Transcriptional Regulation of the Kaposi's Sarcoma-Associated Herpesvirus Viral Interferon Regulatory Factor Gene

Abstract

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 ␤-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.

Transcription mapping of human herpesvirus 8 genes encoding viral interferon regulatory factors

“…The situation regarding the use of alternative promoters to generate minor vIRF-1 transcripts in uninduced cells remains unresolved. We were unable to confirm the minor end reported by Chen et al (2000), and instead detected a different minor end, but with only one kit. The pattern of vIRF-1 expression in uninduced cells would thus bear greater scrutiny.…”

“…In nested PCR experiments using the Smart RACE kit with uninduced RNA (data not shown), a minor RACE product (denoted L in Table 1) was generated that was 60-70 bp larger than the product corresponding to the major 59-end (denoted S). As shown in Table 1, the corresponding 59-ends were distributed over a region upstream from the major end, but none corresponded to the minor end of Chen et al (2000). No distinct product indicating 59-ends upstream of the major end were identified using the GeneRacer kit followed by nested PCR, and cloned products from the relevant region of the gel exhibited 59-ends no different from those of the major product.…”

“…Inagi et al (1999) employed a RACE kit (Invitrogen) involving priming of the cDNA by a genespecific primer, addition of a poly(dC) tract by terminal transferase, priming of the second cDNA strand by an oligonucleotide with a dG tract at the 39-end, and PCR using adaptor-specific and gene-specific primers. Chen et al (2000), from the same group, also used primer extension. Wang et al (2001) utilized first-strand cDNA priming with a gene-specific primer, addition of a poly(dA) tract to the 39-end and PCR using an oligo(dT) primer and a second gene-specific primer.…”

“…Wang et al (2001) utilized first-strand cDNA priming with a gene-specific primer, addition of a poly(dA) tract to the 39-end and PCR using an oligo(dT) primer and a second gene-specific primer. Wang et al (2001) mapped the major 59-end to an A residue in the sequence TATCTGA and Chen et al (2000) to two closely adjacent nucleotides (TATCTGA). These 59-ends are located 21-25 nucleotides downstream from a TATA box (TATATA).…”

“…The mRNA is 1?5 kb in size Sarid et al, 1998) and appears to be unspliced. The 59-end of the predominant transcript has been mapped by RACE and primer extension, and the core promoter and regulatory regions have been dissected functionally (Inagi et al, 1999;Chen et al, 2000;Wang et al, 2001Wang et al, , 2002Ueda et al, 2002). The 50 kDa vIRF-1 protein is predominantly nuclear (Inagi et al, 1999).…”