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Herpes simplex virus (HSV) infections cause many lesions, including labialis, keratitis, pharyngotonsillitis, and encephalitis. A major characteristic of HSV infection is that the virus replicates within the infected cells resulting in cell death or viral latency. During the lytic phase of HSV infection, the virus uses the cellular host machinery to replicate and express viral proteins. Viral gene expression is mediated by both host and viral factors, and thus these factors are required for effective viral infection.  Based on the time course of gene expression and the sensitivity to the chemical inhibitors of viral DNA and protein synthesis, HSV genes have been divided into four classes:α(immediate early),β(early),γ₁(leaky late),andγ₂(true late). In a normal lytic infection expression of α,β,and γ₁  genes occurs prior to viral DNA synthesis, however, expression of γ₂ genes is stringently dependent on viral DNA synthesis. In contrast, when theγ₂ genes are integrated into the cellular genome or are present on a plasmid, they are regulated like β genes independent of viral DNA synthesis. The mechanism behind the location dependent differential expression of the γ₂  genes remain unclear. Two hypotheses have been proposed to explain γ₂   gene regulation; the repressor-mediated mechanism and the lack of cis-action elements upstream of the TATA box. However, direct evidence to support either hypothesis is still lacking. The purpose of this investigation was to identify and characterize the regulatory elements present upstream of a γ₂   (gC) promoter TATA box involved in gene regulation. We characterized the gC promoter region upstream of the TATA box(-31 to –28 relative to the transcription start site) and found that sequences –72 to –35 can act to activate or repress expression in transient expression assays or the viral genome. Recombinant viruses, containing the luciferase reporter gene driven by wild-type and truncated gC promoters, indicated that the sequence –66 to –35 acts as a repressor site which dominated gene expression in the viral genome. However, the sequence –72 to –56 contains an activator domain which increases expression in transient expression assays and in the viral genome. Further, an E2F-like site was characterized in the sequence –72 to –56 and an Ap2 consensus sequence was characterized in the sequence –55 to –35. These results not only demonstrate that functional cis-acting elements are present upstream of aγ₂   promoter but also provide direct evidence supporting the repressor-mediated hypothesis of gC gene expression.