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 transduction. Consequently, this gene is thought to play an important role in the tumorigenicity of KSHV. To understand the molecular mechanisms underlying vIRF expression, we studied the transcriptional regulation of this gene. Experiments using 5' rapid amplification of cDNA ends and primer extension revealed that vIRF had different transcriptional patterns during the latent and lytic phases. The promoter region of the minor transcript, which was mainly 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 expressed in tetradecanoyl phorbol acetate-induced BCBL-1 cells, contained a canonical TATA box. A luciferase reporter assay using a deletion mutant of the vIRF promoter and a mutation in the TATA box showed that the TATA box was critical 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 than 10% of the activity in the lytic phase. Therefore, KSHV may use different functional promoter elements to regulate the expression of vIRF and to antagonize the cell's interferon-mediated antiviral activity. We have also identified a functional domain in the ORF 50 protein, an immediate-early gene product that is mainly encoded by ORF 50. The ORF 50 protein transactivated the vIRF and DNA polymerase promoters in BCBL-1, 293T, and CV-1 cells. Deleting one of its two putative nuclear localization signals (NLSs) resulted in failure of the ORF 50 protein to localize to the nucleus and consequently abrogated its transactivating activity. We further confirmed that the N-terminal region of the ORF 50 protein included an NLS domain. We found that this domain was sufficient to translocate beta-galactosidase to the nucleus. Analysis of deletions within the vIRF promoter suggested that two sequence domains were important for its transactivation by the ORF 50 protein, both of which included putative SP-1 and AP-1 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 transactivation by ORF 50.
Chen
J
J
Department of Microbiology, Osaka University Medical School, Osaka 565-0871, Japan.
Ueda
K
K
Sakakibara
S
S
Okuno
T
T
Yamanishi
K
K
D001483:Base Sequence; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D015967:Gene Expression Regulation, Viral; D017930:Genes, Reporter; D019288:Herpesvirus 8, Human / Q000235:genetics* / Q000502:physiology; D006801:Humans; D017874:Immediate-Early Proteins / Q000378:metabolism; D050835:Interferon Regulatory Factors; D007372:Interferons / Q000037:antagonists & inhibitors*; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D019913:Nuclear Localization Signals; D011401:Promoter Regions, Genetic; D017434:Protein Structure, Tertiary; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D015398:Signal Transduction; D015534:Trans-Activators / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014764:Viral Proteins
D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D050835:Interferon Regulatory Factors; D019913:Nuclear Localization Signals; C116742:ORF 50 transactivator; D012097:Repressor Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D014764:Viral Proteins; C413486:viral interferon regulatory factors; D007372:Interferons; D008156:Luciferases