Barrier-to-autointegration Factor 1 (BAF) Promotes Gammaherpesvirus Reactivation from Latency
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE200781 | GEO | 2022/12/12
REPOSITORIES: GEO
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Project description:Recent studies have identified circular RNAs (circRNAs) expressed from the Epstein-Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV) human DNA tu- mor viruses. To gain initial insights into the potential relevance of EBV circRNAs in virus biology and disease, we assessed the circRNAome of the interspecies homologue rhesus macaque lymphocryptovirus (rLCV) in a naturally occurring lymphoma from a simian immunodeficiency virus (SIV)-infected rhesus macaque. This analysis revealed rLCV orthologues of the latency-associated EBV circular RNAs circRPMS1_E4_E3a and circEBNA_U. Also identified in two samples displaying unusually high lytic gene expres- sion was a novel rLCV circRNA that contains both conserved and rLCV-specific RPMS1 exons and whose backsplice junctions flank an rLCV lytic origin of replication (OriLyt). Analysis of a lytic infection model for the murid herpesvirus 68 (MHV68) rhadinovirus identified a cluster of circRNAs near an MHV68 lytic origin of replication, with the most abundant of these, circM11_ORF69, spanning the OriLyt. Lastly, analysis of KSHV la- tency and reactivation models revealed the latency associated circRNA originating from the vIRF4 gene as the predominant viral circRNA. Together, the results of this study broaden our appreciation for circRNA repertoires in the Lymphocryptovirus and Rhadinovirus genera of gammaherpesviruses and provide evolutionary support for vi- ral circRNA functions in latency and viral replication.
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Project description:Epstein-Barr virus (EBV) and Kaposi’s sarcoma herpesvirus (KSHV) cause ~2% of all human cancers. RNase R-resistant RNA sequencing revealed that both gammaherpesviruses encode multiple, uniquely stable, circular RNAs (circRNA). EBV abundantly expressed both exon-only and exon-intron circRNAs from the BART locus (circBARTs) formed from a spliced BART transcript and excluding the EBV miRNA region. CircBARTs were expressed in all verified EBV latency types, including EBV- positive post-transplant lymphoproliferative disease (PTLD), Burkitt lymphoma, nasopharyngeal carcinoma, and AIDS-associated lymphoma tissues and cell lines. Only cells infected with the B95-8 EBV strain, with a 12-kb BART locus deletion, were negative for EBV circBARTs. Less abundant levels of EBV circRNAs originating from LMP2 and BHLF1-encoding genes were also identified. CircRNA sequencing of KSHV- infected PEL cells revealed a KSHV-encoded circRNA from the vIRF4 locus (circvIRF4) that was constitutively expressed. In addition, KSHV polyadenylated nuclear (PAN) RNA locus generated a swarm (>100) of multiply backspliced, low-abundance RNase R- resistant circRNAs originating in both sense and antisense directions consistent with a novel hyper-backsplicing mechanism. In EBV and KSHV co-infected cells, exon-only EBV circBARTS were located more in the cytoplasm, whereas the intron-retaining circBARTs were found in the nuclear fraction. KSHV circvIRF4 and circPANs were detected in both nuclear and cytoplasmic fractions. Among viral circRNAs tested, none were found in polysome fractions from KSHV-EBV co-infected BC1 cells although low abundance protein translation from viral circRNAs could not be excluded. CircRNAs are a new class of viral transcripts expressed in gammaherpesvirus-related tumors that might contribute to viral oncogenesis.
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Project description:Gammaherpesviruses, including the human pathogens Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus, establish lifelong latent infection in B cells and are associated with a variety of tumors. In addition to protein coding genes, these viruses encode numerous microRNAs (miRNAs) within their genomes. While putative host targets of EBV and KSHV miRNAs have been previously identified, the specific functions of these miRNAs during in vivo infection are largely unknown. Murine gammaherpesvirus 68 is a natural pathogen of rodents that is genetically related to both EBV and KSHV, and thus serves as an excellent model for the study of EBV and KSHV elements such as miRNAs in the context of infection and disease. However, MHV68 the specific targets of miRNAs remain unknown. Using a technique known as quick CLASH (crosslinking, ligation, and sequencing of hybrids), we have now identified specific, Ago-associated mRNA targets of MHV68 miRNAs during lytic infection, latent infection and reactivation from latency.
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Project description:Kaposi’s sarcoma-associated herpesvirus (KSHV) is the etiologic agent of primary effusion lymphoma (PEL). All PEL cell lines are infected with KSHV, and 70% are co-infected with Epstein-Barr Virus (EBV). KSHV reactivation from latency requires promoter-specific transactivation by the KSHV Rta protein through interactions with RBP-Jk (CSL), the cellular DNA binding component of the Notch signal transduction pathway. EBV transformation of primary B cells requires EBV nuclear antigen (EBNA)-2 to interact with RBP-Jk to direct the latent viral and cellular gene expression program. Although KSHV Rta and EBV EBNA-2 both require RBP-Jk for transactivation, previous studies have suggested that RBP-Jk-dependent transactivators do not function identically. We have found that the EBV latent protein LMP-1 is expressed in less than 5% of KSHV+/EBV+ PEL cells, but is induced in an Rta-dependent fashion when KSHV reactivates. KSHV Rta transactivates the EBV latency promoters in an RBP-Jk-dependent fashion and forms a ternary complex with RBP-Jk on the promoters. In B cells that are conditionally transformed by EBV alone, we show that KSHV Rta complements a short-term EBNA2 growth deficiency in an autocrine/paracrine manner. Complementaton of EBNA2-deficiency by Rta depends on RBP-Jk and LMP-1, and Rta transactivation is required for optimal growth of KSHV+/EBV+ PEL lines. Our data suggest that Rta can contribute to EBV-driven cellular growth by transactivating RBP-Jk-dependent EBV latency genes. However, our data also suggest that EBNA2 and Rta induce distinct alterations in the cellular proteomes that contribute to growth of infected cells. EREB2-5 cells were transfected and grown in the presence or absence of β-estradiol, as described. Seven days post-transfection, protein extracts were prepared, and 200 ugs. of each were analyzed using the RayBio Human Apoptosis Antibody Array Kit (RayBiotech) as per manufacturers suggestions. The membranes were exposed to autoradiography film for different times to detect the chemiluminescent signals. Images with signals in linear range were quantitated using the program ImageJ [59]. For each membrane, signals from the negative control spots were averaged, and then subtracted from each of the other spots. A signal was considered valid if its value exceeded both its average local background, and the average of all valid negative control values. Valid signals were normalized using the positive control spots (for cellular BID protein). Fold change in signals for each spot were quantitated by dividing by the valid signals for each corresponding spot on the minus β-estradiol membrane. Average fold change, and standard deviation, were calculated for each protein.
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Project description:lncRNAs regulate protein functions via formation of protein-RNA complexes. Previous studies have shown that expression of viral lncRNA, polyadenylated nuclear RNA (PAN RNA) is essential for inducible viral genomic looping and distal gene activation during Kaposi's sarcoma-associated herpesvirus (KSHV) reactivation. Here we show an underlying molecular mechanism and regulation of KSHV latency by a viral lncRNA-CHD4 (chromodomain helicase DNA binding protein 4) interaction. Knock-out of the viral RNA binding protein, ORF57 protein, leads to decreased inducible and static viral genomic loops in latent chromatin and a failure to form RNA polymerase II aggregates in the nucleus during reactivation. We identified that CHD4's enzymatic activity silences viral gene expression by preventing nuclear aggregate formation. Furthermore, integrated genomic and proteomic studies together show that KSHV episomes frequently tether near the host cell centromeres and colocalize with a CHD4 protein complex, ChAHP. KSHV episomes detached from these sites when reactivation is triggered, and PAN RNA binds and inhibits CHD4 DNA binding in vitro. Our studies suggest that CHD4 exhibits strong repressor function by preventing inducible enhancer-promoter looping, and is therefore important for the ability of KSHV to establish and maintain latency in a “poised” state at specific host genomic loci.
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