Mutations that increase DNA binding by the processivity factor of herpes simplex virus affect virus production and DNA replication fidelity.

The interactions of the herpes simplex virus processivity factor UL42 with the catalytic subunit of the viral polymerase (Pol) and DNA are critical for viral DNA replication. Previous studies, including one showing that substitution of glutamine residue 282 with arginine (Q282R) results in an increase of DNA binding in vitro, have indicated that the positively charged back surface of UL42 interacts with DNA. To investigate the biological consequences of increased DNA binding by UL42 mutations, we constructed two additional UL42 mutants, including one with a double substitution of alanine for aspartic acid residues (D270A/D271A) and a triple mutant with the D270A/D271A and Q282R substitutions.

Finger domain mutation affects enzyme activity, DNA replication efficiency, and fidelity of an exonuclease-deficient DNA polymerase of herpes simplex virus type 1.

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.

The enhanced DNA replication fidelity of a mutant herpes simplex virus type 1 DNA polymerase is mediated by an improved nucleotide selectivity and reduced mismatch extension ability.

We previously demonstrated that a recombinant herpes simplex virus containing a mutation within the finger domain of DNA polymerase replicated DNA with increased fidelity. In this study, we demonstrate that, compared with wild-type polymerase, the mutant enzyme exhibited improved nucleotide selectivity and a reduced ability to extend from mismatched primer termini, which would contribute to the increased DNA replication fidelity.

Herpes simplex virus mutants with multiple substitutions affecting DNA binding of UL42 are impaired for viral replication and DNA synthesis.

Herpes simplex virus mutants with single substitutions that decrease DNA binding by the DNA polymerase processivity subunit UL42 are only modestly impaired for viral replication. In this study, recombinant viruses harboring two or four of these mutations were constructed. The more substitutions, the more severe the defects in viral replication and DNA synthesis, suggesting that DNA binding by UL42 is important for these processes.

Mutations that decrease DNA binding of the processivity factor of the herpes simplex virus DNA polymerase reduce viral yield, alter the kinetics of viral DNA replication, and decrease the fidelity of DNA replication.

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.

Effects of substitutions of arginine residues on the basic surface of herpes simplex virus UL42 support a role for DNA binding in processive DNA synthesis.

The way that UL42, the processivity subunit of the herpes simplex virus DNA polymerase, interacts with DNA and promotes processivity remains unclear. A positively charged face of UL42 has been proposed to participate in electrostatic interactions with DNA that would tether the polymerase to a template without preventing its translocation via DNA sliding. An alternative model proposes that DNA binding by UL42 is not important for processivity.

On the mutation rate of herpes simplex virus type 1.

All seven DNA-based microbes for which carefully established mutation rates and mutational spectra were previously available displayed a genomic mutation rate in the neighborhood of 0.003 per chromosome replication. The pathogenic mammalian DNA virus herpes simplex type 1 has an estimated genomic mutation rate compatible with that value.

A point mutation within conserved region VI of herpes simplex virus type 1 DNA polymerase confers altered drug sensitivity and enhances replication fidelity.

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.

Exonuclease-deficient polymerase mutant of herpes simplex virus type 1 induces altered spectra of mutations.

The effect of exonuclease activity of the herpes simplex virus DNA polymerase (Pol) on DNA replication fidelity was examined by using the supF mutagenesis assay. The recombinants with exonuclease-deficient Pol, containing an integrated supF gene in the thymidine kinase locus (tk), exhibited supF mutation frequencies ranging from 0.14 to 5.

Thymidine kinase of herpes simplex virus type 1 strain KOS lacks mutator activity.

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.

Mutation spectra of herpes simplex virus type 1 thymidine kinase mutants.

To examine whether the exonuclease activity intrinsic to the polymerase (Pol) of herpes simplex virus type 1 can influence the mutational spectra, we applied the denaturing gradient gel electrophoresis (DGGE) system combined with sequencing to characterize thymidine kinase mutants isolated from both the wild-type virus and a mutant deficient in exonuclease activity, Y7. Wild-type viruses produced predominantly frameshift mutations (67%), whereas Y7 replicated a significantly lower proportion of frameshifts (21%; P < 0.005).

Replication fidelity of the supF gene integrated in the thymidine kinase locus of herpes simplex virus type 1.

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.

Detection of mutations within the thymidine kinase gene of herpes simplex virus type 1 by denaturing gradient gel electrophoresis.

Denaturing gradient gel electrophoresis has been applied widely for the detection of mutation(s) and polymorphisms of various genes. It is shown that this system is also feasible for analyzing mutations in the thymidine kinase (tk) gene of herpes simplex virus type 1 (HSV-1). Thus, this system is applicable for examining whether a drug-resistant HSV-1 contains a tk mutation(s) or otherwise.