scholarly journals Transcriptional Downregulation of ORF50/Rta by Methotrexate Inhibits the Switch of Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 from Latency to Lytic Replication

2002 ◽  
Vol 76 (10) ◽  
pp. 5208-5219 ◽  
Author(s):  
Francesca Curreli ◽  
Francesca Cerimele ◽  
Sumitra Muralidhar ◽  
Leonard J. Rosenthal ◽  
Ethel Cesarman ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Cultured Cells ◽  
Primary Effusion Lymphoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Early Gene ◽  
Transcription Activator ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a cellular dihydrofolate reductase (DHFR) homologue. Methotrexate (MTX), a potent anti-inflammatory agent, inhibits cellular DHFR activity. We investigated the effect of noncytotoxic doses of MTX on latency and lytic KSHV replication in two KSHV-infected primary effusion lymphoma cell lines (BC-3 and BC-1) and in MTX-resistant BC-3 cells (MTX-R-BC-3 cells). Treatment with MTX completely prevented tetradecanoyl phorbol acetate-induced viral DNA replication and strongly decreased viral lytic transcript levels, even in MTX-resistant cells. However, the same treatment had no effect on transcription of cellular genes and KSHV latent genes. One of the lytic transcripts inhibited by MTX, ORF50/Rta (open reading frame), is an immediate-early gene encoding a replication-transcription activator required for expression of other viral lytic genes. Therefore, transcription of genes downstream of ORF50/Rta was inhibited, including those encoding the viral G-protein-coupled receptor (GPCR), viral interleukin-6, and K12/kaposin, which have been shown to be transforming in vitro and oncogenic in mice. Resistance to MTX has been documented in cultured cells and also in patients treated with this drug. However, MTX showed an inhibitory activity even in MTX-R-BC-3 cells. Two currently available antiherpesvirus drugs, cidofovir and foscarnet, had no effect on the transcription of these viral oncogenes and ORF50/Rta. MTX is the first example of a compound shown to downregulate the expression of ORF50/Rta and therefore prevent viral transforming gene transcription. Given that the expression of these genes may be important for tumor development, MTX could play a role in the future management of KSHV-associated malignancies.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus Latent and Lytic Gene Expression as Revealed by DNA Arrays

2001 ◽  
Vol 75 (2) ◽  
pp. 891-902 ◽  
Author(s):  
Richard G. Jenner ◽  
M. Mar Albà ◽  
Chris Boshoff ◽  
Paul Kellam
Keyword(s):  
Gene Expression ◽  
Kaposi's Sarcoma ◽  
Primary Effusion Lymphoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Dna Arrays ◽  
Nylon Membrane ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV; human herpesvirus 8) is associated with three human tumors, Kaposi's sarcoma, primary effusion lymphoma (PEL), and multicentric Castleman's disease. KSHV encodes a number of homologs of cellular proteins involved in the cell cycle, signal transduction, and modulation of the host immune response. Of the virus complement of over 85 open reading frames (ORFs), the expression of only a minority has been characterized individually. We have constructed a nylon membrane-based DNA array which allows the expression of almost every ORF of KSHV to be measured simultaneously. A PEL-derived cell line, BC-3, was used to study the expression of KSHV during latency and after the induction of lytic replication. Cluster analysis, which arranges genes according to their expression profile, revealed a correlation between expression and assigned gene function that is consistent with the known stages of the herpesvirus life cycle. Furthermore, latent and lytic genes thought to be functionally related cluster into groups. The correlation between gene expression and function also infers possible roles for KSHV genes yet to be characterized.

scholarly journals Activation of the Kaposi's Sarcoma-Associated Herpesvirus Major Latency Locus by the Lytic Switch Protein RTA (ORF50)

2005 ◽  
Vol 79 (13) ◽  
pp. 8493-8505 ◽  
Author(s):  
Satoko Matsumura ◽  
Yuriko Fujita ◽  
Evan Gomez ◽  
Naoko Tanese ◽  
Angus C. Wilson
Keyword(s):  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Early Gene ◽  
Viral Gene ◽  
Transcription Activator ◽  
Potent Inducer ◽  
Alternative Mrna Splicing ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) maintains a latent infection in primary effusion lymphoma cells but can be induced to enter full lytic replication by exposure to a variety of chemical inducing agents or by expression of the KSHV-encoded replication and transcription activator (RTA) protein. During latency, only a few viral genes are expressed, and these include the three genes of the so-called latency transcript (LT) cluster: v-FLIP (open reading frame 71 [ORF71]), v-cyclin (ORF72), and latency-associated nuclear antigen (ORF73). During latency, all three open reading frames are transcribed from a common promoter as part of a multicistronic mRNA. Subsequent alternative mRNA splicing and internal ribosome entry allows for the expression of each protein. Here, we show that transcription of LT cassette mRNA can be induced by RTA through the activation of a second promoter (LTi) immediately downstream of the constitutively active promoter (LTc). We identified a minimal cis-regulatory region, which overlaps with the promoter for the bicistronic K14/v-GPCR delayed early gene that is transcribed in the opposite direction. In addition to a TATA box at −30 relative to the LTi mRNA start sites, we identified three separate RTA response elements that are also utilized by the K14/v-GPCR promoter. Interestingly, LTi is unresponsive to sodium butyrate, a potent inducer of lytic replication. This suggests there is a previously unrecognized class of RTA-responsive promoters that respond to direct, but not indirect, induction of RTA. These studies highlight the fact that induction method can influence the precise program of viral gene expression during early events in reactivation and also suggest a mechanism by which RTA contributes to establishment of latency during de novo infections.

scholarly journals Kaposi's Sarcoma-Associated Herpesvirus ori-Lyt-Dependent DNA Replication: DualRole of Replication and Transcription Activator

2006 ◽  
Vol 80 (24) ◽  
pp. 12171-12186 ◽  
Author(s):  
Yan Wang ◽  
Qiyi Tang ◽  
Gerd G. Maul ◽  
Yan Yuan
Keyword(s):  
Dna Replication ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Transcription Activator ◽  
Binding Motifs ◽  
Viral Propagation ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
Lytic Dna Replication

ABSTRACT Lytic replication of Kaposi's sarcoma-associated herpesvirus (KSHV) is essential for viral propagation and pathogenicity. In Kaposi's sarcoma lesions, constant lytic replication plays a role in sustaining the population of latently infected cells that otherwise are quickly lost by segregation of latent viral episomes as spindle cells divide. Lytic DNA replication initiates from an origin (ori-Lyt) and requires trans-acting elements. Two functional ori-Lyts have been identified in the KSHV genome. Some cis-acting and trans-acting elements for ori-Lyt-dependent DNA replication have been found. Among these, K8 binding sites, a cluster of C/EBP binding motifs, and a replication and transcription activator (RTA) responsive element (RRE) are crucial cis-acting elements. Binding of K8 and RTA proteins to these motifs in ori-Lyt DNA was demonstrated to be absolutely essential for DNA replication. In the present study, functional roles of RTA in ori-Lyt-dependent DNA replication have been investigated. Two distinct functions of RTA were revealed. First, RTA activates an ori-Lyt promoter and initiates transcription across GC-rich tandem repeats. This RTA-mediated transcription is indispensable for DNA replication. Second, RTA is a component of the replication compartment, where RTA interacts with prereplication complexes composed of at least six core machinery proteins and K8. The prereplication complexes are recruited to ori-Lyt DNA through RTA, which interacts with the RRE, as well as K8, which binds to a cluster of C/EBP binding motifs with the aid of C/EBP α. The revelation of these two functions of RTA, together with its role in initiation of a transcriptional cascade that leads to transcription of all viral lytic genes, shows that RTA is a critical initiator and regulator of KSHV lytic DNA replication and viral propagation.

scholarly journals Lytic Replication-Defective Kaposi's Sarcoma-Associated Herpesvirus: Potential Role in Infection and Malignant Transformation

2004 ◽  
Vol 78 (20) ◽  
pp. 11108-11120 ◽  
Author(s):  
Jian-Hong Deng ◽  
Yan-Jin Zhang ◽  
Xin-Ping Wang ◽  
Shou-Jiang Gao
Keyword(s):  
Malignant Transformation ◽  
Cell Lines ◽  
Kaposi's Sarcoma ◽  
Potential Role ◽  
Primary Effusion Lymphoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Screening Methods ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Defective viruses often have pivotal roles in virus-induced diseases. Although Kaposi's sarcoma-associated herpesvirus (KSHV) is etiologically associated with Kaposi's sarcoma (KS) and primary effusion lymphoma (PEL), defective KSHV has not been reported. Using differential genetic screening methods, we show that defective KSHV is present in KS tumors and PEL cell lines. To investigate the role of defective viruses in KSHV-induced pathogenesis, we isolated and characterized a lytic replication-defective KSHV, KV-1, containing an 82-kb genomic deletion of solely lytic genes. Cells harboring KV-1 escaped G0/G1 apoptosis induced by spontaneous lytic replication occurred in cells infected with regular KSHV but maintained efficient latent replication. Consequently, KV-1-infected cells had phenotypes of enhanced cell proliferation and transformation potentials. Importantly, KV-1 was packaged as infectious virions by using regular KSHV as helpers, and KV-1-like variants were detected in cultures of two of five KSHV cell lines and 1 of 18 KS tumors. These results point to a potential role for defective viruses in the regulation of KSHV infection and malignant transformation.

scholarly journals Activation of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Lytic Replication by Human Cytomegalovirus

2001 ◽  
Vol 75 (3) ◽  
pp. 1378-1386 ◽  
Author(s):  
Jeffrey Vieira ◽  
Patricia O'Hearn ◽  
Louise Kimball ◽  
Bala Chandran ◽  
Lawrence Corey
Keyword(s):  
Human Cytomegalovirus ◽  
Kaposi's Sarcoma ◽  
Human Fibroblasts ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Nuclear Antigen ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT The majority of Kaposi's sarcoma-associated herpesvirus (KSHV)-infected cells identified in vivo contain latent KSHV, with lytic replication in only a few percent of cells, as is the case for the cells of Kaposi's sarcoma (KS) lesions. Factors that influence KSHV latent or lytic replication are not well defined. Because persons with KS are often immunosuppressed and susceptible to many infectious agents, including human cytomegalovirus (HCMV), we have investigated the potential for HCMV to influence the replication of KSHV. Important to this work was the construction of a recombinant KSHV, rKSHV.152, expressing the green fluorescent protein (GFP) andneo (conferring resistance to G418). The expression of GFP was a marker of KSHV infection in cells of both epithelial and endothelial origin. The rKSHV.152 virus was used to establish cells, including human fibroblasts (HF), containing only latent KSHV, as demonstrated by latency-associated nuclear antigen expression and Gardella gel analysis. HCMV infection of KSHV latently infected HF activated KSHV lytic replication with the production of infectious KSHV. Dual-color immunofluorescence detected both the KSHV lytic open reading frame 59 protein and the HCMV glycoprotein B in coinfected cells, and UV-inactivated HCMV did not activate the production of infectious KSHV-GFP. In addition, HCMV coinfection increased the production of KSHV from endothelial cells and activated lytic cycle gene expression in keratinocytes. These data demonstrate that HCMV can activate KSHV lytic replication and suggest that HCMV could influence KSHV pathogenesis.

scholarly journals Identification and Characterization of the Orf49 Protein of Kaposi's Sarcoma-Associated Herpesvirus

2006 ◽  
Vol 80 (6) ◽  
pp. 3062-3070 ◽  
Author(s):  
Carlos M. González ◽  
Emily L. Wong ◽  
Brian S. Bowser ◽  
Gregory K. Hong ◽  
Shannon Kenney ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Transcriptional Activation ◽  
Kaposi's Sarcoma ◽  
Primary Effusion Lymphoma ◽  
Kaposi’S Sarcoma ◽  
Lytic Cycle ◽  
Transcription Activator ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Factor C ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiological agent of Kaposi's sarcoma, primary effusion lymphoma, and multicentric Castleman's disease. Kaposi's sarcoma is the most common neoplasm among human immunodeficiency virus-positive individuals. Like other herpesviruses, KSHV is able to establish a predominantly latent, life-long infection in its host. The KSHV lytic cycle can be triggered by a number of stimuli that induce the expression of the key lytic switch protein, the replication and transcription activator (RTA) encoded by Orf50. The expression of Rta is necessary and sufficient to trigger the full lytic program resulting in the ordered expression of viral proteins, release of viral progeny, and host cell death. We have characterized an unknown open reading frame, Orf49, which lies adjacent and in the opposite orientation to Orf50. Orf49 is expressed during the KSHV lytic cycle and shows early transcription kinetics. We have mapped the 5′ and 3′ ends of the unspliced Orf49 transcript, which encodes a 30-kDa protein that is localized to both the nucleus and the cytoplasm. Interestingly, we found that Orf49 was able to cooperate with Rta to activate several KSHV lytic promoters containing AP-1 sites. The Orf49-encoded protein was also able to induce transcriptional activation through c-Jun but not the ATF1, ATF2, or CREB transcription factor. We found that Orf49 could induce phosphorylation and activation of the transcription factor c-Jun, the Jun N-terminal kinase (JNK), and p38. Our data suggest that Orf49 functions to activate the JNK and p38 pathways during the KSHV lytic cycle.

scholarly journals Suberoyl bis-hydroxamic acid reactivates Kaposi’s sarcoma-associated herpesvirus through histone acetylation and induces apoptosis in lymphoma cells

2020 ◽  
Author(s):  
Shun Iida ◽  
Sohtaro Mine ◽  
Keiji Ueda ◽  
Tadaki Suzuki ◽  
Hideki Hasegawa ◽  
...  
Keyword(s):  
Histone Acetylation ◽  
Hydroxamic Acid ◽  
Kaposi's Sarcoma ◽  
Primary Effusion Lymphoma ◽  
Kaposi’S Sarcoma ◽  
Mitochondrial Pathway ◽  
Dependent Manner ◽  
Transcription Activator ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an etiologic agent of Kaposi’s sarcoma as well as primary effusion lymphoma (PEL), an aggressive B-cell neoplasm which mostly arises in immunocompromised individuals. Lytic replication of KSHV is also associated with a subset of multicentric Castleman diseases. At present, there is no specific treatment available for PEL and its prognosis is poor. In this study, we found that the histone deacetylase inhibitor suberoyl bis-hydroxamic acid (SBHA) induced KSHV reactivation in PEL cells in a dose-dependent manner. Next-generation sequencing analysis showed that more than 40% of all transcripts expressed in SBHA-treated PEL cells originated from the KSHV genome compared with less than 1% in untreated cells. Chromatin immunoprecipitation assays demonstrated that SBHA induced histone acetylation targeting the promoter region of the KSHV replication and transcription activator gene. However, there was no significant change in methylation status of the promoter region of this gene. In addition to its effect of KSHV reactivation, this study revealed that SBHA induces apoptosis in PEL cells in a dose-dependent manner, inducing acetylation and phosphorylation of p53, cleavage of caspases, and expression of pro-apoptotic factors such as Bim and Bax. These findings suggest that SBHA reactivates KSHV from latency and induces apoptosis through the mitochondrial pathway in PEL cells. Therefore, SBHA can be considered a new tool for induction of KSHV reactivation, and could provide a novel therapeutic strategy against PEL. IMPORTANCE Kaposi’s sarcoma and primary effusion lymphoma cells are latently infected with Kaposi’s sarcoma-associated herpesvirus (KSHV), whereas KSHV replication is frequently observed in multicentric Castleman disease. Although KSHV replication can be induced by some chemical reagents (e.g. 12-O-tetradecanoylphorbol-13-acetate), the mechanism of KSHV replication is not fully understood. We found that the histone deacetylase inhibitor suberoyl bis-hydroxamic acid (SBHA) induced KSHV reactivation with high efficiency, through histone acetylation in the promoter of the replication and transcription activator gene, compared with 12-O-tetradecanoylphorbol-13-acetate. SBHA also induced apoptosis through the mitochondrial pathway in KSHV-infected cells, with a lower EC50 than measured for viral reactivation. SBHA could be used in a highly efficient replication system for KSHV in vitro, and as a tool to reveal the mechanism of replication and pathogenesis of KSHV. The ability of SBHA to induce apoptosis at lower levels than needed to stimulate KSHV reactivation, indicates its therapeutic potential.

scholarly journals The Kaposi's Sarcoma-Associated Herpesvirus G Protein-Coupled Receptor Has Broad Signaling Effects in Primary Effusion Lymphoma Cells

2003 ◽  
Vol 77 (1) ◽  
pp. 57-67 ◽  
Author(s):  
Mark Cannon ◽  
Nicola J. Philpott ◽  
Ethel Cesarman
Keyword(s):  
G Protein ◽  
Kaposi's Sarcoma ◽  
Primary Effusion Lymphoma ◽  
Cell Cycle Control ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
G Protein Coupled Receptor ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
G Protein Coupled

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV-8]) is a gamma-2-herpesvirus responsible for Kaposi's sarcoma as well as primary effusion lymphoma (PEL). KSHV is a lymphotropic virus that has pirated many mammalian genes involved in inflammation, cell cycle control, and angiogenesis. Among these is the early lytic viral G protein-coupled receptor (vGPCR), a homologue of the human interleukin-8 (IL-8) receptor. When expressed, vGPCR is constitutively active and can signal via mitogen- and stress-activated kinases. In certain models it activates the transcriptional potential of NF-κB and activator protein 1 (AP-1) and induces vascular endothelial growth factor (VEGF) production. Despite its importance to the pathogenesis of all KSHV-mediated disease, little is known about vGPCR activity in hematopoietic cells. To study the signaling potential and downstream effects of vGPCR in such cells, we have developed PEL cell lines that express vGPCR under the control of an inducible promoter. The sequences required for tetracycline-mediated induction were cloned into a plasmid containing adeno-associated virus type 2 elements to enhance integration efficiency. This novel plasmid permitted studies of vGPCR activity in naturally infected KSHV-positive lymphocytes. We show that vGPCR activates ERK-2 and p38 in PEL cells. In addition, it increases the transcription of reporter genes under the control of AP-1, NF-κB, CREB, and NFAT, a Ca2+-dependent transcription factor important to KSHV lytic gene expression. vGPCR also increases the transcription of KSHV open reading frames 50 and 57, thereby displaying broad potential to affect viral transcription patterns. Finally, vGPCR signaling results in increased PEL cell elaboration of KSHV vIL-6 and VEGF, two growth factors involved in KSHV-mediated disease pathogenesis.

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