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

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.

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

Journal of Virology ◽

10.1128/jvi.79.5.3217-3222.2005 ◽

2005 ◽

Vol 79 (5) ◽

pp. 3217-3222 ◽

Cited By ~ 15

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.

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Kaposi's Sarcoma-Associated Herpesvirus Lytic Switch Protein Stimulates DNA Binding of RBP-Jk/CSL To Activate the Notch Pathway

Journal of Virology ◽

10.1128/jvi.00746-06 ◽

2006 ◽

Vol 80 (19) ◽

pp. 9697-9709 ◽

Cited By ~ 44

Author(s):

Kyla Driscoll Carroll ◽

Wei Bu ◽

Diana Palmeri ◽

Sophia Spadavecchia ◽

Stephen J. Lynch ◽

...

Keyword(s):

Dna Binding ◽

Transcriptional Activation ◽

Kaposi's Sarcoma ◽

Notch Pathway ◽

Kaposi’S Sarcoma ◽

Lytic Cycle ◽

Viral Reactivation ◽

Transactivation Domain ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV) lytic switch protein, Rta, is a ligand-independent inducer of the Notch signal transduction pathway, and KSHV cannot reactivate from latency in cells null for the Notch target protein RBP-Jk. Here we show that Rta promotes DNA binding of RBP-Jk, a mechanism that is fundamentally different from that established for the RBP-Jk-activating proteins, Notch intracellular domain (NICD) and Epstein-Barr virus EBNA2. Although constitutively active RBP-Jk and NICD do not transactivate KSHV promoters independently, cotransfection of an Rta mutant lacking its transactivation domain robustly restores transcriptional activation. Cooperation requires intact DNA binding sites for Rta and RBP-Jk and trimeric complex formation between the three molecules in vitro. In infected cells, RBP-Jk is virtually undetectable on a series of viral and cellular promoters during KSHV latency but is significantly enriched following Rta expression during viral reactivation. Accordingly, Rta, but not EBNA2 and NICD, reactivates the complete viral lytic cycle.

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Involvement of SSRP1 in Latent Replication of Kaposi's Sarcoma-Associated Herpesvirus

Journal of Virology ◽

10.1128/jvi.00907-09 ◽

2009 ◽

Vol 83 (21) ◽

pp. 11051-11063 ◽

Cited By ~ 23

Author(s):

Jianhong Hu ◽

Eugene Liu ◽

Rolf Renne

Keyword(s):

Dna Replication ◽

Kaposi's Sarcoma ◽

Latent Infection ◽

Mutational Analysis ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Dominant Negative ◽

Nuclear Antigen ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (also named human herpesvirus 8) is a γ-herpesvirus that undergoes both lytic and latent infection. During latent infection, two viral elements are required: latency-associated nuclear antigen (LANA), which functions as an origin binding protein, and the latent origin, which resides within the terminal repeats (TRs) of the viral genome. Previously, we identified two cis-elements within the TRs which are required for latent DNA replication: two LANA binding sites (LBS1 and LBS2 [LBS1/2]) and a GC-rich replication element (RE) upstream of LBS1/2. To further characterize the RE, we constructed a 71-bp minimal replicon (MR) and performed a detailed mutational analysis. Our data indicate that the first 8 nucleotides within the RE are critical for replication. Moreover, both the position and the distance between the RE and LBS1/2 can affect origin replication activity, suggesting that the RE may function as a loading pad for cellular proteins involved in replication. Using biotinylated DNA fragments of wild-type or mutant MRs as probes, we identified 30 proteins that preferentially bind to the origin. Among these proteins, structure-specific recognition protein 1 (SSRP1), a subunit of the FACT complex, and telomeric repeat binding factor 2 (TRF2) formed complexes with LANA at the MR region. Furthermore, the small interfering RNA-based knockdown of SSRP1, but not the dominant-negative-based knockdown of TRF2, significantly decreased the efficiency of LANA-dependent DNA replication. These results indicate that SSRP1 is a novel cellular protein involved in LANA-dependent DNA replication.

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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

Journal of Virology ◽

10.1128/jvi.76.23.12044-12054.2002 ◽

2002 ◽

Vol 76 (23) ◽

pp. 12044-12054 ◽

Cited By ~ 43

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.

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Identification of a Cellular Protein That Interacts and Synergizes with the RTA (ORF50) Protein of Kaposi's Sarcoma-Associated Herpesvirus in Transcriptional Activation

Journal of Virology ◽

10.1128/jvi.75.24.11961-11973.2001 ◽

2001 ◽

Vol 75 (24) ◽

pp. 11961-11973 ◽

Cited By ~ 65

Author(s):

Shizhen Wang ◽

Shuhong Liu ◽

Ming-Hoi Wu ◽

Yunqi Geng ◽

Charles Wood

Keyword(s):

Zinc Finger ◽

Transcriptional Activation ◽

Kaposi's Sarcoma ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Cellular Protein ◽

Viral Gene ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Lytic reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV), or human herpesvirus 8, from latency requires transcriptional transactivation by the viral protein RTA encoded by the ORF50 gene. Very little is known about how RTA functions and the cellular factors that may be involved in its transactivation function. Using the yeast two-hybrid system, we have identified a human cellular protein that can interact with KSHV RTA. The cellular protein, referred to as the human hypothetical protein MGC2663 by GenBank, is encoded by human chromosome 19. This protein is 554 amino acids (aa) in size and displays sequence similarity with members of the Krueppel-associated box–zinc finger proteins (KRAB-ZFPs). MGC2663 expression could be detected in all primate cell lines tested, and its expression level was neither stimulated nor inhibited by RTA. MGC2663 specifically synergizes with RTA to activate viral transcription, and overexpression of MGC2663 in the presence of RTA further enhances RTA transactivation of several viral promoters that were identified as targets for RTA. Coimmunoprecipitation and pull-down assays further demonstrated that MGC2663 interacts with RTA both in vivo and in vitro, and the N-terminal 273 aa of KSHV RTA and the potential zinc finger domain of MGC2663 are required for their interaction. Our results indicate that this novel human cellular protein, MGC2663, named K-RBP (KSHV RTA binding protein) due to its RTA binding feature, specifically interacts with the KSHV RTA protein and functions as a cellular RTA cofactor to activate viral gene expression. Though its normal cellular function needs to be further studied, K-RBP may play a significant role in mediating RTA transactivation in vivo.

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Kaposi's Sarcoma-Associated Herpesvirus-Encoded Latency-Associated Nuclear Antigen Inhibits Lytic Replication by Targeting Rta: a Potential Mechanism for Virus-Mediated Control of Latency

Journal of Virology ◽

10.1128/jvi.78.12.6585-6594.2004 ◽

2004 ◽

Vol 78 (12) ◽

pp. 6585-6594 ◽

Cited By ~ 146

Author(s):

Ke Lan ◽

Daniel A. Kuppers ◽

Subhash C. Verma ◽

Erle S. Robertson

Keyword(s):

Kaposi's Sarcoma ◽

Critical Role ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Lytic Replication ◽

Nuclear Antigen ◽

Viral Progeny ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Like other herpesviruses, Kaposi's sarcoma-associated herpesvirus (KSHV, also designated human herpesvirus 8) can establish a latent infection in the infected host. During latency a small number of genes are expressed. One of those genes encodes latency-associated nuclear antigen (LANA), which is constitutively expressed in cells during latent as well as lytic infection. LANA has previously been shown to be important for the establishment of latent episome maintenance through tethering of the viral genome to the host chromosomes. Under specific conditions, KSHV can undergo lytic replication, with the production of viral progeny. The immediate-early Rta, encoded by open reading frame 50 of KSHV, has been shown to play a critical role in switching from viral latent replication to lytic replication. Overexpression of Rta from a heterologous promoter is sufficient for driving KSHV lytic replication and the production of viral progeny. In the present study, we show that LANA down-modulates Rta's promoter activity in transient reporter assays, thus repressing Rta-mediated transactivation. This results in a decrease in the production of KSHV progeny virions. We also found that LANA interacts physically with Rta both in vivo and in vitro. Taken together, our results demonstrate that LANA can inhibit viral lytic replication by inhibiting expression as well as antagonizing the function of Rta. This suggests that LANA may play a critical role in maintaining latency by controlling the switch between viral latency and lytic replication.

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Kaposi's Sarcoma-Associated Herpesvirus Latency-Associated Nuclear Antigen 1 Mimics Epstein-Barr Virus EBNA1 Immune Evasion through Central Repeat Domain Effects on Protein Processing

Journal of Virology ◽

10.1128/jvi.00411-07 ◽

2007 ◽

Vol 81 (15) ◽

pp. 8225-8235 ◽

Cited By ~ 70

Author(s):

Hyun Jin Kwun ◽

Suzane Ramos da Silva ◽

Ishita M. Shah ◽

Neil Blake ◽

Patrick S. Moore ◽

...

Keyword(s):

Antigen Processing ◽

Kaposi's Sarcoma ◽

Epstein Barr Virus ◽

Human Herpesvirus ◽

Kaposi’S Sarcoma ◽

Nuclear Antigen ◽

Barr Virus ◽

Kaposi's Sarcoma Associated Herpesvirus ◽

Epstein Barr ◽

Kaposi’S Sarcoma Associated Herpesvirus

ABSTRACT Kaposi's sarcoma-associated herpesvirus (KSHV/human herpesvirus 8 [HHV8]) and Epstein-Barr virus (EBV/HHV4) are distantly related gammaherpesviruses causing tumors in humans. KSHV latency-associated nuclear antigen 1 (LANA1) is functionally similar to the EBV nuclear antigen-1 (EBNA1) protein expressed during viral latency, although they have no amino acid similarities. EBNA1 escapes cytotoxic lymphocyte (CTL) antigen processing by inhibiting its own proteosomal degradation and retarding its own synthesis to reduce defective ribosomal product processing. We show here that the LANA1 QED-rich central repeat (CR) region, particularly the CR2CR3 subdomain, also retards LANA1 synthesis and markedly enhances LANA1 stability in vitro and in vivo. LANA1 isoforms have half-lives greater than 24 h, and fusion of the LANA1 CR2CR3 domain to a destabilized heterologous protein markedly decreases protein turnover. Unlike EBNA1, the LANA1 CR2CR3 subdomain retards translation regardless of whether it is fused to the 5′ or 3′ end of a heterologous gene construct. Manipulation of sequence order, orientation, and composition of the CR2 and CR3 subdomains suggests that specific peptide sequences rather than RNA structures are responsible for synthesis retardation. Although mechanistic differences exist between LANA1 and EBNA1, the primary structures of both proteins have evolved to minimize provoking CTL immune responses. Simple strategies to eliminate these viral inhibitory regions may markedly improve vaccine effectiveness by maximizing CTL responses.

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

Journal of Virology ◽

10.1128/jvi.75.3.1378-1386.2001 ◽

2001 ◽

Vol 75 (3) ◽

pp. 1378-1386 ◽

Cited By ~ 134

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.

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