Herpes simplex virus type 1 (HSV-1) DNA polymerase contains several conserved regions within the polymerase domain. The conserved regions I, II, III, V, and VII have been shown to have functional roles in the interaction with deoxynucleoside triphosphates (dNTPs) and DNA. However, the role of conserved region VI in DNA replication has remained unclear due, in part, to the lack of a well-characterized region VI mutant. In this report, recombinant viruses containing a point mutation (L774F) within the conserved region VI were constructed. These recombinant viruses were more susceptible to aphidicolin and resistant to both foscarnet and acyclovir, compared to the wild-type KOS strain. Marker transfer experiments demonstrated that the L774F mutation conferred the altered drug sensitivities. Furthermore, mutagenesis assays demonstrated that L774F recombinant viruses containing the supF marker gene, which was integrated within the thymidine kinase locus (tk), exhibited increased fidelity of DNA replication. These data indicate that conserved region VI, together with other conserved regions, forms the polymerase active site, has a role in the interaction with deoxyribonucleotides, and regulates DNA replication fidelity. The possible effect of the L774F mutation in altering the polymerase structure and activity is discussed.DNA polymerase (Pol) is the pivotal enzyme involved in DNA replication and plays a central role in controlling the accuracy of DNA replication. Amino acid sequence alignment of various Pols reveals that all Pols contain conserved residues distributed within several regions of the Pol domain. Previous studies demonstrated that these conserved residues play an important role in the polymerization reaction (reviewed in reference 38 and references therein). These conserved residues interact with incoming deoxyribonucleotides (deoxynucleoside triphosphates [dNTPs]) and the primer-template DNA. Therefore, mutations within these residues could result in altered polymerase activity and/or replication fidelity.Herpes simplex virus (HSV) has proven to be a good model for genetic, molecular biological, and biochemical studies of Pol, since it can be manipulated to contain desired mutations for further characterization regardless of whether the mutated Pol is lethal for viral replication (reviewed in reference 11 and references therein). Examination of the HSV-1 Pol also reveals seven conserved regions within the carboxyl-terminal half of the Pol, which has been defined as the polymerase domain (Fig. 1). Indeed, mutations within most of these conserved regions confer altered sensitivities to certain nucleoside and pyrophosphate analog antiviral drugs, indicating that they are involved in the interactions with dNTPs and DNA (11).Since only a few region VI mutants of HSV have been described (4, 5, 34), and the effects of these mutations on replication fidelity have not been characterized, we constructed recombinant HSV-1 viruses containing a region VI mutation (L774F) for characterizing the effects of this mutatio...

The processivity subunit of the herpes simplex virus DNA polymerase, UL42, is essential for viral replication and possesses both Pol-and DNA-binding activities. Previous studies demonstrated that the substitution of alanine for each of four arginine residues, which reside on the positively charged surface of UL42, resulted in decreased DNA binding affinity and a decreased ability to synthesize long-chain DNA by the polymerase. In this study, the effects of each substitution on the production of viral progeny, viral DNA replication, and DNA replication fidelity were examined. Each substitution mutant was able to complement the replication of a UL42 null mutant in transient complementation assays and to support the replication of plasmid DNA containing herpes simplex virus type 1 (HSV-1) origin sequences in transient DNA replication assays. Mutant viruses containing each substitution and a lacZ insertion in a nonessential region of the genome were constructed and characterized. In single-cycle growth assays, the mutants produced significantly less progeny virus than the control virus containing wild-type UL42. Real-time PCR assays revealed that these UL42 mutants synthesized less viral DNA during the early phase of infection. Interestingly, during the late phase of infection, the mutant viruses synthesized larger amounts of viral DNA than the control virus. The frequencies of mutations of the virus-borne lacZ gene increased significantly in the substitution mutants compared to those observed for the control virus. These results demonstrate that the reduced DNA binding of UL42 is associated with significant effects on virus yields, viral DNA replication, and replication fidelity. Thus, a processivity factor can influence replication fidelity in mammalian cells.

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 gangciclovir, 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.

The herpes simplex virus DNA polymerase catalytic subunit, which has intrinsic polymerase and 3-5 exonuclease activities, contains sequence motifs that are homologous to those important for 3-5 exonuclease activity in other polymerases. The role of one such motif, Exo III, was examined in this study. Mutated polymerases containing either a single tyrosine-to-histidine change at residue 577 or this change plus an aspartic acid-to-alanine at residue 581 in the Exo III motif exhibited defective or undetectable exonuclease activity, respectively, yet retained substantial polymerase activity. Despite the defects in exonuclease activity, the mutant polymerases were able to support viral replication in transient complementation assays, albeit inefficiently. Viruses replicated via the action of these mutant polymerases exhibited substantially increased frequencies of mutants resistant to ganciclovir. Furthermore, when the Exo III mutations were incorporated into the viral genome, the resulting mutant viruses displayed only modestly defect in replication in Vero cells and exhibited substantially increased mutation frequencies. The results suggest that herpes simplex virus can replicate despite severely impaired exonuclease activity and that the 3-5 exonuclease contributes substantially to the fidelity of viral DNA replication.

Recombinant viruses were constructed to have an Escherichia coli replicon containing a mutagenesis marker, the supF gene, integrated within the thymidine kinase locus (tk) of herpes simplex virus type 1. These viruses expressed either wild-type or mutant DNA polymerase (Pol) and were tested in a mutagenesis assay for the fidelity of their replication of the supF gene. A mutation frequency of approximately 10 ؊4 was observed for wild-type strain KOS-derived recombinants in their replication of the supF gene. However, recombinants derived from the PAA r 5 Pol mutant, which has been demonstrated to have an antimutator phenotype in replicating the tk gene, had three-to fourfold increases in supF mutation frequency (P < 0.01), a result similar to that exhibited when the supF gene was induced to replicate as episomal DNA (Y. T. Hwang, B.-Y. Liu, C.-Y. Hong, E. J. Shillitoe, and C. B. C. Hwang, J. Virol. 73:5326-5332, 1999). Thus, the PAA r 5 Pol mutant had an antimutator function in replicating the tk gene and was less accurate in replicating the supF gene than was the wild-type strain. The spectra of mutations and distributions of substituted bases within the supF genes that replicated as genomic DNA were different from those in the genes that replicated as episomal DNA. Therefore, the differences in sequence contents between the two target genes influenced the accuracy of the Pol during viral replication. Furthermore, the replication mode of the target gene also affected the mutational spectrum.DNA replication in mammalian cells is a complicated process (reviewed in references 2 and 25). It requires intricate DNA replication machinery that both duplicates chromosomal DNA and maintains genomic integrity. As the pivotal enzymes, DNA polymerases (Pols) are critical for ensuring the accuracy of DNA synthesis via their roles in the selection of nucleotides to be incorporated during DNA replication (polymerization) and in the removal of misinserted nucleotides from the 3Ј-OH end of the primer strand (proofreading) (reviewed in references 20 and 23