Dataset Information

TREx-BCBL1-RTA GPRC5A-FLAG Immunoprecipitation LC-MS

ABSTRACT: The epitranscriptomic modification m6A is a ubiquitous feature of the mammalian transcriptome. It modulates mRNA fate and dynamics to exert regulatory control over numerous cellular processes and disease pathways, including viral infection. Kaposi’s sarcoma-associated herpesvirus (KSHV) reactivation from the latent phase leads to redistribution of m6A topology upon both viral and cellular mRNAs within infected cells. Here we investigate the role of m6A in cellular transcripts upregulated during KSHV lytic replication. Results show that m6A is crucial for the stability of the GPRC5A mRNA, whose expression is induced by the KSHV latent-lytic switch master regulator, the replication and transcription activator (RTA) protein. Moreover, we demonstrate that GPRC5A is essential for efficient KSHV lytic replication by directly regulating NFκB signalling. Overall, this work highlights the central importance of m6A in modulating cellular gene expression to influence viral infection.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Homo Sapiens (human) Human Gammaherpesvirus 8

TISSUE(S): B Cell, Cell Culture

SUBMITTER: Timothy Mottram

LAB HEAD: Adrian Whitehouse

PROVIDER: PXD039669 | Pride | 2023-07-20

REPOSITORIES: Pride

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Publications

m6A Regulates the Stability of Cellular Transcripts Required for Efficient KSHV Lytic Replication.

Manners Oliver O Baquero-Perez Belinda B Mottram Timothy J TJ Yonchev Ivaylo D ID Trevelyan Christopher J CJ Harper Katherine L KL Menezes Sarah S Patterson Molly R MR Macdonald Andrew A Wilson Stuart A SA Aspden Julie L JL Whitehouse Adrian A

Viruses 20230616 6

The epitranscriptomic modification N6-methyladenosine (m6A) is a ubiquitous feature of the mammalian transcriptome. It modulates mRNA fate and dynamics to exert regulatory control over numerous cellular processes and disease pathways, including viral infection. Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation from the latent phase leads to the redistribution of m6A topology upon both viral and cellular mRNAs within infected cells. Here we invest ...[more]

PMID: 37376680

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Project description:The Kaposi's sarcoma associated herpesvirus (KSHV) is an oncogenic virus that causes Kaposi's sarcoma, primary effusion lymphoma (PEL), and some forms of multicentric Castleman's disease. The KSHV ORF57 protein is a conserved posttranscriptional regulator of gene expression that is essential for virus replication. ORF57 is multifunctional, but most of its activities are directly linked to its ability to bind RNA. We globally identified virus and host RNAs bound by ORF57 during lytic reactivation in PEL cells using high-throughput sequencing of RNA isolated by crosslinking immunoprecipitation (HITS-CLIP). As expected, ORF57-bound RNA fragments mapped throughout the KSHV genome, including the known ORF57 ligand PAN RNA. In agreement with previously published ChIP results, we observed that ORF57 bound RNAs near the oriLyt regions of the genome. Examination of the host RNA fragments revealed that a subset of the ORF57-bound RNAs was derived from transcript 5´ ends. The position of these 5´-bound fragments correlated closely with the 5´-most exonintron junction of the pre-mRNA. We selected four candidates (BTG1, EGR1, ZFP36, and TNFSF9) and analyzed their pre-mRNA and mRNA levels during lytic phase. Analysis of both steady-state and newly made RNAs revealed that these candidate ORF57-bound pre-mRNAs persisted for longer periods of time throughout infection than control RNAs, consistent with a role for ORF57 in pre-mRNA metabolism. In addition, exogenous expression of ORF57 was sufficient to increase the pre-mRNA levels and, in one case, the mRNA levels of the putative ORF57 targets. These results demonstrate that ORF57 interacts with specific host pre-mRNAs during lytic reactivation and alters their processing, likely by stabilizing pre-mRNAs. These data suggest that ORF57 is involved in modulating host gene expression in addition to KSHV gene expression during lytic reactivation. HITS-CLIP was performed on TREx BCBL-Rta cells 20 hpi using antibodies against ORF57. Three biological replicates were performed.

Project description:Chromatin-organizing factors, like CTCF and cohesins, have been implicated in the control of complex viral regulatory programs. We investigated the role of CTCF and cohesin in the control of the latent to lytic switch for Kaposi's Sarcoma-Associated Herpesvirus (KSHV). We found that cohesin subunits, but not CTCF, were required for the repression of KSHV immediate early gene transcription. Depletion of cohesin subunits Rad21, SMC1, or SMC3 resulted in lytic cycle gene transcription and viral DNA replication. In contrast, depletion of CTCF failed to induce lytic transcription or DNA replication. ChiP-Seq analysis revealed that cohesins and CTCF bound to several sites within the immediate early control regions for ORF50 and more distal 5' sites that also regulate the divergently transcribed ORF45-46-47 gene cluster. Rad21 depletion led to a robust increase in ORF45 and ORF47 transcripts, with similar kinetics to that observed with chemical induction by sodium butyrate. During latency, the chromatin between the ORF45 and ORF50 transcription start sites was enriched in histone H3K4me3 with elevated H3K9ac at the ORF45 promoter and elevated H3K27me3 at the ORF50 promoter. A paused form of RNA pol II was loosely associated with the ORF45 promoter region during latency, but was converted to an active elongating form upon reactivation induced by Rad21 depletion. Butyrate-induced transcription of ORF45 and ORF47 was resistant to cyclohexamide, suggesting that these genes have immediate early features similar to ORF50. Butyrate-treatment caused the rapid dissociation of cohesins and loss of CTCF binding at the immediate early gene locus, suggesting that cohesins may be a direct target of butyrate-mediated lytic induction. Our findings implicate cohesins as a major repressor of KSHV lytic gene activation, and function coordinately with CTCF to regulate the switch between latent and lytic gene activity. Study of chromatin-organizing factors, like CTCF and cohesins.

Project description:The establishment of latency is an essential step for the life-long persistent infection and pathogenesis of KaposiM-bM-^@M-^Ys sarcoma-associated herpesvirus (KSHV). While the KSHV genome is chromatin-free in the virions, the viral DNA in latently infected cells has a chromatin structure that is characterized by a specific pattern of activating and repressive histone modifications that ultimately promote latent gene expression while suppressing lytic gene expression. To investigate the molecular events involved in the establishment of the latent chromatin structure during the pre-latency phase of KSHV infection, we performed a comprehensive epigenetic study to analyze the recruitment of chromatin regulatory factors onto the KSHV genome at various time-points following de novo infection of SLK and TIME cells. This showed that the KSHV genome undergoes a biphasic chromatinization following de novo infection. Initially, a transcriptionally active chromatin (euchromatin), characterized by high levels of the H3K4me3 and acetylated H3K27 (H3K27ac) activating histone marks, was deposited on the viral episome and was accompanied by the temporary induction of a limited number of lytic genes. Interestingly, transient expression of the RTA protein facilitated the increases of H3K4me3 and H3K27ac occupancy on the KSHV episome during de novo infection. Between 24-72 hours post-infection, as the levels of these activating histone marks declined on the KSHV genome, the levels of the repressive H3K27me3 and H2AK119ub histone marks increased concomitantly with the decline of lytic gene expression. Importantly, this transition to heterochromatin was dependent on both the Polycomb Repressive Complex 2 and 1. In contrast, upon infection of human gingiva-derived epithelial cells, the KSHV genome underwent a continuously transcription-active euchromatinization, resulting in efficient lytic gene expression. Our data demonstrate that the KSHV genome undergoes a temporally ordered biphasic euchromatin-to-heterochromatin transition in endothelial cells, leading to latent infection, whereas KSHV preferentially adopts a transcriptionally active euchromatin in oral epithelial cells, resulting in lytic gene expression. Our results suggest that the differential epigenetic modification of the KSHV genome in distinct cell types is a potential determining factor for latent infection vs. lytic replication of KSHV. Please see above. 16 hybridizations: ChIP and Input DNA

Dataset Information

TREx-BCBL1-RTA GPRC5A-FLAG Immunoprecipitation LC-MS

Publications

m<sup>6</sup>A Regulates the Stability of Cellular Transcripts Required for Efficient KSHV Lytic Replication.

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