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KSHV Terminal Repeat Regulates Inducible Lytic Gene Promoters

ABSTRACT: 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.

ORGANISM(S): Homo sapiens

PROVIDER: GSE241949 | GEO | 2023/09/08

REPOSITORIES: GEO

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Project description:Kaposi’s sarcoma-associated herpesvirus (KSHV) is a g-herpesvirus which persists as circular extrachromasomal DNA in the nucleus during latent infection. During latency a limited number of viral proteins are expressed, including LANA (latency-associated nuclear antigen). LANA is a multi-functional protein known to interact with transcriptional regulators and chromatin remodelers, and to regulate the LANA and RTA promoters. We hypothesized that LANA may contribute to the establishment of latency through controlling chromatinization and histone modification of KSHV episomes. We performed ChIP-seq analysis and correlated H3K4me3, H3K27me3, polII, and LANA occupancy of the KSHV genome at nucleotide resolution. Epigenetic marks were analyzed in BCBL-1 lymphoid cells and in telomerase-immortalized vein endothelial TIVE-LTC cells. We found that the transcription active mark H3K4me3, but not silencing mark H3K27me3, was enriched at KSHV regions where LANA is bound. Co-occupancy of LANA and H3K4 was also detected on 167 host genes, of which 89 are actively transcribed. By comparing LANA occupancy with the profiling of 43 transcription factors from ENCODE, a subset of transcription factors was enriched at regions where LANA is bound, including znf143, CTCF, and Stat1. These results indicate that LANA also plays a role in modulating expression of host genes. Co-immunoprecipitation of LANA with the H3K4 methyltransferase hSET1 suggests that LANA recruits hSET1 to specific loci on the viral genome, leading to H3K4 methylation and ensuring transcription of latency-associated genes. Host gene expression may be regulated by the same mechanism. LANA binding was correlated with the distribution in the latent KSHV genome of the transcription active histone modification H3K4me3, the silencing mark H3K27me3, and RNA polymerase II, using cells of lymphoid and endothelial origin. A similar analysis was done for the human genome. Biological replicates (BR) and technical replicates (TR) were included.

Project description:Kaposi’s Sarcoma Herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS) and isassociated with primary effusion lymphoma (PEL), multicentric Castleman’s disease (MCD) and two inflammatory diseases. KSHV-associated cancers are primarily associated with genes expressed during latency, while other pathologies are associated with lytic gene expression. The major lytic switch of the virus, RTA, interacts with cellular machinery to co-opt the host ubiquitin proteasome system to evade the immune response as well as activate the program of lytic replication. Through SILAC labeling, ubiquitin remnant enrichment and mass spectrometry, we have analyzed the RTA dependent ubiquitin-modified proteome. We identified RTA dependent changes in the populations of polyubiquitin chains, as well as changes in ubiquitinated proteins in both cells expressing RTA and naturally infected cells following lytic reactivation. We observed an enrichment of proteins that are also reported to be SUMOylated, suggesting that RTA, a SUMO targeting ubiquitin ligase, may function to alleviate a SUMO dependent block to lytic reactivation. RTA targeted substrates directly through a ubiquitin ligase domain dependent mechanism as well as indirectly through cellular ubiquitin ligases, including RAUL. Our ubiquitome analysis revealed an RTA dependent mechanism of immune evasion. We provide evidence of inhibition of TAP dependent peptide transport, resulting in decreased HLA complex stability. The results of this analysis increase our understanding of mechanisms governing the latent to lytic transition in addition to the identification of a novel RTA dependent mechanism of immune evasion.Kaposi’s Sarcoma Herpesvirus (KSHV) is the causative agent of Kaposi’s Sarcoma (KS) and is associated with primary effusion lymphoma (PEL), multicentric Castleman’s disease (MCD) and two inflammatory diseases. KSHV-associated cancers are primarily associated with genes expressed during latency, while other pathologies are associated with lytic gene expression. The major lytic switch of the virus, RTA, interacts with cellular machinery to co-opt the host ubiquitin proteasome system to evade the immune response as well as activate the program of lytic replication. Through SILAC labeling, ubiquitin remnant enrichment and mass spectrometry, we have analyzed the RTA dependent ubiquitin-modified proteome. We identified RTA dependent changes in the populations of polyubiquitin chains, as well as changes in ubiquitinated proteins in both cells expressing RTA and naturally infected cells following lytic reactivation. We observed an enrichment of proteins that are also reported to be SUMOylated, suggesting that RTA, a SUMO targeting ubiquitin ligase, may function to alleviate a SUMO dependent block to lytic reactivation. RTA targeted substrates directly through a ubiquitin ligase domain dependent mechanism as well as indirectly through cellular ubiquitin ligases, including RAUL. Our ubiquitome analysis revealed an RTA dependent mechanism of immune evasion. We provide evidence of inhibition of TAP dependent peptide transport, resulting in decreased HLA complex stability. The results of this analysis increase our understanding of mechanisms governing the latent to lytic transition in addition to the identification of a novel RTA dependent mechanism of immune evasion.

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KSHV Terminal Repeat Regulates Inducible Lytic Gene Promoters

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