scholarly journals Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 RTA Reactivates Murine Gammaherpesvirus 68 from Latency

2005 ◽  
Vol 79 (5) ◽  
pp. 3217-3222 ◽  
Author(s):  
Tammy M. Rickabaugh ◽  
Helen J. Brown ◽  
Ting-Ting Wu ◽  
Moon Jung Song ◽  
Seungmin Hwang ◽  
...  
Keyword(s):  
Kaposi's Sarcoma ◽  
Critical Role ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Cycle ◽  
Murine Gammaherpesvirus ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Murine gammaherpesvirus 68 (MHV-68), Kaposi's sarcoma-associated herpesvirus (HHV-8), and Epstein-Barr virus (EBV) are all members of the gammaherpesvirus family, characterized by their ability to establish latency in lymphocytes. The RTA protein, conserved in all gammaherpesviruses, is known to play a critical role in reactivation from latency. Here we report that HHV-8 RTA, not EBV RTA, was able to induce MHV-68 lytic viral proteins and DNA replication and processing and produce viable MHV-68 virions from latently infected cells at levels similar to those for MHV-68 RTA. HHV-8 RTA was also able to activate two MHV-68 lytic promoters, whereas EBV RTA was not. In order to define the domains of RTA responsible for their functional differences in viral promoter activation and initiation of the MHV-68 lytic cycle, chimeric RTA proteins were constructed by exchanging the N-terminal and C-terminal domains of the RTA proteins. Our data suggest that the species specificity of MHV-68 RTA resides in the N-terminal DNA binding domain.

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 DNA Binding by Kaposi's Sarcoma-Associated Herpesvirus Lytic Switch Protein Is Necessary for Transcriptional Activation of Two Viral Delayed Early Promoters

2001 ◽  
Vol 75 (15) ◽  
pp. 6786-6799 ◽  
Author(s):  
David M. Lukac ◽  
Lilit Garibyan ◽  
Jessica R. Kirshner ◽  
Diana Palmeri ◽  
Don Ganem
Keyword(s):  
Dna Binding ◽  
Transcriptional Activation ◽  
Kaposi's Sarcoma ◽  
Mutational Analysis ◽  
Critical Role ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Amino Terminal ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus-8) establishes latent and lytic infections in both lymphoid and endothelial cells and has been associated with diseases of both cell types. The KSHV open reading frame 50 (ORF50) protein is a transcriptional activator that plays a central role in the reactivation of lytic viral replication from latency. Here we identify and characterize a DNA binding site for the ORF50 protein that is shared by the promoters of two delayed early genes (ORF57 and K-bZIP). Transfer of this element to heterologous promoters confers on them high-level responsiveness to ORF50, indicating that the element is both necessary and sufficient for activation. The element consists of a conserved 12-bp palindromic sequence and less conserved sequences immediately 3′ to it. Mutational analysis reveals that sequences within the palindrome are critical for binding and activation by ORF50, but the presence of a palindrome itself is not absolutely required. The 3′ flanking sequences also play a critical role in DNA binding and transactivation. The strong concordance of DNA binding in vitro with transcriptional activation in vivo strongly implies that sequence-specific DNA binding is necessary for ORF50-mediated activation through this element. Expression of truncated versions of the ORF50 protein reveals that DNA binding is mediated by the amino-terminal 272 amino acids of the polypeptide.

scholarly journals A Kaposi's Sarcoma-Associated Herpesvirus/Human Herpesvirus 8 ORF50 Deletion Mutant Is Defective for Reactivation of Latent Virus and DNA Replication

2005 ◽  
Vol 79 (6) ◽  
pp. 3479-3487 ◽  
Author(s):  
Yiyang Xu ◽  
David P. AuCoin ◽  
Alicia Rodriguez Huete ◽  
Sylvia A. Cei ◽  
Lisa J. Hanson ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Synthesis ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Latent Virus ◽  
Lytic Cycle ◽  
Viral Genes ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (also called human herpesvirus type 8 [HHV8]) latently infects a number of cell types. Reactivation of latent virus can occur by treatment with the phorbol ester tetradecanoyl phorbol acetate (TPA) or with the transfection of plasmids expressing the lytic switch activator protein K-Rta, the gene product of ORF50. K-Rta expression is sufficient for the activation of the entire lytic cycle and the transactivation of viral genes necessary for DNA replication. In addition, recent evidence has suggested that K-Rta may participate directly in the initiation of lytic DNA synthesis. We have now generated a recombinant HHV8 bacterial artificial chromosome (BAC) with a large deletion within the ORF50 locus. This BAC, BAC36Δ50, failed to produce infectious virus upon treatment with TPA and was defective for DNA synthesis. Expression of K-Rta in trans in BAC36Δ50-containing cells was able to abolish both defects. Real-time PCR revealed that K-bZIP, ORF40/41, and K8.1 were not expressed when BAC36Δ50-containing cells were induced with TPA. However, the mRNA levels of ORF57 were over fivefold higher in TPA-treated BAC36Δ50-containing cells than those observed in similarly treated wild-type BAC-containing cells. In addition, immunohistochemical analysis showed that while the latency-associated nuclear antigen (LANA) was expressed in the mutant BAC-containing cells, ORF59 and K8.1 expression was not detected in TPA-induced BAC36Δ50-containing cells. These results showed that K-Rta is essential for lytic viral reactivation and transactivation of viral genes contributing to DNA replication.

scholarly journals Overexpression of the Kaposi's Sarcoma-Associated Herpesvirus Transactivator K-Rta Can Complement a K-bZIP Deletion BACmid and Yields an Enhanced Growth Phenotype

2007 ◽  
Vol 81 (24) ◽  
pp. 13519-13532 ◽  
Author(s):  
Taeko Kato-Noah ◽  
Yiyang Xu ◽  
Cyprian C. Rossetto ◽  
Kelly Colletti ◽  
Iva Papousková ◽  
...  
Keyword(s):  
Subcellular Localization ◽  
Kaposi's Sarcoma ◽  
Gene Expression Pattern ◽  
Human Herpesvirus ◽  
Virus Production ◽  
Kaposi’S Sarcoma ◽  
Lytic Cycle ◽  
Growth Phenotype ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus/human herpesvirus 8 (HHV8) ORF50 encodes a transactivator, K-Rta, which functions as the switch from latent to lytic virus replication. K-bZIP interacts with K-Rta and can repress its transactivation activity for some viral promoters. Both K-Rta and K-bZIP are required for origin-dependent DNA replication. To determine the role of K-bZIP in the context of the viral genome, we generated a recombinant HHV8 bacterial artificial chromosome (BAC) with a deletion in the K-bZIP open reading frame. This BACmid, BAC36ΔK8, displayed an enhanced growth phenotype with respect to virus production and accumulation of virus-encoded mRNAs measured by real-time PCR when K-Rta was used to induce the virus lytic cycle. Conversely, induction of the virus lytic cycle using tetradecanoyl phorbol acetate/n-butyrate resulted in no virus production and an aberrant gene expression pattern from BAC36ΔK8-containing cells compared to wild-type (wt) BAC. This null virus phenotype was efficiently complemented by the expression of K-bZIP in trans, restoring virus production to wt BAC levels. Immunofluorescence staining revealed that subcellular localization of K-Rta was unchanged; however, a disruption of LANA subcellular localization was observed in cells harboring BAC36ΔK8, suggesting that K-bZIP influences LANA localization. Coimmunoprecipitation experiments confirmed that K-bZIP interacts with LANA in BCBL-1 cells and in cotransfection assays. Lastly, the chromatin immunoprecipitation assay revealed that, in an environment where K-Rta is overexpressed and in the absence of K-bZIP, K-Rta binds to CAAT enhancer binding protein α sites within oriLyt, suggesting that it is K-Rta that supplies an essential replication function and that K-bZIP may serve to augment or facilitate the interaction of K-Rta with oriLyt.

scholarly journals Differential Activation of Murine Herpesvirus 68- and Kaposi's Sarcoma-Associated Herpesvirus-Encoded ORF74 G Protein-Coupled Receptors by Human and Murine Chemokines

2004 ◽  
Vol 78 (7) ◽  
pp. 3343-3351 ◽  
Author(s):  
Dennis Verzijl ◽  
Carlos P. Fitzsimons ◽  
Marie van Dijk ◽  
James P. Stewart ◽  
Henk Timmerman ◽  
...  
Keyword(s):  
G Protein ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
G Protein Coupled Receptors ◽  
Mitogen Activated Protein Kinase ◽  
Murine Gammaherpesvirus ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus ◽  
G Protein Coupled

ABSTRACT Infection of mice with murine gammaherpesvirus 68 (MHV-68) is a well-characterized small animal model for the study of gammaherpesvirus infection. MHV-68 belongs to the same herpesvirus family as herpesvirus saimiri (HVS) of New World squirrel monkeys and human herpesvirus 8 (HHV-8) (also referred to as Kaposi's sarcoma-associated herpesvirus [KSHV]). The open reading frame ORF74 of HVS, KSHV, and MHV-68 encodes a protein with homology to G protein-coupled receptors and chemokine receptors in particular. ORF74 of KSHV (human ORF74 [hORF74]) is highly constitutively active and has been implicated in the pathogenesis of Kaposi's sarcoma. MHV-68-encoded ORF74 (mORF74) is oncogenic and has been implicated in viral replication and reactivation from latency. Here, we show that mORF74 is a functional chemokine receptor. Chemokines with an N-terminal glutamic acid-leucine-arginine (ELR) motif (e.g., KC and macrophage inflammatory protein 2) act as agonists on mORF74, activating phospholipase C, NF-κB, p44/p42 mitogen-activated protein kinase, and Akt signaling pathways and inhibiting formation of cyclic AMP. Using 125I-labeled CXCL1/growth-related oncogene α as a tracer, we show that murine CXCL10/gamma interferon-inducible protein 10 binds mORF74, and functional assays show that it behaves as an antagonist for this virally encoded G protein-coupled receptor. Profound differences in the upstream activation of signal transduction pathways between mORF74 and hORF74 were found. Moreover, in contrast to hORF74, no constitutive activity of mORF74 could be detected.

scholarly journals Molecular Genetics of Kaposi's Sarcoma-Associated Herpesvirus (Human Herpesvirus 8) Epidemiology and Pathogenesis

2003 ◽  
Vol 67 (2) ◽  
pp. 175-212 ◽  
Author(s):  
Lyubomir A. Dourmishev ◽  
Assen L. Dourmishev ◽  
Diana Palmeri ◽  
Robert A. Schwartz ◽  
David M. Lukac
Keyword(s):  
Genomic Dna ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Cycle ◽  
Human Herpesvirus 8 ◽  
Pathogenic Potential ◽  
Herpesvirus 8 ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

SUMMARY Kaposi's sarcoma had been recognized as unique human cancer for a century before it manifested as an AIDS-defining illness with a suspected infectious etiology. The discovery of Kaposi's sarcoma-associated herpesvirus (KSHV), also known as human herpesvirus-8, in 1994 by using representational difference analysis, a subtractive method previously employed for cloning differences in human genomic DNA, was a fitting harbinger for the powerful bioinformatic approaches since employed to understand its pathogenesis in KS. Indeed, the discovery of KSHV was rapidly followed by publication of its complete sequence, which revealed that the virus had coopted a wide armamentarium of human genes; in the short time since then, the functions of many of these viral gene variants in cell growth control, signaling apoptosis, angiogenesis, and immunomodulation have been characterized. This critical literature review explores the pathogenic potential of these genes within the framework of current knowledge of the basic herpesvirology of KSHV, including the relationships between viral genotypic variation and the four clinicoepidemiologic forms of Kaposi's sarcoma, current viral detection methods and their utility, primary infection by KSHV, tissue culture and animal models of latent- and lytic-cycle gene expression and pathogenesis, and viral reactivation from latency. Recent advances in models of de novo endothelial infection, microarray analyses of the host response to infection, receptor identification, and cloning of full-length, infectious KSHV genomic DNA promise to reveal key molecular mechanisms of the candidate pathogeneic genes when expressed in the context of viral infection.

scholarly journals Lack of Heparan Sulfate Expression in B-Cell Lines: Implications for Kaposi's Sarcoma-Associated Herpesvirus and Murine Gammaherpesvirus 68 Infections

2008 ◽  
Vol 82 (24) ◽  
pp. 12591-12597 ◽  
Author(s):  
Nadine Jarousse ◽  
Bala Chandran ◽  
Laurent Coscoy
Keyword(s):  
Cell Surface ◽  
Heparan Sulfate ◽  
B Cell ◽  
Cell Lines ◽  
Kaposi's Sarcoma ◽  
Kaposi’S Sarcoma ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) and its murine homolog, murine gammaherpesvirus 68 (MHV68), are lymphotropic viruses that establish latent infection in their host. Surprisingly, while B cells are the main viral reservoir in vivo, B-cell lines are poorly permissive to infection by either MHV68 or KSHV. Here, we report that most B-cell lines express very little to no cell surface heparan sulfate (HS), a glycosaminoglycan that is essential for infection by these viruses. We found that Ext1, a key enzyme in the biosynthesis of HS, was expressed at a low level in these cells. Transfection of B-cell lines with Ext1 restored high HS expression at the cell surface. Overexpression of Ext1 in murine A20 and M12 B-cell lines increased MHV68 surface binding and enhanced the efficiency of infection. Finally, although it was not sufficient to allow efficient infection, the expression of HS on BJAB cells promoted KSHV binding at the cell surface. Thus, our results indicate that MHV68 and KSHV cycles are blocked in B-cell lines at the binding step due to a lack of surface HS.

scholarly journals Identification of the Physiological Gene Targets of the Essential Lytic Replicative Kaposi's Sarcoma-Associated Herpesvirus ORF57 Protein

2014 ◽  
Vol 89 (3) ◽  
pp. 1688-1702 ◽  
Author(s):  
Dinesh Verma ◽  
Da-Jiang Li ◽  
Brian Krueger ◽  
Rolf Renne ◽  
Sankar Swaminathan
Keyword(s):  
Nuclear Export ◽  
Kaposi's Sarcoma ◽  
Human Herpesvirus ◽  
Kaposi’S Sarcoma ◽  
Lytic Replication ◽  
Lytic Cycle ◽  
Wild Type ◽  
Mrna Stabilization ◽  
Kaposi's Sarcoma Associated Herpesvirus ◽  
Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACTThe Kaposi's sarcoma-associated herpesvirus (KSHV) ORF57 gene product is essential for lytic KSHV replication and virion production. Recombinant ORF57-null mutants fail to accumulate several lytic cycle mRNAs at wild-type levels, leading to decreased production of lytic proteins necessary for efficient replication. Several mechanisms by which ORF57 may enhance expression of lytic KSHV mRNAs have been proposed, including mRNA stabilization, mRNA nuclear export, increased polyadenylation, and transcriptional activation. ORF57 activity is also gene specific, with some genes being highly dependent on ORF57, whereas others are relatively independent. Most experiments have utilized transfection models for ORF57 and have not systematically examined the gene specificity and potential mechanisms of action of ORF57 in the context of KSHV-infected cells. In this study, the KSHV genes that are most highly upregulated by ORF57 during KSHV lytic replication were identified by a combination of high-throughput deep RNA sequencing, quantitative PCR, Northern blotting, and rapid amplification of cDNA ends methods. Comparison of gene expression from a ΔORF57 KSHV recombinant, a rescued ΔORF57 KSHV recombinant, and wild-type KSHV revealed that two clusters of lytic genes are most highly dependent on ORF57 for efficient expression. Despite contiguous location in the genome and shared polyadenylation of several of the ORF57-dependent genes, ORF57 regulation was promoter and polyadenylation signal independent, suggesting that the mRNAs are stabilized by ORF57. The eight genes identified to critically require ORF57 belong to both early and late lytic temporal classes, and seven are involved in DNA replication, virion assembly, or viral infectivity, explaining the essential role of ORF57 in infectious KSHV production.IMPORTANCEKaposi's sarcoma-associated herpesvirus (KSHV) is a human herpesvirus involved in the causation of several human cancers. The KSHV ORF57 protein is required for KSHV to replicate and produce infectious virus. We have identified several KSHV genes whose expression is highly dependent on ORF57 and shown that ORF57 increases expression of these genes specifically. These genes code for proteins that are required for the virus to replicate its DNA and to infect other cells. Identifying the targets and mechanism of action of ORF57 provides further approaches to discover antiviral therapy.

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