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

Abstract

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.

DNA Replication Fidelity of Herpes Simplex Virus

“…As expected the mutants are more mutagenic than the control virus expressing wild type UL42 (Jiang et al, 2009). Similarly mutants with decreased DNA binding also have defective phenotypes and are highly mutagenic (Jiang et al, 2007a), and that mutants with no detectable DNA binding are deleterious in viral growth and DNA synthesis (Jiang et al, 2007b). These studies provide information that UL42 cannot bind DNA too tightly or too weakly and that any perturbation of DNA binding can impact virus production and replication fidelity.…”

Finger Domain Mutation Affects Enzyme Activity, DNA Replication Efficiency, and Fidelity of an Exonuclease-Deficient DNA Polymerase of Herpes Simplex Virus Type 1

“…The recombinant YD12 Pol exhibited resistance to PAA (20). We thought to examine whether the L774F mutant Pol and the L774F mutation in the context of YD12 Pol also exhibit altered drug sensitivity.…”

“…The single-cycle growth curve assay was performed as described previously (20). Briefly, the 1 ϫ 10 5 cells preseeded onto 12-well plates were infected with HP66 at a multiplicity of infection of 3.…”

“…Total RNA was prepared from infected cells, using Trizol reagent (Invitrogen). The cDNA was synthesized using the iScript cDNA synthesis kit (Bio-Rad) and subjected to real-time PCR quantification as described previously (20), except that two pol primers, pol-1333 (5Ј-GAGATGCTGTTGGCCTTCAT-3Ј) and pol-1418 (5Ј-GGCCAGTCGAAGTTGATGAT-3Ј), were used to amplify an 86-bp target sequence of the pol.…”

“…The amount of viral DNA synthesized during the single-cycle growth curve assays was quantified by real-time PCR as described previously (20), using the 73-bp target sequences of the HSV-1 UL29 gene.…”