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Expression Profiling of Cellular Genes Modulated by Kaposi’s Sarcoma Associated Herpesvirus (KSHV) RTA/ORF50 in HEK293 Cells

ABSTRACT: KSHV RTA (K-RTA) is a transcriptional activator that functions to disrupt KSHV latency and activates specific sets of viral promoters in the lytic cycle. Structure-function studies indicate that K-RTA possesses a very potent transactivation domain locating at the C-terminus. To further characterize the biological functions of K-RTA, we have established three doxycycline-inducible K-RTA 293 cell lines using RevTRE/Tet-On system (Clontech). Comparing to two control lines in which K-RTA was replaced with luciferase reporter, a total of 88 host genes were identified to be modulated by 24 h doxycycline-induced K-RTA synthesized in 293 cells. Designations for the three K-RTA inducible cell lines are KRta_92, KRta_116 and KRta_124; for the control line is RevTRE_Luc_1.

ORGANISM(S): Homo sapiens

PROVIDER: GSE21987 | GEO | 2010/05/26

SECONDARY ACCESSION(S): PRJNA127203

REPOSITORIES: GEO

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Expression Profiling of Cellular Genes Modulated by Kaposiâs Sarcoma Associated Herpesvirus (KSHV) RTA/ORF50 in HEK293 Cells

Project description:KSHV RTA (K-RTA) is a transcriptional activator that functions to disrupt KSHV latency and activates specific sets of viral promoters in the lytic cycle. Structure-function studies indicate that K-RTA possesses a very potent transactivation domain locating at the C-terminus. To further characterize the biological functions of K-RTA, we have established three doxycycline-inducible K-RTA 293 cell lines using RevTRE/Tet-On system (Clontech). Comparing to two control lines in which K-RTA was replaced with luciferase reporter, a total of 88 host genes were identified to be modulated by 24 h doxycycline-induced K-RTA synthesized in 293 cells. Designations for the three K-RTA inducible cell lines are KRta_92, KRta_116 and KRta_124; for the control line is RevTRE_Luc_1. All the four inducible cell lines were grown to log-phase before doxycycline induction. Same number of cells from the four cell lines were treated with doxycycline for 24 h . One duplicate of control RevTRE_Luc_1 was left untreated for inducer control. Total RNAs from the five samples were extracted and subjected to microarray analysis (Affymetrix HG-U133A, n=22,283). We sought to identify genes up- or down-regulated in the three doxycycline-treated K-RTA cells, but not in the treated or untreated RevTRE_Luc_1 cells.

2010-06-08 | E-GEOD-21987 | biostudies-arrayexpress

Expression profiling of host genes modulated by Epstein-Barr virus (EBV) Rta in HEK293 cells

2011-05-22 | GSE24585 | GEO

Epstein–Barr virus (EBV) Rta-mediated cell cycle arrest enables permissive replication of EBV and Kaposi’s sarcoma-associated herpesvirus in 293 cells

Project description:Epstein-Barr virus (EBV) Rta is a latent-lytic molecular switch evolutionarily conserved in all gamma-herpesviruses. In previous studies, doxycycline-inducible Rta was shown to potently produce an irreversible G1 arrest followed by cellular senescence in 293 cells. Here, we demonstrate that in this system the inducible Rta not only reactivates resident genome of EBV but also that of Kaposi’s sarcoma-associated herpesvirus (KSHV), to similar efficiency. However, Rta-induced senescence program was terminated by the robust viral lytic cycle replication that eventually caused cell death. Furthermore, prior to the abrupt expression of immediate-early protein (EBV BZLF1 or KSHV RTA), Rta simultaneously down-regulates cell cycle activators (c-Myc, CDK6, CCND2) and up-regulates senescence-related genes (p21, 14-3-3s). Since Rta is a viral immediate-early transcriptional activator, it is envisioned that during the initial stage of viral reactivation, Rta may engage to modulate the host transcriptome, to halt cell cycle progression, and to maintain an ideal environment for manufacturing infectious virions. Refer to individual Series. This SuperSeries is composed of the following subset Series: GSE24585: Expression profiling of host genes modulated by Epstein-Barr virus (EBV) Rta in HEK293 cells GSE24586: Expression profiling of host genes modulated by Epstein-Barr virus Rta in nasopharyngeal carcinoma cells

2011-03-31 | E-GEOD-24587 | biostudies-arrayexpress

Epstein–Barr virus (EBV) Rta-mediated cell cycle arrest enables permissive replication of EBV and Kaposi’s sarcoma-associated herpesvirus in 293 cells

Project description:Epstein-Barr virus (EBV) Rta is a latent-lytic molecular switch evolutionarily conserved in all gamma-herpesviruses. In previous studies, doxycycline-inducible Rta was shown to potently produce an irreversible G1 arrest followed by cellular senescence in 293 cells. Here, we demonstrate that in this system the inducible Rta not only reactivates resident genome of EBV but also that of Kaposi’s sarcoma-associated herpesvirus (KSHV), to similar efficiency. However, Rta-induced senescence program was terminated by the robust viral lytic cycle replication that eventually caused cell death. Furthermore, prior to the abrupt expression of immediate-early protein (EBV BZLF1 or KSHV RTA), Rta simultaneously down-regulates cell cycle activators (c-Myc, CDK6, CCND2) and up-regulates senescence-related genes (p21, 14-3-3s). Since Rta is a viral immediate-early transcriptional activator, it is envisioned that during the initial stage of viral reactivation, Rta may engage to modulate the host transcriptome, to halt cell cycle progression, and to maintain an ideal environment for manufacturing infectious virions. This SuperSeries is composed of the SubSeries listed below.

2011-03-31 | GSE24587 | GEO

Expression profiling of host genes modulated by Epstein-Barr virus Rta in nasopharyngeal carcinoma cells

2011-05-22 | GSE24586 | GEO

Expression profiling of host genes modulated by Epstein-Barr virus (EBV) Rta in HEK293 cells

Project description:EBV Rta is a transcriptional activator that functions to disrupt EBV latency in cells of epithelial origin. This series of experiment is to identify host genes that are moduated by the expression of doxycycline-inducible EBV Rta in HEK293 cells. Designations for the pooled EBV Rta inducible cell lines is 293TetER; pooled luciferase inducible lines is 293TetLuc (control). Both 293TetER and 293TetLuc cells were grown to log-phase before doxycycline induction. Same number of cells from the two cell lines were treated with doxycycline for 48 h. Experiments were performed in duplicates. Total RNAs from the four samples were extracted and subjected to microarray analysis (Affymetrix Human Gene 1.0 ST Array , n=33,297). Compared to doxycycline treated 293TetLuc, we sought to identify genes up- or down-regulated in by EBV Rta in doxycycline treated 293TetER cells..

2011-05-22 | E-GEOD-24585 | biostudies-arrayexpress

Expression profiling of host genes modulated by Epstein-Barr virus Rta in nasopharyngeal carcinoma cells

Project description:EBV Rta is a transcriptional activator that functions to disrupt EBV latency in cells of epithelial origin. This series of experiment is to identify host genes that are moduated by the expression of doxycycline-inducible EBV Rta in nasopharyngeal carcinoma cells. Designations for the two EBV Rta inducible cell lines are TW01TetER_cl7 (lower expression level) and TW01TetER_cl19 (higher expression level); for the control line is TW01Tet. All the three inducible cell lines were grown to log-phase before doxycycline induction. Same number of cells from the three cell lines were treated with doxycycline for 24 h. Total RNAs from the three samples were extracted and subjected to microarray analysis (Affymetrix Human Gene 1.0 ST Array , n=33,297). We sought to identify genes up- or down-regulated in the two doxycycline-treated EBV Rta cells, but not in the treated control cells.

2011-05-22 | E-GEOD-24586 | biostudies-arrayexpress

KSHV chromatin looping facilitates effective gene expression: high-resolution mapping of K-Rta binding sites in the KSHV genome [ChIP-seq]

Project description:The three-dimensional structure of chromatin via formation of genomic loops allows cellular RNA polymerase II to access distal promoters, thus it has a significant impact on the outcome of gene expression. The Kaposi’s sarcoma-associated herpesvirus (KSHV) entire lytic transcriptional program of 80 plus genes is initiated by a single viral transactivator, K-Rta. Here we examined the relationship between the KSHV genomic structure and K-Rta recruitment sites with unbiased Capture Hi-C analyses. The studies showed that KSHV forms a unique genomic structure, which coordinates a domain-specific viral gene expression program via K-Rta recruitment, and identified a number of direct physical, long-range, and dynamic genomic interactions. Chromatin immunoprecipitation-sequencing analyses identified a limited number of K-Rta recruitment sites in the KSHV genome, including the K12 and PAN RNA promoters. KSHV engineered to harbor point mutations in the K-Rta-responsive elements (RE) at these promoters significantly attenuated not only the direct downstream gene, but also distal viral gene expression in a domain-specific manner. Despite the long distance between two RE sites in the linear orientation, efficient recruitment of K-Rta to either promoter required intact K-Rta REs at both promoters. Finally, inducible genomic loops generated during KSHV reactivation occurred preferentially at K-Rta recruitment sites. Mutation of a K-Rta RE inhibited formation of inducible genomic loops, gene expression of its destination of looping genes, and KSHV replication in de novo infected human gingival epithelial cells. These results suggest that the KSHV genome is programed to form both stable and inducible chromatin hubs for effective KSHV gene expression, and partly explains why K-Rta has such a dominant effect on KSHV lytic gene transcription.

2018-01-22 | GSE99943 | GEO

Identification of KSHV RTA and K8 binding sites on viral genome

Project description:The objective of this study was to identify the binding sites of KSHV encoded imemdiate early protein, RTA and early protein, K8 on KSHV genome by chromatin immunoprecipitation assay and sequencing of the DNA bound to RTA and K8

2016-06-07 | GSE82283 | GEO

Direct transcriptional targets of the HHV 8/KSHV Rta Lytic Switch Protein

Project description:Lytic reactivation from latency is critical for the pathogenesis of KSHV. We previously demonstrated that the 691 amino acid KSHV Rta transcriptional transactivator is necessary and sufficient to reactivate the virus from latency. Viral lytic cycle genes, including those expressing additional transactivators and putative oncogenes, are induced in a cascade fashion following Rta expression. In this study, we sought to define Rta’s direct targets during reactivation by generating a conditionally nuclear variant of Rta. WT Rta protein is constitutively localized to cell nuclei, and contains two putative nuclear localization signals (NLSs). Only one NLS (NLS-2; aa 516-530) was required for nuclear localization of Rta, and relocalized eGFP exclusively to cell nuclei. Analyses of Rta NLS mutants demonstrated that proper nuclear localization of Rta was required for transactivation and stimulation of viral reactivation. Fusion of Rta_NLS-1,2 to the hormone binding domain of the murine estrogen receptor generated a variant of Rta whose nuclear localization and ability to transactivate and induce reactivation were tightly controlled post-translationally by the synthetic hormone tamoxifen. We used this strategy in KSHV-infected cells treated with protein synthesis inhibitors to identify direct transcriptional targets of Rta. Only eight KSHV genes were activated by Rta in the absence of de novo protein synthesis. These direct transcriptional targets of Rta were transactivated to different magnitudes, and included the genes nut-1/PAN, ORF57/Mta, ORF56/Primase, K2/vIL-6, ORF37/SOX, K14/vOX, K9/vIRF1, and ORF52. Our data suggest that induction of most of the KSHV lytic cycle genes requires additional protein expression post-Rta. Keywords: Comparative transcriptome analysis by oligonucleotide microarray

2008-08-31 | GSE11432 | GEO

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