Translesional DNA Polη Bypass of LANA-DNA Crosslinks Ensures KSHV Episome Persistence and Tumor Growth
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ABSTRACT:
ORGANISM(S): Human gammaherpesvirus 8 Homo sapiens
PROVIDER: GSE329039 | GEO | 2026/04/29
REPOSITORIES: GEO
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Project description:Latency-associated nuclear antigen (LANA), a multifunctional protein expressed by the Kaposi sarcoma-associated herpesvirus (KSHV) in latently-infected cells, is required for stable maintenance of the viral episome. This is mediated by two interactions: LANA binds to specific sequences (LBS1 and 2) on viral DNA, and also engages host histones, tethering the viral genome to host chromosomes in mitosis. LANA has also been suggested to affect host gene expression, but both the mechanism(s) and role of this dysregulation in KSHV biology remain unclear. Here we have examined LANA interactions with host chromatin on a genome-wide scale using ChIP-seq, and show that LANA predominantly targets human genes near their transcriptional start sites (TSSs). These host LANA-binding sites are generally found within transcriptionally active promoters and display striking overrepresentation of a consensus DNA sequence virtually identical to the LBS1 motif in KSHV DNA. Comparison of the ChIP-seq profile with whole transcriptome (RNA-seq) data reveals that few of the genes that are differentially regulated in latent infection are occupied by LANA at their promoters. This suggests that direct LANA binding to promoters is not the prime determinant of altered host transcription in KSHV-infected cells. Most surprisingly, the association of LANA to both host and viral DNA is strongly disrupted during the lytic cycle of KSHV. This disruption can be prevented by the inhibition of viral DNA synthesis, suggesting the existence of novel and potent regulatory mechanisms linked to either viral DNA replication or late gene expression. Profiling of KSHV LANA positioning on the host genome and examination of gene expression from promoters bound by KSHV LANA.
2014-04-01 | E-GEOD-56144 | biostudies-arrayexpress
Project description:Latency-associated nuclear antigen (LANA), a multifunctional protein expressed by the Kaposi sarcoma-associated herpesvirus (KSHV) in latently-infected cells, is required for stable maintenance of the viral episome. This is mediated by two interactions: LANA binds to specific sequences (LBS1 and 2) on viral DNA, and also engages host histones, tethering the viral genome to host chromosomes in mitosis. LANA has also been suggested to affect host gene expression, but both the mechanism(s) and role of this dysregulation in KSHV biology remain unclear. Here we have examined LANA interactions with host chromatin on a genome-wide scale using ChIP-seq, and show that LANA predominantly targets human genes near their transcriptional start sites (TSSs). These host LANA-binding sites are generally found within transcriptionally active promoters and display striking overrepresentation of a consensus DNA sequence virtually identical to the LBS1 motif in KSHV DNA. Comparison of the ChIP-seq profile with whole transcriptome (RNA-seq) data reveals that few of the genes that are differentially regulated in latent infection are occupied by LANA at their promoters. This suggests that direct LANA binding to promoters is not the prime determinant of altered host transcription in KSHV-infected cells. Most surprisingly, the association of LANA to both host and viral DNA is strongly disrupted during the lytic cycle of KSHV. This disruption can be prevented by the inhibition of viral DNA synthesis, suggesting the existence of novel and potent regulatory mechanisms linked to either viral DNA replication or late gene expression.
2014-04-01 | GSE56144 | GEO
Project description:Translesional DNA Polη Bypass of LANA-DNA Crosslinks Ensures KSHV Episome Persistence and Tumor Growth
| PRJNA1457412 | ENA
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:LANA is essential for tethering the KSHV genome to metaphase chromosomes and for modulating host-cell gene expression, but the binding sites in the host-chromosome remain unknown. Here, we use LANA-specific chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to identify LANA binding sites in the viral and host-cell genomes of a latently infected pleural effusion lymphoma cell line BCBL1. LANA bound with high occupancy to the KSHV genome terminal repeats (TR), and to a few minor binding sites within the latency control region encoding that LANA transcript. We identified 256 LANA binding peaks with p < 0.01 and overlap in two independent ChIP-Seq experiments. We validated several of the high-occupancy binding sites by conventional ChIP assays and quantitative PCR. Two candidate DNA sequence motifs were identified, and confirmed to bind purified LANA protein, although with weaker affinity compared to viral TR binding site. More than half of the LANA binding sites (170/256) could be mapped to within 2.5 kb of a cellular gene transcript. Pathways and Gene Ontogeny (GO) analysis revealed that LANA binds to genes within the p53 and TNF regulatory network. Further analysis revealed partial overlap of LANA binding sites with STAT1 binding sites in several interferon (IFN)-g regulated genes. We show that ectopic expression of LANA can down-modulate IFN-g mediated activation of a subset of genes, including the TAP1 peptide transporter and proteasome subunit beta type 9 (PSMB9) required for class I antigen presentation. Our data provides a potential mechanism through which LANA may regulate several host cell pathways by direct binding to gene regulatory elements. Study of KSHV LANA
2012-01-06 | E-GEOD-34890 | biostudies-arrayexpress
Project description:Kaposi's sarcoma (KS), is an AIDS-associated neoplasm caused by the KS herpesvirus (KSHV/ HHV-8). KSHV-sarcomagenesis is the consequence of oncogenic viral gene expression as well as host genetic and epigenetic alterations. Although KSHV is invariably found in all KS lesions, the percentage of KSHV-infected (LANA+) spindle-cells of the lesion is variable, suggesting the existence of paracrine oncogenic mechanisms and/or spindle cells that have lost KSHV. A mouse model of KSHVBac36-driven tumorigenesis allowed us to induce KSHV-episome loss before and after tumor development. Although cells that lose the KSHV episome prior to tumor formation lose their tumorigenicity, explanted KSHV positive tumor cells that lost the KSHV episome remained tumorigenic. This pointed to the existence of virally-induced irreversible oncogenic alterations occurring during KSHV-tumor formation that support the possibility of hit and run viral sarcomagenesis. RNA sequencing and CpG-methylation analysis were performed on KSHV positive and negative cells, KSHV positive and KSHV negative tumors that developed following KSHV-episome loss from explanted tumor cells. When KSHV positive cells form KSHV-driven tumors, along with viral-gene upregulation there is a tendency for hypo-methylation in genes from oncogenic and differentiation pathways. In contrast, KSHV-negative tumors formed after KSHV-episome loss, show a tendency towards gene hyper-methylation when compared to KSHV-positive tumors. Regarding occurrence of host-mutations, we found the same set of innate-immunity related mutations undetected in KSHV-infected cells but present in all KSHV positive tumors, indicating that pre-existing host mutations that provide an in vivo growth advantage are clonally-selected and contribute to KSHV-tumorigenesis. In addition, KSHV negative tumors display de novo mutations related to cell proliferation that, together with the PDGFRAD842V, were responsible for driving tumorigenesis in absence of the KSHV-episomes. KSHV-induced irreversible genetic and epigenetic oncogenic alterations support the possibility of “hit and run” KSHV-sarcomagenesis consistent with the presence of LANA-negative spindle-cells in KS lesions.
2020-04-16 | GSE148741 | GEO
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.
2014-10-27 | E-GEOD-52421 | biostudies-arrayexpress
Project description:Kaposi's sarcoma (KS), is an AIDS-associated neoplasm caused by the KS herpesvirus (KSHV). A mouse model of KSHV-dependent tumorigenicity, allowed us to induce KSHV viral-episome loss following tumor development to test the plausibility of “hit and run” mechanism by KSHV. RNA-seq-transcriptome analysis and CpG-methylation were performed on KSHV positive cells, KSHV positive tumors and tumors that developed following viral-episome loss. During KSHV tumorigenesis, hypo-methylation was detected of oncogenic and differentiation pathways. In contrast, during tumorigenesis following KSHV-episome loss, a tendency towards hyper-methylation was detected. We found the same set of innate-immunity related mutations undetected in KSHV-infected cells but present in all KSHV-positive tumors, indicating that pre-existing host mutations that provide an in vivo growth advantage are clonally-selected and contribute to KSHV-tumorigenesis. We found de novo mutations related to cell proliferation that, together with the PDGFRAD842V, were responsible for driving tumorigenesis in absence of the KSHV-episomes. Virally-induced irreversible genetic and epigenetic oncogenic alteration supports the possibility of “hit and run” KSHV-sarcomagenesis consistent with the existence of LANA-negative spindle-cells in KS lesions.
2020-01-23 | GSE144101 | GEO
Project description:LANA is essential for tethering the KSHV genome to metaphase chromosomes and for modulating host-cell gene expression, but the binding sites in the host-chromosome remain unknown. Here, we use LANA-specific chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) to identify LANA binding sites in the viral and host-cell genomes of a latently infected pleural effusion lymphoma cell line BCBL1. LANA bound with high occupancy to the KSHV genome terminal repeats (TR), and to a few minor binding sites within the latency control region encoding that LANA transcript. We identified 256 LANA binding peaks with p < 0.01 and overlap in two independent ChIP-Seq experiments. We validated several of the high-occupancy binding sites by conventional ChIP assays and quantitative PCR. Two candidate DNA sequence motifs were identified, and confirmed to bind purified LANA protein, although with weaker affinity compared to viral TR binding site. More than half of the LANA binding sites (170/256) could be mapped to within 2.5 kb of a cellular gene transcript. Pathways and Gene Ontogeny (GO) analysis revealed that LANA binds to genes within the p53 and TNF regulatory network. Further analysis revealed partial overlap of LANA binding sites with STAT1 binding sites in several interferon (IFN)-g regulated genes. We show that ectopic expression of LANA can down-modulate IFN-g mediated activation of a subset of genes, including the TAP1 peptide transporter and proteasome subunit beta type 9 (PSMB9) required for class I antigen presentation. Our data provides a potential mechanism through which LANA may regulate several host cell pathways by direct binding to gene regulatory elements.
2012-01-06 | GSE34890 | GEO
Project description:Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of Kaposi's sarcoma (KS) and two human lymphoproliferative diseases: primary effusion lymphoma and AIDS-related multicentric Castleman's disease. KSHV latency-associated nuclear antigen (LANA) is expressed in KSHV-infected cancer cells and is responsible for maintaining viral genomes in infected cells. Thus, LANA is an attractive target for therapeutic intervention for KSHV-associated diseases. Here, we devised a gene therapy vector using the adeno-associated virus (AAV), which capitalizes the LANA's function to tether terminal repeat (TR) containing circular genome in latently infected cells and the TR's enhancer function for KSHV inducible gene promoters. By including two TR copies with a lytic inducible gene promoter (TR2-Orip), we generated an AAV vector, which expresses an engineered thymidine kinase (TK) selectively in KSHV-infected cells. Ganciclovir (GCV), an anti-herpesvirus drug, effectively eradicated multiple KSHV-infected cells that include iPSC derived epithelial colony forming cells, but not non-KSHV-infected counterparts in the presence of AAV8-TR2-Orip-TK. In addition, AAV8-TR2-Orip-TK prevents KSHV virion production from reactivated cells, hence spreading KSHV infections from reactivated cells. Anti-cancer drugs, known to reactivate KSHV, stimulated TK expression from the vector and, therefore, synergized with AAV8 TR2-Orip-TK to induce KSHV-infected cancer cell death. Finally, the AAV8-TR2-Orip-TK with GCV completely diminished KSHV-infected cancer cells in the xenograft tumor model. Our new cancer gene therapy should augment the current clinical protocol for KS.
2025-02-19 | GSE289947 | GEO