The catalytic subunit of herpes simplex virus DNA polymerase (Pol), a member of the B family polymerases, possesses both polymerase and exonuclease activities. We previously demonstrated that a recombinant virus (YD12) containing a double mutation within conserved exonuclease motif III of the Pol was highly mutagenic and rapidly evolved to contain an additional leucine-to-phenylalanine mutation at residue 774 (L774F), which is located within the finger subdomain of the polymerase domain. We further demonstrated that the recombinant L774F virus replicated DNA with increased fidelity and that the L774F mutant Pol exhibited altered enzyme kinetics and impaired polymerase activity to extension from mismatched primer termini. In this study, we demonstrated that addition of the L774F mutation to the YD12 Pol did not restore the exonuclease deficiency. However, the polymerase activity of the YD12 Pol to extension from mismatched primer termini and on the nucleotide incorporation pattern was altered upon addition of the L774F mutation. The L774F mutation-containing YD12 Pol also supported the growth of viral progeny and replicated DNA more efficiently and more accurately than did the YD12 Pol. Together, these studies demonstrate that a herpes simplex virus Pol mutant with a highly mutagenic ability can rapidly acquire additional mutations, which may be selected for their survival and outgrowth. Furthermore, the studies demonstrate that the polymerase activity of HSV-1 Pol on primer extension is influenced by sequence context and that herpes simplex virus type 1 Pol may dissociate more frequently at G ⅐ C sites during the polymerization reaction. The implications of the findings are discussed.Herpes simplex virus (HSV) DNA polymerase consists of the catalytic subunit of the polymerase (Pol) and the processivity factor UL42. The Pol subunit contains three well-defined activities: polymerization (replication), exonuclease proofreading (editing), and UL42 binding (5, 6, 28). The UL42 binding activity is mediated by amino acid residues located at the C terminus (5, 6). Although the UL42 binding residues are unique to certain alphaherpesvirus DNA polymerases, the sequences comprising the polymerase and exonuclease domains are conserved among the B family (or the ␣-like) polymerases (2-4). The exonuclease domain of the HSV type 1 (HSV-1) Pol contains conserved exonuclease I (Exo I), II, and III motifs, whereas the polymerase domain contains seven conserved regions (I to VII); conserved region IV overlaps with the Exo II motif. The Exo III motif is located within the ␦ region C, which is highly conserved among the B family polymerases (Fig. 1A). These conserved regions are located within the palm, the thumb, and the finger subdomains, which comprise the structural components of the polymerase domain. The crystal structure of the HSV-1 Pol subunit revealed three grooves that form the putative polymerase, exonuclease, and DNA binding sites. The putative exonuclease site is defined as a groove formed between the exonuclease domain...

The effect of thymidine kinase (TK) encoded by herpes simplex virus type 1(HSV-1) strain KOS in DNA replication fidelity was examined by two different mutagenesis assays. Mutagenesis assay of the LacZ reporter gene present in recombinant tkLTRZ1, which contained the integrated LacZ gene in the tk locus, revealed a less than 0.05% mutation frequency of the LacZ gene regardless of whether the viruses were propagated in TK-expressing cells or control cells, conflicting an earlier report that a HSV-1 TK(+) strain replicated a 0.5% mutation frequency of the LacZ gene (R. B. Pyles and R. L. Thompson, 1994, J. Virol. 68, 4514-4524). Furthermore, TK-proficient and -deficient recombinant viruses replicated with similar mutation frequencies (0.027 and 0.026%, respectively) of the LacZ gene, which was integrated in the polymerase locus. Results of SupF mutagenesis assay demonstrated that neither the spectra of mutation nor the mutation frequencies of SupF gene, which was integrated in the tk locus of recombinant, were significantly different (P > 0.05) in progeny viruses grown in TK-expressing cells and control cells. Therefore, both LacZ and SupF mutagenesis assays demonstrated that TK of the HSV-1 strain KOS did not have detectable mutator activity.