Transcriptional regulation of Kaposi's sarcoma-associated herpesvirus-encoded oncogene viral interferon regulatory factor by a novel transcriptional silencer, Tis

Viral interferon regulatory factor (vIRF) encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) has been shown to transform NIH3T3 and Rat-1 cells, inhibit interferon signal transduction, and regulate the expression of KSHV genes. We had previously characterized the vIRF core promoter and defined a 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive region in the upstream regulatory sequence of vIRF gene. Here, we have further identified a novel transcriptional silencer, named Tis in this region. Tis represses the promoter activities of vIRF and heterologous herpes simplex virus thymidine kinase genes in both position- and orientation-independent manners. Deletion analysis has identified a cis-element of 23 nucleotides that is essential for the negative regulation. Two Tis-binding protein complexes, named vR1 and vR2, were observed by electrophoretic mobility shift assays using nuclear extracts from both KSHV-negative and -positive cell lines. A sequence fragment GAGTTAATAGGTAGAG in the cis-element was shown to be required for the DNA-protein interactions as well as the repression of vIRF promoter activity. Point-mutation analysis identified TTAAT and GTTAATAG as the core sequence motifs for the binding of vR1 and vR2, respectively. These results define the function of a novel transcriptional silencer in the regulation of vIRF gene expression.