ACTIVATION OF HUMAN HERPESVINUS 8 (HHV-8) THYMIDINE KINASE (TK) TATAA-LESS PROMOTER BY HHV-8 ORF50 GENE-PRODUCT IS SP1 DEPENDENT

Human herpesvirus 8 (HHV-8) is a newly discovered virus closely associ ated with Kaposi's sarcoma and primary effusion lymphomas, When they o ccur in patients with AIDS, these B-cell lymphomas frequently harbor a nother human herpesvirus, Epstein-Barr virus (EBV), To determine the m olecular mechanisms of the regulation of early gene expression by the immediate-early gene products of HHV-8 and to assess possible molecula r interactions between HHV-8 and EBV, we studied the regulation of the HHV-8 thymidine kinase (TK) promoter in cell lines harboring either o r both viruses. The constitutive chloramphenicol acetyltransferase (CA T) activity of the TK promoter was low in all six cell lines tested, A putative immediate-early gene product of HHV-8 ORF50, which is a homo log of EBV BRLF1, was cloned into an expression vector and tested for its transactivating capacity. In the presence of 12-O-tetradecanoyl-ph orbol-13-acetate (TPA), the CAT activity of the TK promoter was increa sed 7- to 720-fold by cotransfection with the ORF50 clone in EBV-produ cing cell lines (Ramos/AW, P3HR-1, and BC-1) but not in EBV-negative c ell lines (BCBL-1 and Ramos), nor in the latently EBV-infected cell li ne Raji. The TK promoter contains three consensus SP1- and two AP1-bin ding sites. In electrophoretic mobility shift assays, the cellular fac tor SP1, but not AP1, was found to bind specifically to the TK promote r. To determine whether the increased CAT activity resulted from the i nteraction of SP1 with the ORF50 gene product, we introduced mutations into two SP1-binding sites. Both mutated SP1 sites had reduced SP1-bi nding activity and greatly decreased TK promoter responsiveness to ORF 50 transactivation, suggesting that upregulation of TK promoter by ORF 50 is SP1 dependent.