VGPCR and lytic KSHV time course in TIME cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE1406 | GEO | 2004/05/18
SECONDARY ACCESSION(S): PRJNA90193
REPOSITORIES: GEO
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Project description:TIME cells either expressing KSHV-RTA or KSHV-vGPCR alone, or TIME cells infected with KSHV and lytically reactivated with RTA for 6, 12, or 20 hours. Keywords = KSHV Keywords = host shutoff Keywords = vGPCR
2010-06-09 | E-GEOD-1406 | biostudies-arrayexpress
Project description:Expression profiling of latently infected cells using a custom tiling microarray HUVEC and TIME cells were infected BCBL-1-derived KSHV. Mock infected HUVEC and TIME cells served as controls for each of these two stably infected cells, respectively. BJAB cells served as uninfected controls for the BCBL-1 cells. KSHV-infected cells are induced to enter lytic cycle with valproate or Adenovirus-RTA. Cells were harvested at indicated time points and analyzed. Three condition experiment: mock infected, latently infected cells and lytically infected. Three cell types (BJAB cells served as uninfected controls for the BCBL-1 cells).
2010-07-07 | E-GEOD-21972 | biostudies-arrayexpress
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
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. Here, we performed a comprehensive time course transcriptome analysis during KSHV reactivation in KSHV+ primary effusion B-cell lymphoma cells (PEL). For this we used a recombinant PEL cell line called TRExBCBL1-3xFLAG-RTA. The expression of the N-terminally 3xFLAG-tagged RTA was induced by adding 1 μg/ml doxycycline (Dox) to the medium. Total gene expression changes were identified in TRExBCBL1-3xFLAG-RTA cells at 0, 6, 12, and 24 hours post-induction (hpi).
2019-01-09 | GSE123898 | GEO
Project description:The Kaposi's sarcoma-associated herpesvirus (KSHV) genome consists of an approximately 140 kb unique coding region flanked by 30-40 copies of 0.8 kb terminal repeat (TR) sequence. KSHV genomes persist in latently infected cells as episomes via tethering to the host cell chromosomes, and KSHV latency associated nuclear antigen (LANA) plays a crucial role in latent episomal DNA replication and segregation during host cell mitosis by binding to TR. While TR's function in plasmid maintenance is well-established, TR’s transcription regulatory roles as gene enhancer has not been fully explored. Gene enhancer harbors transcription enzymes via arrays of transcription factors bindings and often forms phase separate nuclear body in part through recruitment of BRD4 and MED1 that contain intrinsically disordered domain. Here we show KSHV TR possesses transcription regulatory function with LANA. A series of Cleavage Under Targets & Release Using Nuclease (CUT&RUN) demonstrated that TR fragments are occupied by histone modifying enzymes that are known to interact with LANA in naturally infected cells, and the TR possessed characteristic enhancer histone modifications. The H3K4me3 and H3K27Ac modification were also conserved in unique region of the KSHV genome among three PEL cells, and the KSHV Origin of lytic replication (Ori-Lyt) showed similar protein and histone modification occupancies with TR's. In the Ori-Lyt region, the LANA protein complex colocalizes with H3K27Ac-modified nucleosome along with paused RNA polymerase II, and the nucleosome is franked by two K-Rta recruitment sites. The isolated reporter assays demonstrated that neighboring TR fragments enhanced viral lytic gene promoter activity independent of orientation in KSHV-infected and non-infected 293FT cells. K-Rta transactivation function was drastically enhanced with TR, while LANA acquired promoter repression function when the reporter was ligated with TR. The deletion of LANA acidic repeat sequence, a highly-disordered protein domain, further increased gene repression functions. Combined, the TR region is (i) an epigenetically active DNA element that is stitched within 12.5 kb (20-40 copies), (ii) has an array of transcription factor (LANA) binding sites, (iii) recruited by transcription related enzymes including BRD4 (bromodomain containing 4), (iv) decorated by histone H3K27Ac marks, and (v) possesses orientation-independent transcription activation function. KSHV TR is therefore an enhancer domain for KSHV inducible genes. However, in contrast to cellular enhancers that are bound by multiple transcription factors, perhaps KSHV enhancer is predominantly regulated by the LANA nuclear body on the TR. We suggest that KSHV evolved a clever mechanism to tightly control the latency-lytic switch with the TR/LANA complex.
2023-09-08 | GSE241949 | GEO
Project description:Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) analysis was performed during Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation in KSHV+ recombinant primary effusion B-cell lymphoma cells (PEL). RTA binding sites were identified genome-wide in a recombinant PEL cell line called TRExBCBL1-3xFLAG-RTA cells at 12 hours post-induction (hpi) of RTA expression.
2019-01-09 | GSE123897 | GEO
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 | GSE99946 | GEO
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
Project description:Expression profiling of latently infected cells using a custom tiling microarray HUVEC and TIME cells were infected BCBL-1-derived KSHV. Mock infected HUVEC and TIME cells served as controls for each of these two stably infected cells, respectively. BJAB cells served as uninfected controls for the BCBL-1 cells. KSHV-infected cells are induced to enter lytic cycle with valproate or Adenovirus-RTA. Cells were harvested at indicated time points and analyzed.
2010-07-07 | GSE21972 | GEO
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
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