Transcriptional regulation of the Kaposi's sarcoma-associated herpesvirus viral interferon regulatory factor gene

The Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, open reading frame (ORF) K9 encodes a viral interferon regulatory factor ( vIRF) that functions as a repressor for interferon-mediated signal transduc tion. Consequently, this gene is thought to play an important role in the t umorigenicity of KSHV. To understand the molecular mechanisms underlying vI RF expression, we studied the transcriptional regulation of this gene. Expe riments using 5' rapid amplification of cDNA ends and primer extension reve aled that vIRF had different transcriptional patterns during the latent and lytic phases. The promoter region of the minor transcript, which was mainl y expressed in uninduced BCBL-1 cells, did not contain a canonical TATA box , but a cap-like element and an initiator element flanked the transcription start site. The promoter of the major transcript, which was mainly express ed in tetradecanoyl phorbol acetate-induced BCBL-1 cells, contained a canon ical TATA box. A luciferase reporter assay using a deletion mutant of the v IRF promoter and a mutation in the TATA box showed that the TATA box was cr itical for the lytic activity of vIRF. The promoter activity in the latent phase was eight times stronger than that of the empty vector but was less t han 10% of the activity in the lytic phase. Therefore, KSHV may use differe nt functional promoter elements to regulate the expression of vIRF and to a ntagonize the cell's interferon-mediated antiviral activity. We have also i dentified a functional domain in the ORF 50 protein, an immediate-early gen e product that is mainly encoded by ORF 50. The ORF 50 protein transactivat ed the vIRF and DNA polymerase promoters in BCBL-1, 293T, and CV-1 cells. D eleting one of its two putative nuclear localization signals (NLSs) resulte d in failure of the ORF 50 protein to localize to the nucleus and consequen tly abrogated its transactivating activity. We further confirmed that the N -terminal region of the ORF 50 protein included an NLS domain. We found tha t this domain was sufficient to translocate beta-galactosidase to the nucle us. Analysis of deletions within the vIRF promoter suggested that two seque nce domains were important for its transactivation by the ORF 50 protein, b oth of which included putative SP-1 and AP-l binding sites. Competition gel shift assays demonstrated that SP-1 bound to these two domains, suggesting that the SP-1 binding sites in the vIRF promoter are involved in its trans activation by ORF 50.