scholarly journals Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Contains Hypoxia Response Elements: Relevance to Lytic Induction by Hypoxia

2003 ◽  
Vol 77 (12) ◽  
pp. 6761-6768 ◽  
Author(s):  
Muzammel Haque ◽  
David A. Davis ◽  
Victoria Wang ◽  
Isabelle Widmer ◽  
Robert Yarchoan
Keyword(s):  
Promoter Region ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Human Herpesvirus 8 ◽  
Viral Gene ◽  
Hypoxia Response ◽  
Herpesvirus 8 ◽  
Response Elements

ABSTRACT Kaposi's sarcoma (KS)-associated herpesvirus (KSHV), also known as human herpesvirus 8, is an etiologic agent of KS, primary effusion lymphoma (PEL), and multicentric Castleman's disease. We recently demonstrated that hypoxia can induce lytic replication of KSHV in PEL cell lines. Hypoxia induces the accumulation of hypoxia-inducible factors (HIF), and we hypothesized that the KSHV genome may respond to hypoxia through functional hypoxia response elements (HREs). Here, we demonstrate the presence of at least two promoters within the KSHV genome that are activated by hypoxia or hypoxia mimics. One is in the promoter region of the gene for Rta, the main lytic switch gene, and the other is within the promoter region of ORF34, a lytic gene of unknown function. The ORF34 promoter contains three putative consensus HREs oriented in the direction of the gene. Dissection and site-directed mutagenesis studies confirmed that one of the HREs of the ORF34 promoter is functional. Under conditions of hypoxia, the ORF34 promoter was strongly upregulated by HIF-1α and HIF-2α. By contrast, the promoter of the gene for Rta appeared to be preferentially upregulated by HIF-2α. Reverse transcription-PCR analysis revealed that specific messages for ORF34 and ORF50 are upregulated in BCBL-1 cells exposed to hypoxia. An HIF-1 binding and competition assay demonstrated that the HRE sequence from the ORF34 promoter can compete for HIF-1α binding to an erythropoietin HRE oligonucleotide while a mutant sequence cannot. Thus, we demonstrated that a viral gene can be activated by hypoxia through activation of a functional viral HRE. To our knowledge, this is the first example of a functional HRE in a viral promoter.

scholarly journals Transcription Program of Human Herpesvirus 8 (Kaposi's Sarcoma-Associated Herpesvirus)

2001 ◽  
Vol 75 (10) ◽  
pp. 4843-4853 ◽  
Author(s):  
Mini Paulose-Murphy ◽  
Nguyen-Khoi Ha ◽  
Chunsheng Xiang ◽  
Yidong Chen ◽  
Laura Gillim ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Primary Effusion Lymphoma ◽  
Human Herpesvirus ◽  
Viral Gene Expression ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Human Herpesvirus 8 ◽  
Viral Gene ◽  
Herpesvirus 8 ◽  
Transcription Program

ABSTRACT Human herpesvirus 8 (HHV-8), a gammaherpesvirus implicated in Kaposi's sarcoma, primary effusion lymphoma, and Castleman's disease, encodes several pathogenically important cellular homologs. To define the HHV-8 transcription program, RNA obtained from latently infected body cavity-based lymphoma 1 cells induced to undergo lytic replication was used to query a custom HHV-8 DNA microarray containing nearly every known viral open reading frame. The patterns of viral gene expression offer insights into the replication and pathogenic strategies of HHV-8.

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 Human Herpesvirus 8 Infects and Replicates in Langerhans Cells and Interstitial Dermal Dendritic Cells and Impairs Their Function

2017 ◽  
Vol 91 (20) ◽  
Author(s):  
Giovanna Rappocciolo ◽  
Mariel Jais ◽  
Paolo A. Piazza ◽  
Diana C. DeLucia ◽  
Frank J. Jenkins ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Langerhans Cells ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Productive Infection ◽  
Receptor Protein ◽  
Human Herpesvirus 8 ◽  
Herpesvirus 8

ABSTRACT The predominant types of dendritic cells (DC) in the skin and mucosa are Langerhans cells (LC) and interstitial dermal DC (iDDC). LC and iDDC process cutaneous antigens and migrate out of the skin and mucosa to the draining lymph nodes to present antigens to T and B cells. Because of the strategic location of LC and iDDC and the ability of these cells to capture and process pathogens, we hypothesized that they could be infected with human herpesvirus 8 (HHV-8) (Kaposi's sarcoma [KS]-associated herpesvirus) and have an important role in the development of KS. We have previously shown that HHV-8 enters monocyte-derived dendritic cells (MDDC) through DC-SIGN, resulting in nonproductive infection. Here we show that LC and iDDC generated from pluripotent cord blood CD34+ cell precursors support productive infection with HHV-8. Anti-DC-SIGN monoclonal antibody (MAb) inhibited HHV-8 infection of iDDC, as shown by low expression levels of viral proteins and DNA. In contrast, blocking of both langerin and the receptor protein tyrosine kinase ephrin A2 was required to inhibit HHV-8 infection of LC. Infection with HHV-8 did not alter the cell surface expression of langerin on LC but downregulated the expression of DC-SIGN on iDDC, as we previously reported for MDDC. HHV-8-infected LC and iDDC had a reduced ability to stimulate allogeneic CD4+ T cells in the mixed-lymphocyte reaction. These results indicate that HHV-8 can target both LC and iDDC for productive infection via different receptors and alter their function, supporting their potential role in HHV-8 pathogenesis and KS. IMPORTANCE Here we show that HHV-8, a DNA tumor virus that causes Kaposi's sarcoma, infects three types of dendritic cells: monocyte-derived dendritic cells, Langerhans cells, and interstitial dermal dendritic cells. We show that different receptors are used by this virus to infect these cells. DC-SIGN is a major receptor for infection of both monocyte-derived dendritic cells and interstitial dermal dendritic cells, yet the virus fully replicates only in the latter. HHV-8 uses langerin and the ephrin A2 receptor to infect Langerhans cells, which support full HHV-8 lytic replication. This infection of Langerhans cells and interstitial dermal dendritic cells results in an impaired ability to stimulate CD4+ helper T cell responses. Taken together, our data show that HHV-8 utilizes alternate receptors to differentially infect and replicate in these tissue-resident DC and support the hypothesis that these cells play an important role in HHV-8 infection and pathogenesis.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Infection of Human Fibroblast Cells Occurs through Endocytosis

2003 ◽  
Vol 77 (14) ◽  
pp. 7978-7990 ◽  
Author(s):  
Shaw M. Akula ◽  
Pramod P. Naranatt ◽  
Neelam-Sharma Walia ◽  
Fu-Zhang Wang ◽  
Barbara Fegley ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Human Herpesvirus 8 ◽  
Target Cells ◽  
Fibroblast Cells ◽  
Coated Pits ◽  
Herpesvirus 8 ◽  
Endocytic Vesicles

ABSTRACT Kaposi's sarcoma (KS)-associated herpesvirus or human herpesvirus 8 (HHV-8) DNA and transcripts have been detected in the B cells, macrophages, keratinocytes, and endothelial and epithelial cells of KS patients. In vitro, HHV-8 infects human B, endothelial, epithelial, and fibroblast cells, as well as animal cells, and the infection is characterized by (i) absence of lytic replication by the input virus and (ii) latent infection. For its initial binding to target cells, HHV-8 uses ubiquitous heparan sulfate molecules via its envelope-associated glycoproteins gB and gpK8.1A. HHV-8 also interacts with the α3β1 integrin via its glycoprotein gB, and virus binding studies suggest that α3β1 is one of the HHV-8 entry receptors (S. M. Akula, N. P. Pramod, F. Z. Wang, and B. Chandran, Cell 108:407-419, 2002). In this study, morphological and biochemical techniques were used to examine the entry of HHV-8 into human foreskin fibroblasts (HFF). HHV-8 was detected in coated vesicles and in large, smooth-surfaced endocytic vesicles. Fusion of viral envelope with the vesicle wall was also observed. In immune electron microscopy, anti-HHV-8 gB antibodies colocalized with virus-containing endocytic vesicles. In fluorescence microscopic analyses, transferrin was colocalized with HHV-8. HHV-8 infection was significantly inhibited by preincubation of cells with chlorpromazine HCl, which blocks endocytosis via clathrin-coated pits, but not by nystatin and cholera toxin B, which blocks endocytosis via caveolae and induces the dissociation of lipid rafts, respectively. Infection was also inhibited by blocking the acidification of endosomes by NH4Cl and bafilomycin A. Inhibition of HHV-8 open reading frame 73 gene expression by chlorpromazine HCl, bafilomycin A, and NH4Cl demonstrated that the virions in the vesicles could proceed to cause an infection. Taken together, these findings suggest that for its infectious entry into HFF, HHV-8 uses clathrin-mediated endocytosis and a low-pH intracellular environment.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 Replication and Transcription Activator Regulates Viral and Cellular Genes via Interferon-Stimulated Response Elements

2005 ◽  
Vol 79 (9) ◽  
pp. 5640-5652 ◽  
Author(s):  
Jun Zhang ◽  
Jinzhong Wang ◽  
Charles Wood ◽  
Dongsheng Xu ◽  
Luwen Zhang
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Human Herpesvirus 8 ◽  
Transcription Activator ◽  
Reporter Construct ◽  
Herpesvirus 8 ◽  
Response Elements

ABSTRACT Kaposi's sarcoma-associated herpesvirus (also called human herpesvirus 8 [HHV-8]) replication and transcription activator (RTA) is apparently necessary and sufficient for the switch from viral latency to lytic replication. RTA may regulate open reading frame (ORF) K14 (viral OX-2 homologue) and ORF74 (viral G-protein-coupled receptor homologue) genes through an interferon-stimulated response element (ISRE)-like sequence (K14 ISRE) in the promoter region. RTA strongly activated a K14 ISRE-containing K14-ORF74 promoter reporter construct and a heterologous promoter reporter construct containing K14 ISRE. RTA could bind to K14 ISRE and other ISREs, activate promoter reporter constructs from interferon-simulated genes (ISGs), and selectively induce three endogenous ISGs in primary endothelial cells: ISG-54, myxovirus resistance protein 1 (MxA), and stimulated trans-acting factor of 50 kDa. In addition, a region in the RTA DNA-binding domain has been identified with certain sequence similarity to the DNA-binding domains of the interferon regulatory factor (IRF) family. Mutation in one conserved amino acid within this region reduced the ability of RTA to bind to ISRE as well as other RTA response elements. Furthermore, the mutant failed to activate RTA-responsive promoters and to induce viral lytic gene expression. The mutation at the same conserved amino acid residue in IRF-7 drastically reduced its ability to bind to DNA and to activate the beta interferon promoter. The sequence and functional similarities between RTA and IRFs suggest that the HHV-8 RTA may usurp the cellular IRF pathway.

scholarly journals RNA Editing of the Human Herpesvirus 8 Kaposin Transcript Eliminates Its Transforming Activity and Is Induced during Lytic Replication

2007 ◽  
Vol 81 (24) ◽  
pp. 13544-13551 ◽  
Author(s):  
Sharon Z. Gandy ◽  
Sarah D. Linnstaedt ◽  
Sumitra Muralidhar ◽  
Kathleen A. Cashman ◽  
Leonard J. Rosenthal ◽  
...  
Keyword(s):  
Rna Editing ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Human Herpesvirus 8 ◽  
Viral Dna ◽  
Herpesvirus 8 ◽  
Sequence Heterogeneity ◽  
Transforming Activity

ABSTRACT Human herpesvirus 8 is the etiologic agent associated with Kaposi's sarcoma and primary effusion lymphoma (PEL). The K12 RNA, which produces as many as three variants of the kaposin protein, as well as a microRNA, is the most abundant transcript expressed in latent Kaposi's sarcoma-associated herpesvirus infection, and yet it is also induced during lytic replication. The portion of the transcript that includes the microRNA and the kaposin A sequence has been shown to have tumorigenic potential. Genome coordinate 117990, which is within this transcript, has been found to be heterogeneous, primarily in RNAs but also among viral DNA sequences. This sequence heterogeneity affects an amino acid in kaposins A and C and the microRNA. The functional effects of this sequence heterogeneity have not been studied, and its origin has not been definitively settled; both RNA editing and heterogeneity at the level of the viral genome have been proposed. Here, we show that transcripts containing A at position 117990 are tumorigenic, while those with G at this position are not. Using a highly sensitive quantitative assay, we observed that, in PEL cells under conditions where more than 60% of cDNAs derived from K12 RNA transcripts have G at coordinate 117990, there is no detectable G in the viral DNA sequence at this position, only A. This result is consistent with RNA editing by one of the host RNA adenosine deaminases (ADARs). Indeed, we observed that purified human ADAR1 efficiently edits K12 RNA in vitro. Remarkably, the amount of editing correlated with the replicative state of the virus; editing levels were nearly 10-fold higher in cells treated to induce lytic viral replication. These results suggest that RNA editing controls the function of one segment of the kaposin transcript, such that it has transforming activity during latent replication and possibly another, as-yet-undetermined, function during lytic replication.

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 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.

scholarly journals Characterization of Monoclonal Antibodies Raised against the Latent Nuclear Antigen of Human Herpesvirus 8

1999 ◽  
Vol 73 (6) ◽  
pp. 5149-5155 ◽  
Author(s):  
Paul Kellam ◽  
Dimitra Bourboulia ◽  
Nicolas Dupin ◽  
Chris Shotton ◽  
Cyril Fisher ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Human Herpesvirus 8 ◽  
Nuclear Antigen ◽  
Viral Gene ◽  
Herpesvirus 8 ◽  
Latent Nuclear Antigen

ABSTRACT Human herpesvirus 8 (HHV-8; also designated Kaposi’s sarcoma-associated herpesvirus) is the likely etiological agent of Kaposi’s sarcoma (KS). HHV-8 encodes a latent nuclear antigen (LNA) which is the product of the viral gene orf 73. LNA is recognized by most infected patient sera and is the basis of current immunofluorescence assays used in epidemiological studies of HHV-8 infection. Here we describe the characterization of four monoclonal antibodies raised to the C-terminal third of LNA–glutathioneS-transferase fusion proteins. These monoclonal antibodies recognized discrete linear epitopes within the C terminus and repetitive region of LNA, detected antigen in primary effusion lymphoma (PEL) cells, and precipitated a 220- to 230-kDa protein doublet corresponding to LNA from HHV-8-infected PEL cell lines. In situ immunocytochemistry of KS lesions with these antibodies show that LNA is extensively expressed in KS spindle cells.

Sign in / Sign up

Export Citation Format

Share Document