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Journal of Virology, July 2002, p. 7020-7029, Vol. 76, No. 14
0022-538X/02/$04.00+0     DOI: 10.1128/JVI.76.14.7020-7029.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Herpes Simplex Virus Type 1 Origins of DNA Replication Play No Role in the Regulation of Flanking Promoters

Bretton C. Summers^1, and David A. Leib^1,2*

Departments of Ophthalmology and Visual Sciences,¹ Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri²

Received 11 February 2002/ Accepted 24 April 2002

Herpes simplex virus (HSV) exhibits altered gene regulation in neuronal compared to nonneuronal tissues. It has been hypothesized that initiation of DNA synthesis at the viral origins of replication (oriS and oriL) is a critical step in the upregulation of transcriptional activity of flanking divergent promoters, thereby increasing productive gene expression in neurons. Notably, oriS is flanked by the immediate-early (IE) ICP4 and ICP22/47 promoters, and oriL is flanked by the early (E) UL29 and UL30 promoters. To test this hypothesis further, a series of constructs were generated in which these promoters were placed upstream of luciferase genes. In addition, DNA replication origins were deleted in the context of these promoter constructs. All cassettes were recombined into the viral genome of HSV type 1 strain KOS at a site distal to its native origins. Recombinant reporter expression was monitored in vitro and in vivo to determine the role of viral origins of DNA replication in the regulation of their flanking promoters. Reporter gene expression was unaffected by the presence or absence of oriS or oriL, with the exception of a twofold increase in ICP22/47 promoter activity in the absence of oriS. DNA synthesis inhibitors resulted in a decrease of both IE- and E-promoter activity in primary cells but not continuous cell cultures. Reporter activity was readily assayed in vivo during acute infection and reactivation from latency and was also sensitive to DNA synthesis inhibitors. In all assays, reporter gene expression was unaffected by the presence or absence of either oriS or oriL. These data support the requirement of DNA synthesis for full viral gene expression in vivo but suggest that the origin elements play no role in the regulation of their flanking promoters.

Present address: ChemoCentryx, Inc., San Carlos, CA 94070.

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