Identification of KSHV RTA and K8 binding sites on viral genome
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE82283 | GEO | 2016/06/07
SECONDARY ACCESSION(S): PRJNA324549
REPOSITORIES: GEO
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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.
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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
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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.
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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
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