scholarly journals Transcription Activation of Polyadenylated Nuclear RNA by Rta in Human Herpesvirus 8/Kaposi's Sarcoma-Associated Herpesvirus

2001 ◽  
Vol 75 (7) ◽  
pp. 3129-3140 ◽  
Author(s):  
Moon Jung Song ◽  
Helen J. Brown ◽  
Ting-Ting Wu ◽  
Ren Sun
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Human Herpesvirus 8 ◽  
Rna Expression ◽  
Herpesvirus 8 ◽  
Infected Cells ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Human herpesvirus 8 (HHV-8) (also known as Kaposi's sarcoma-associated herpesvirus) encodes a novel noncoding polyadenylated nuclear (PAN) RNA (also known as T1.1 or nut-1) during the early phase of lytic replication. PAN RNA is the most abundant transcript of HHV-8, comprising 80% of total poly(A)-selected transcripts in HHV-8-infected cells during lytic replication. We directly measured the abundance of PAN RNA by visualizing 1.1- to 1.2- kb PAN RNA in an ethidium bromide-stained gel from poly(A)-selected RNA. We further pursued the mechanisms by which PAN RNA expression is induced to such high levels.rta, an immediate-early gene of HHV-8, is a transactivator that is sufficient and necessary to activate lytic gene expression in latently infected cells. Ectopic expression of Rta was previously shown to induce PAN RNA expression from the endogenous viral genome and activate the PAN promoter in a reporter system. Here, we have identified the Rta-responsive element (RRE) in the PAN promoter. Deletion analysis revealed that the RRE is present in a region between nucleotides −69 and −38 of the PAN promoter. A promoter construct containing the 69 nucleotides upstream of the transcription start site of the PAN promoter was activated by Rta in the absence or presence of the HHV-8 genome. Rta activated the PAN promoter up to 7,000-fold in 293T cells and 2,000-fold in B cells. Electrophoretic mobility shift assays demonstrated that Rta formed a highly stable complex with the RRE of the PAN promoter. Our study suggests that Rta can induce PAN RNA expression by direct binding of Rta to the RRE of the PAN promoter. This study has highlighted an important mechanism controlling PAN RNA expression and also provides a model system for investigating how Rta transactivates gene expression during lytic replication.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Replication and Transcription Factor Activates the K9 (vIRF) Gene through Two Distinct cis Elements by a Non-DNA-Binding Mechanism

2002 ◽  
Vol 76 (23) ◽  
pp. 12044-12054 ◽  
Author(s):  
Keiji Ueda ◽  
Kayo Ishikawa ◽  
Ken Nishimura ◽  
Shuhei Sakakibara ◽  
Eunju Do ◽  
...  
Keyword(s):  
Dna Binding ◽  
Promoter Region ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8 ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, a homologue of Epstein-Barr virus BRLF1 or Rta, is a strong transactivator and inducer of lytic replication. RTA acting alone can induce lytic replication of KSHV in infected cell lines that originated from primary effusion lymphomas, leading to virus production. During the lytic replication process, RTA activates many kinds of genes, including polyadenylated nuclear RNA, K8, K9 (vIRF), ORF57, and so on. We focused here on the mechanism of how RTA upregulates the K9 (vIRF) promoter and identified two independent cis-acting elements in the K9 (vIRF) promoter that responded to RTA. These elements were finally confined to the sequence 5′-TCTGGGACAGTC-3′ in responsive element (RE) I-2B and the sequence 5′-GTACTTAAAATA-3′ in RE IIC-2, both of which did not share sequence homology. Multiple factors bound specifically with these elements, and their binding was correlated with the RTA-responsive activity. Electrophoretic mobility shift assay with nuclear extract from infected cells and the N-terminal part of RTA expressed in Escherichia coli, however, did not show that RTA interacted directly with these elements, in contrast to the RTA responsive elements in the PAN/K12 promoter region, the ORF57/K8 promoter region. Thus, it was likely that RTA could transactivate several kinds of unique cis elements without directly binding to the responsive elements, probably through cooperation with other DNA-binding factors.

scholarly journals Transcriptional Regulation of the Interleukin-6 Gene of Human Herpesvirus 8 (Kaposi's Sarcoma-Associated Herpesvirus)

2002 ◽  
Vol 76 (16) ◽  
pp. 8252-8264 ◽  
Author(s):  
Hongyu Deng ◽  
Moon Jung Song ◽  
Julia T. Chu ◽  
Ren Sun
Keyword(s):  
Gene Expression ◽  
Interleukin 6 ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8 ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Responsive Element ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Human herpesvirus 8 (HHV-8; Kaposi's sarcoma-associated herpesvirus is linked to Kaposi's sarcoma (KS), primary effusion lymphoma (PEL), and multicentric Castleman's disease (MCD), all of which are viewed as cytokine-driven malignancies. In particular, interleukin-6 (IL-6) has been found to promote the growth and proliferation of cells from KS and PEL. HHV-8 encodes a homologue of IL-6 (viral IL-6 [vIL-6]), which functions similarly to the cellular IL-6. Therefore, vIL-6 has been proposed to play an important role in tumor progression. Several groups have reported that vIL-6 is expressed from the HHV-8 genome at higher levels in PEL and MCD lesions than in KS lesions. However, it is not clear how vIL-6 expression is regulated. We characterized the transcription at the vIL-6 gene locus by Northern blot analysis and, in contrast to previous reports, we observed two distinct transcripts from induced PEL cell lines. This observation was confirmed by primer extension, as well as 5′ and 3′ rapid amplification of cDNA ends. Two transcription initiation sites and putative TATA boxes were mapped. A luciferase reporter system was used to show that each of the two putative TATA boxes contributed to vIL-6 promoter activity. Since virally encoded transcriptional activator Rta potently activates the viral lytic gene expression cascade, we examined the role of Rta in controlling vIL-6 gene expression and found that Rta activated the vIL-6 promoter. The Rta-responsive element was further mapped through a series of deletion constructs. Electrophoretic mobility shift assays demonstrated that Rta binds directly to the vIL-6 Rta-responsive element, and the core Rta-responsive element was mapped to a 26-bp region spanning from nucleotide 18315 to 18290 on the viral genome. We propose that the existence of two vIL-6 promoters offers opportunities for differential regulation of vIL-6 gene expression in different tissue types and may account for the variable vIL-6 levels observed in KS, PEL, and MCD.

scholarly journals Characterization of Interactions between RTA and the Promoter of Polyadenylated Nuclear RNA in Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8

2002 ◽  
Vol 76 (10) ◽  
pp. 5000-5013 ◽  
Author(s):  
Moon Jung Song ◽  
Xudong Li ◽  
Helen J. Brown ◽  
Ren Sun
Keyword(s):  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Human Herpesvirus 8 ◽  
Rna Expression ◽  
Specific Interactions ◽  
Herpesvirus 8 ◽  
Reporter Assays ◽  
Direct Binding

ABSTRACT RTA (replication and transcription activator; also referred to as ORF50, Lyta, and ART), an immediate-early gene product of Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus 8, disrupts latency and drives lytic replication. RTA activates the expression of polyadenylated nuclear (PAN) RNA (also known as T1.1 or nut-1) of KSHV. This novel noncoding PAN RNA is the most abundant lytic transcript of KSHV; therefore, studying PAN RNA expression serves as a model system for understanding how RTA transactivates target genes during lytic replication. The RTA-responsive element of the PAN promoter (pPAN RRE) was previously identified, and our data suggested direct binding of full-length RTA to the pPAN RRE. Here, we present a detailed analysis of specific interactions between RTA and the PAN promoter. We expressed and purified the DNA-binding domain of RTA (Rdbd) to near homogeneity and measured its affinity for the pPAN RRE. In electrophoretic mobility shift assays (EMSAs), the dissociation constant (Kd ) of Rdbd on the pPAN RRE was determined to be approximately 8 × 10−9 M, suggesting a strong interaction between RTA and DNA. The specificity of RTA binding to the PAN promoter was confirmed with supershift assays. The Rdbd binding sequences on the PAN promoter were mapped within a 16-bp region of the pPAN RRE by methylation interference assays. However, the minimal DNA sequence for Rdbd binding requires an additional 7 bp on both sides of the area mapped by interference assays, suggesting that non-sequence-specific as well as sequence-specific interactions between RTA and DNA contribute to high-affinity binding. To better understand the molecular interactions between RTA and the PAN promoter, an extensive mutagenesis study on the pPAN RRE was carried out by using EMSAs and reporter assays. These analyses revealed base pairs critical for both Rdbd binding in vitro and RTA transactivation in vivo of the PAN promoter. The results from methylation interference, deletion analysis, and mutagenesis using EMSAs and reporter assays were closely correlated and support the hypothesis that RTA activates PAN RNA expression through direct binding to DNA.

Lytic growth of Kaposi's sarcoma–associated herpesvirus (human herpesvirus 8) in culture

1996 ◽  
Vol 2 (3) ◽  
pp. 342-346 ◽  
Author(s):  
Rolf Renne ◽  
Weidong Zhong ◽  
Brian Herndier ◽  
Michael Mcgrath ◽  
Nancy Abbey ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8 ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus
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