AP-1 CIS-RESPONSE ELEMENTS ARE INVOLVED IN BASAL EXPRESSION AND VMW-110 TRANSACTIVATION OF THE LARGE SUBUNIT OF HERPES-SIMPLEX VIRUS TYPE-2RIBONUCLEOTIDE REDUCTASE (ICP10)

The promoter of the large subunit of herpes simplex virus type 2 ribon ucleotide reductase (ICP10) has two AP-I cis-response elements, respec tively located at positions -62 and -94 relative to the transcription start site (Wymer et al., 1989, J. Virol. 63, 2773-2784). Chlorampheni col acetyl transferase (CAT) analysis with hybrid constructions of the CAT structural gene and the ICP10 promoter or its mutants and gel ret ardation studies were used to examine the role of the AP-1 cis-respons e elements in expression from the ICP10 promoter. Basal expression fro m the wild-type promoter was significantly (75-90%) reduced by mutatio n of the upstream or downstream AP-1 element. Mutation in the upstream AP-1 element also caused a 60% reduction in c-Jun-mediated activation . Activation was decreased 40% by mutation in the downstream AP-1 elem ent and it was abrogated by mutation of both elements. Similar results were obtained for ACT-deleted mutants and mutants in which CT was mut ated to AG. The Irans-activation by Vmw110 was also reduced by mutatio n of the AP-1 elements (10- and 2-fold for the upstream and downstream element, respectively) and it was abrogated by mutation of both AP-1 elements. Mutation of nucleotides adjacent to the AP-1 cis-response el ements had no effect on trans-activation. Gel retardation assays with a DNA probe representing the wild-type ICP10 promoter and nuclear extr acts from HSV-1-infected cells identified one complex that was not see n with mock-infected cells or with cells infected with a Vmw110-delete d mutant. The complex was not seen when HSV-1-infected cells were reac ted with an AP-1-mutant DNA probe, and its formation was competed by a n AP-1 but not a mutant AP-1 oligonucleotide. The migration of this co mplex was retarded by c-Fos antibody, suggesting that both AP-1 and Vm w110 are involved in its formation. A mutant deleted in all sequences upstream of the TATA box was also activated by Vmw110, but this activa tion was only 2-fold lower than that seen for the wild type and signif icantly higher (10-fold) than that seen for the double AP-1 mutants. T he data suggest that AP-1 elements play a crucial role in ICP10 gene e xpression/activation. (C) 1997 Academic Press.