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Journal of Virology, April 2007, p. 3495-3502, Vol. 81, No. 7
0022-538X/07/$08.00+0     doi:10.1128/JVI.02359-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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{triangledown}

Changying Jiang,1 Ying T. Hwang,1 John C. W. Randell,2,{dagger} Donald M. Coen,2 and Charles B. C. Hwang1*

Department of Microbiology and Immunology, State University of New York Upstate Medical University, Syracuse, New York 13210,1 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 021152

Received 27 October 2006/ Accepted 9 January 2007

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.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, SUNY, Upstate Medical University, 750 E. Adams St., Syracuse, NY 13210. Phone: (315) 464-8739. Fax: (315) 464-4417. E-mail: hwangc{at}upstate.edu.

{triangledown} Published ahead of print on 17 January 2007.

{dagger} Present address: Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.

Journal of Virology, April 2007, p. 3495-3502, Vol. 81, No. 7
0022-538X/07/$08.00+0     doi:10.1128/JVI.02359-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Jiang, C., Komazin-Meredith, G., Tian, W., Coen, D. M., Hwang, C. B. C. (2009). Mutations That Increase DNA Binding by the Processivity Factor of Herpes Simplex Virus Affect Virus Production and DNA Replication Fidelity. J. Virol. 83: 7573-7580 [Abstract] [Full Text]  
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