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Transcriptional regulation of the Kaposi's sarcoma-associated herpesvirus K15 gene.

ABSTRACT: The K15 gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) is a transmembrane protein that is encoded by the last open reading frame of the KSHV genome. The K15 protein has been implicated in modulation of B-cell signal transduction and activation of the Ras/mitogen-activated protein kinase and NF-kappaB signal transduction pathways. Here we report the identification of the transcriptional start site of the full-length K15 gene in KSHV-positive BCBL-1 cells. We have mapped the K15 transcriptional start site to a position 152 nucleotides upstream from the translation start site by rapid amplification of cDNA ends and RNase protection assays. We have also characterized the K15 promoter element. To analyze the cis-acting elements necessary to regulate K15 gene expression, a series of 5' promoter deletion constructs were generated and subcloned upstream of the luciferase reporter gene. Transcriptional assays with these mutant promoters demonstrated that chemical induction in latently infected KSHV-positive BCBL-1 cells activated K15 transcription. In addition, K15 promoter transactivation was also mediated by the viral immediate-early protein Orf50/Rta, suggesting that the K15 gene is actively transcribed during lytic replication.

SUBMITTER: Wong EL

PROVIDER: S-EPMC1346939 | biostudies-literature | 2006 Feb

REPOSITORIES: biostudies-literature

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Transcriptional regulation of the Kaposi's sarcoma-associated herpesvirus K15 gene.

Wong Emily L EL Damania Blossom B

Journal of virology 20060201 3

The K15 gene product of Kaposi's sarcoma-associated herpesvirus (KSHV) is a transmembrane protein that is encoded by the last open reading frame of the KSHV genome. The K15 protein has been implicated in modulation of B-cell signal transduction and activation of the Ras/mitogen-activated protein kinase and NF-kappaB signal transduction pathways. Here we report the identification of the transcriptional start site of the full-length K15 gene in KSHV-positive BCBL-1 cells. We have mapped the K15 tr ...[more]

PMID: 16415016

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Project description: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.

Project description:Herpesvirus gene expression can be classified into four distinct kinetic stages: latent, immediate early, early, and late. Here we characterize the kinetic class of a group of 16 Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8 genes in a cultured primary effusion cell line and examine the expression of a subset of these genes in KS biopsies. Expression of two latent genes, LANA and vFLIP, was constitutive and was not induced by chemicals that induce the lytic cycle in primary effusion lymphoma (PEL) cell lines. An immediate-early gene, Rta (open reading frame 50 [ORF50]), was induced within 4 h of the addition of n-butyrate, and its 3.6-kb mRNA was resistant to inhibition by cycloheximide. Early genes, including K3 and K5 that are homologues of the "immediate-early" gene of bovine herpesvirus 4, K8 that is a positional homologue of Epstein-Barr virus BZLF1, vMIP II, vIL-6, and polyadenylated nuclear (PAN) RNA, appeared 8 to 13 h after chemical induction. A second group of early genes that were slightly delayed in their appearance included viral DHFR, thymidylate synthase, vMIP I, G protein-coupled receptor, K12, vBcl2, and a lytic transcript that overlapped LANA. The transcript of sVCA (ORF65), a late gene whose expression was abolished by Phosphonoacetic acid, an inhibitor of KSHV DNA replication, did not appear until 30 h after induction. Single-cell assays indicated that the induction of lytic cycle transcripts resulted from the recruitment of additional cells into the lytic cycle. In situ hybridization of KS biopsies showed that about 3% of spindle-shaped tumor cells expressed Rta, ORF K8, vIL-6, vMIP I, vBcl-2, PAN RNA, and sVCA. Our study shows that several KSHV-encoded homologues of cellular cytokines, chemokines, and antiapoptotic factors are expressed during the viral lytic cycle in PEL cell lines and in KS biopsies. The lytic cycle of KSHV, probably under the initial control of the KSHV/Rta gene, may directly contribute to tumor pathogenesis.

Dataset Information

Transcriptional regulation of the Kaposi's sarcoma-associated herpesvirus K15 gene.

Publications

Transcriptional regulation of the Kaposi's sarcoma-associated herpesvirus K15 gene.

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