Activation of Human Herpesvirus 8 (HHV-8) Thymidine Kinase (TK) TATAA-Less Promoter by HHV-8 ORF50 Gene Product Is SP1 Dependent

Abstract

Human herpesvirus 8 (HHV-8) is a newly discovered virus closely associated with Kaposi's sarcoma and primary effusion lymphomas. When they occur in patients with AIDS, these B-cell lymphomas frequently harbor another human herpesvirus, Epstein-Barr virus (EBV). To determine the molecular mechanisms of the regulation of early gene expression by the immediate-early gene products of HHV-8 and to assess possible molecular interactions between HHV-8 and EBV, we studied the regulation of the HHV-8 thymidine kinase (TK) promoter in cell lines harboring either or both viruses. The constitutive chloramphenicol acetyltransferase (CAT) 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 homolog of EBV BRLF1, was cloned into an expression vector and tested for its transactivating capacity. In the presence of 12-O-tetradecanoyl-phorbol-13-acetate (TPA), the CAT activity of the TK promoter was increased 7- to 720-fold by cotransfection with the ORF50 clone in EBV-producing cell lines (Ramos/AW, P3HR-1, and BC-1) but not in EBV-negative cell lines (BCBL-1 and Ramos), nor in the latently EBV-infected cell line Raji. The TK promoter contains three consensus SP1- and two AP1-binding sites. In electrophoretic mobility shift assays, the cellular factor SP1, but not AP1, was found to bind specifically to the TK promoter. To determine whether the increased CAT activity resulted from the interaction of SP1 with the ORF50 gene product, we introduced mutations into two SP1-binding sites. Both mutated SP1 sites had reduced SP1-binding activity and greatly decreased TK promoter responsiveness to ORF50 transactivation, suggesting that upregulation of TK promoter by ORF50 is SP1 dependent.

Differential Regulation of the Overlapping Kaposi's Sarcoma-Associated Herpesvirus vGCR (orf74) and LANA (orf73) Promoters

“…7 and 8). Ectopic expression of orf50 induces KSHV early gene transcription as well as complete lytic replication (15,25,51,55,58,62,64), whereas a dominant-negative orf50 counteracts induction of the lytic cycle (25). This establishes orf50 as necessary and sufficient to induce KSHV replication, a property that it shares with MHV68 orf50 (58).…”

Molecular Genetics of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Epidemiology and Pathogenesis

“…The ORF73 IRD is highly variable among individual KSHV isolates, with variations due to insertions, deletions, and point mutations but never frameshifts or nonsense codons (538). As a result, LANA-1 from different isolates contains variable numbers of acidic repeats, with the glutamine-rich region (region II), in particular, being a hot spot for amino acid variation (177,537).…”

Early Activation of the Kaposi's Sarcoma-Associated Herpesvirus RTA, RAP, and MTA Promoters by the Tetradecanoyl Phorbol Acetate-Induced AP1 Pathway

“…Although RTA transactivates the PAN promoter through direct binding to the type I RRE, mutations in the C/EBP sites outside the originally defined RRE also lead to a partial decrease in RTA activation of the PAN promoter (55). It has been suggested that, besides C/EBP␣ and CBF1, cellular factors such as OCT, CREB, K-RBP, and SP1 play roles in RTA transactivation on certain target promoters (21,44,53,61).…”