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A comprehensive analysis of recruitment and transactivation potential of K-Rta and K-bZIP during reactivation of Kaposi's sarcoma-associated herpesvirus.

ABSTRACT: Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma. K-Rta and K-bZIP are two major viral transcription factors that control reactivation of this virus. Here we report a genome-wide analysis of transcriptional capacity by evaluation of a comprehensive library of 83 putative KSHV promoters. In reporter assays, 34 viral promoters were activated by K-Rta, whereas K-bZIP activated 21 promoters. When K-Rta and K-bZIP were combined, 3 K-Rta responsive promoters were repressed by K-bZIP. The occupancy of K-Rta and K-bZIP across KSHV promoters was analyzed by chromatin immunoprecipitation with a viral promoter-chip in BCBL-1 cells. In addition, acetylation of local histones was examined to determine accessibility of promoters during latency and reactivation. Finally, 10 promoters were selected to study the dynamics of transcription factor recruitment. This study provides a comprehensive overview of the responsiveness of KSHV promoters to K-Rta and K-bZIP, and describes key chromatin changes during viral reactivation.

SUBMITTER: Ellison TJ

PROVIDER: S-EPMC4327937 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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A comprehensive analysis of recruitment and transactivation potential of K-Rta and K-bZIP during reactivation of Kaposi's sarcoma-associated herpesvirus.

Ellison Thomas J TJ Izumiya Yoshihiro Y Izumiya Chie C Luciw Paul A PA Kung Hsing-Jien HJ

Virology 20090309 1

Kaposi's sarcoma-associated herpesvirus (KSHV) is the etiologic agent of Kaposi's sarcoma. K-Rta and K-bZIP are two major viral transcription factors that control reactivation of this virus. Here we report a genome-wide analysis of transcriptional capacity by evaluation of a comprehensive library of 83 putative KSHV promoters. In reporter assays, 34 viral promoters were activated by K-Rta, whereas K-bZIP activated 21 promoters. When K-Rta and K-bZIP were combined, 3 K-Rta responsive promoters we ...[more]

PMID: 19269659

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Project description:The replication and transcription activator (RTA) of Kaposi's sarcoma-associated herpesvirus (KSHV), K-RTA, is a lytic switch protein that moderates the reactivation process of KSHV latency. By mass spectrometric analysis of affinity purified K-RTA, we showed that Thr-513 or Thr-514 was the primary in vivo phosphorylation site. Thr-513 and Thr-514 are proximal to the nuclear localization signal ((527)KKRK(530)) and were previously hypothesized to be target sites of Ser/Thr kinase hKFC. However, substitutions of Thr with Ala at 513 and 514 had no effect on K-RTA subcellular localization or transactivation activity. By contrast, replacement of Ser with Ala at Ser-634 and Ser-636 located in a Ser/Pro-rich region of K-RTA, designated as S634A/S636A, produced a polypeptide with ∼10 kDa shorter in molecular weight and reduced transactivation in a luciferase reporter assay relative to the wild type. In contrast to prediction, the decrease in molecular weight was not due to lack of phosphorylation because the overall Ser and Thr phosphorylation state in K-RTA and S634A/S636A were similar, excluding that Ser-634 or Ser-636 motif served as docking sites for consecutive phosphorylation. Interestingly, S634A/S636A lost ∼30% immuno-reactivity to MPM2, an antibody specific to pSer/pThr-Pro motif, indicating that (634)SPSP(637) motif was in vivo phosphorylated. By in vitro kinase assay, we showed that K-RTA is a substrate of CDK9, a Pro-directed Ser/Thr kinase central to transcriptional regulation. Importantly, the capability of K-RTA in associating with endogenous CDK9 was reduced in S634A/S636A, which suggested that Ser-634 and Ser-636 may be involved in CDK9 recruitment. In agreement, S634A/S636A mutant exhibited ∼25% reduction in KSHV lytic cycle reactivation relative to that by the wild type K-RTA. Taken together, our data propose that Ser-634 and Ser-636 of K-RTA are phosphorylated by host transcriptional kinase CDK9 and such a process contributes to a full transcriptional potency of K-RTA.

Project description:Kaposi's sarcoma-associated herpesvirus (KSHV; also called human herpesvirus 8 [HHV-8]), upon being reactivated, causes serious diseases in immunocompromised individuals. Its reactivation, especially how the cellular regulating mechanisms play roles in KSHV gene expression and viral DNA replication, is not fully understood. In searching for the cellular factors that regulate KSHV gene expression, we found that several histone deacetylases (HDACs) and sirtuins (SIRTs), including HDACs 2, 7, 8, and 11 and SIRTs 4 and 6, repress KSHV ori-Lyt promoter activity. Interestingly, the nuclear protein SIRT6 presents the greatest inhibitory effect on ori-Lyt promoter activity. A more detailed investigation revealed that SIRT6 exerts repressive effects on multiple promoters of KSHV. As a consequence of inhibiting the KSHV promoters, SIRT6 not only represses viral protein production but also inhibits viral DNA replication, as investigated in a KSHV-containing cell line, SLK-iBAC-gfpK52. Depletion of the SIRT6 protein using small interfering RNA could not directly reactivate KSHV from SLK-iBAC-gfpK52 cells but made the reactivation of KSHV by use of a small amount of the reactivator (doxycycline) more effective and enhanced viral DNA replication in the KSHV infection system. We performed DNA chromatin immunoprecipitation (ChIP) assays for SIRT6 in the SLK-iBAC-gfpK52 cell line to determine whether SIRT6 interacts with the KSHV genome in order to exhibit regulatory effects. Our results suggest that SIRT6 interacts with KSHV ori-Lyt and ORF50 promoters. Furthermore, the SIRT6-KSHV DNA interaction is significantly negated by reactivation. Therefore, we identified a cellular regulator, SIRT6, that represses KSHV replication by interacting with KSHV DNA and inhibiting viral gene expression.IMPORTANCE Kaposi's sarcoma-associated herpesvirus (KSHV) is a pathogen causing cancer in the immune-deficient population. The reactivation of KSHV from latency is important for it to be carcinogenic. Our finding that SIRT6 has inhibitory effects on KSHV reactivation by interacting with the viral genome and suppressing viral gene expression is important because it might lead to a strategy of interfering with KSHV reactivation. Overexpression of SIRT6 repressed the activities of several KSHV promoters, leading to reduced gene expression and DNA replication by KSHV in a KSHV bacterial artificial chromosome-containing cell line. Depletion of SIRT6 favored reactivation of KSHV from SLK-iBACV-gfpK52 cells. More importantly, we reveal that SIRT6 interacts with KSHV DNA. Whether the interaction of SIRT6 with KSHV DNA occurs at a global level will be further studied in the future.

Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus that has been implicated in the pathogenesis of Kaposi's sarcoma and B-cell neoplasms. The genomic organization of KSHV is similar to that of Epstein-Barr virus (EBV). EBV encodes two transcriptional factors, Rta and Zta, which functionally interact to transactivate EBV genes during replication and reactivation from latency. KSHV encodes a basic leucine zipper protein (K-bZIP), a homologue of EBV Zta, and K-Rta, the homologue of EBV Rta. EBV Rta and Zta are strong transcriptional transactivators. Although there is ample evidence that K-Rta is a potent transactivator, the role of K-bZIP as a transcriptional factor is much less clear. In this study, we report that K-bZIP modulates K-Rta function. We show that K-bZIP directly interacts with K-Rta in vivo and in vitro. This association is specific, requiring the basic domain (amino acids 122 to 189) of K-bZIP and a specific region (amino acids 499 to 550) of K-Rta, and can be detected with K-bZIP and K-Rta endogenously expressed in BCBL-1 cells treated with tetradecanoyl phorbol acetate. The functional relevance of this association was revealed by the observation that K-bZIP represses the transactivation of the ORF57 promoter by K-Rta in a dose-dependent manner. K-bZIP lacking the interaction domain fails to repress K-Rta-mediated transactivation; this finding attests to the specificity of the repression. Interestingly, this repression is not observed for the promoter of polyadenylated nuclear (PAN) RNA, another target of K-Rta; thus, repression is promoter dependent. Finally, we provide evidence that the modulation of K-Rta by K-bZIP also occurs in vivo during reactivation of the viral genome in BCBL-1 cells. When K-bZIP is overexpressed in BCBL-1 cells, the level of expression of ORF57 but not PAN RNA is repressed. These data support the model that one function of K-bZIP is to modulate the activity of the transcriptional transactivator K-Rta.

Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus, which maintains the persistent infection of the host by intermittently reactivating from latently infected cells to produce viral progenies. While it is established that the replication and transcription activator (RTA) viral transcription factor is required for the induction of lytic viral genes for KSHV lytic reactivation, it is still unknown to what extent RTA alters the host transcriptome to promote KSHV lytic cycle and viral pathogenesis. To address this question, we performed a comprehensive time course transcriptome analysis during KSHV reactivation in B-cell lymphoma cells and determined RTA-binding sites on both the viral and host genomes, which resulted in the identification of the core RTA-induced host genes (core RIGs). We found that the majority of RTA-binding sites at core RIGs contained the canonical RBP-J?-binding DNA motif. Subsequently, we demonstrated the vital role of the Notch signaling transcription factor RBP-J? for RTA-driven rapid host gene induction, which is consistent with RBP-J? being essential for KSHV lytic reactivation. Importantly, many of the core RIGs encode plasma membrane proteins and key regulators of signaling pathways and cell death; however, their contribution to the lytic cycle is largely unknown. We show that the cell cycle and chromatin regulator geminin and the plasma membrane protein gamma-glutamyltransferase 6, two of the core RIGs, are required for efficient KSHV reactivation and virus production. Our results indicate that host genes that RTA rapidly and directly induces can be pivotal for driving the KSHV lytic cycle.IMPORTANCE The lytic cycle of KSHV is involved not only in the dissemination of the virus but also viral oncogenesis, in which the effect of RTA on the host transcriptome is still unclear. Using genomics approaches, we identified a core set of host genes which are rapidly and directly induced by RTA in the early phase of KSHV lytic reactivation. We found that RTA does not need viral cofactors but requires its host cofactor RBP-J? for inducing many of its core RIGs. Importantly, we show a critical role for two of the core RIGs in efficient lytic reactivation and replication, highlighting their significance in the KSHV lytic cycle. We propose that the unbiased identification of RTA-induced host genes can uncover potential therapeutic targets for inhibiting KSHV replication and viral pathogenesis.

Project description:The small ubiquitin-like modifier (SUMO) is a protein that regulates a wide variety of cellular processes by covalent attachment of SUMO moieties to a diverse array of target proteins. Sumoylation also plays an important role in the replication of many viruses. Previously, we showed that Kaposi's sarcoma-associated herpesvirus (KSHV) encodes a SUMO-ligase, K-bZIP, which catalyzes sumoylation of host and viral proteins. We report here that this virus also encodes a gene that functions as a SUMO-targeting ubiquitin-ligase (STUbL) which preferentially targets sumoylated proteins for degradation. K-Rta, the major transcriptional factor which turns on the entire lytic cycle, was recently found to have ubiquitin ligase activity toward a selected set of substrates. We show in this study that K-Rta contains multiple SIMs (SUMO interacting motif) and binds SUMOs with higher affinity toward SUMO-multimers. Like RNF4, the prototypic cellular STUbL, K-Rta degrades SUMO-2/3 and SUMO-2/3 modified proteins, including promyelocytic leukemia (PML) and K-bZIP. PML-NBs (nuclear bodies) or ND-10 are storage warehouses for sumoylated proteins, which negatively regulate herpesvirus infection, as part of the intrinsic immune response. Herpesviruses have evolved different ways to degrade or disperse PML bodies, and KSHV utilizes K-Rta to inhibit PML-NBs formation. This process depends on K-Rta's ability to bind SUMO, as a K-Rta SIM mutant does not effectively degrade PML. Mutations in the K-Rta Ring finger-like domain or SIM significantly inhibited K-Rta transactivation activity in reporter assays and in the course of viral reactivation. Finally, KSHV with a mutation in the Ring finger-like domain or SIM of K-Rta replicates poorly in culture, indicating that reducing SUMO-conjugates in host cells is important for viral replication. To our knowledge, this is the first virus which encodes both a SUMO ligase and a SUMO-targeting ubiquitin ligase that together may generate unique gene regulatory programs.

Dataset Information

A comprehensive analysis of recruitment and transactivation potential of K-Rta and K-bZIP during reactivation of Kaposi's sarcoma-associated herpesvirus.

Publications

A comprehensive analysis of recruitment and transactivation potential of K-Rta and K-bZIP during reactivation of Kaposi's sarcoma-associated herpesvirus.

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