to acyclovir triphosphate and sensitivity to phosphonoacetate can be conferred by this mutation in the absence of other viral factors and that the yeast expression system can be used for structure-function studies on HSV-1 pol.

The herpes simplex virus type 1 (HSV-1) (strain 17) DNA polymerase gene has been cloned into an Escherichia coli-yeast shuttle vector fused to the galactokinase gene (GAL-1) promoter. Genes controlled by the GAL-1 promoter are induced by galactose, uninduced by raffinose, and repressed by glucose. Cell extracts from a strain of Saccharomyces cerevisiae harboring this vector (Y-MH202, expresser cells) grown in the presence of galactose and assayed in high salt (100 mM ammonium sulfate) contained a novel DNA polymerase activity. No significant high-salt DNA polymerase activity was detected in extracts from expresser cells grown in the presence of raffinose or in extracts from control cells containing the E. coli-yeast shuttle vector without the HSV-1 DNA polymerase gene grown in the presence of raffinose of galactose. Immunoblot analysis of the cell extracts by using a polyclonal rabbit antiserum prepared against a highly purified HSV-1 DNA polymerase preparation revealed the specific induction of the HSV-1 approximately 140-kilodalton DNA polymerase polypeptide in expresser cells grown in galactose. Extracts from the same cells grown in raffinose or control cells grown in either raffinose or galactose did not contain this immunoreactive polypeptide. The high-salt DNA polymerase activity in the extracts from expresser cells grown in galactose was inhibited greater than 90% by either acyclovir triphosphate or aphidicolin, as expected for HSV-1 DNA polymerase. In addition, the high-salt polymerase enzyme activity could be depleted from extracts by immunoprecipitation by using purified immunoglobulin G from this same polyclonal rabbit antiserum. These results demonstrate the successful expression of functional HSV-1 DNA polymerase enzyme in S. cerevisiae.

A double-stranded synthetic oligonucleotide that codes for an amino acid sequence specifically recognized and cleaved by the endopeptidase, renin, was inserted into a plasmid expression vector. The double-stranded oligonucleotide was placed at the junction between the sequences coding for two distinct domains of a fusion protein. The vector used for this analysis expressed a 190-kD Epstein-Barr virus membrane antigen (EBV-MA)-beta-galactosidase (beta-gal) fusion protein (Beisel et al., 1985). The resultant novel protein product expressed by the new construction can be cleaved specifically by renin to yield two distinct polypeptides, EBV-MA and beta-gal, corresponding to the two domains of the original fusion protein product.