EBER2 is an abundant nuclear noncoding RNA expressed by the Epstein-Barr virus (EBV). Probing its possible chromatin localization by CHART revealed EBER2’s presence at the terminal repeats (TRs) of the latent EBV genome, overlapping previously identified binding sites for the B cell transcription factor PAX5. EBER2 interacts with PAX5 and is required for the localization of PAX5 to the TRs. EBER2 knockdown phenocopies PAX5 depletion in upregulating the expression of LMP2A/B and LMP1, genes nearest the TRs. Knockdown of EBER2 also decreases EBV lytic replication, underscoring the essential role of the TRs in viral replication. Recruitment of the EBER2-PAX5 complex is mediated by base-pairing between EBER2 and nascent transcripts from the TR locus. The interaction is evolutionarily conserved in the related primate herpesvirus CeHV15 despite great sequence divergence. Using base-pairing with nascent RNA to guide an interacting transcription factor to its DNA target site is a previously undescribed function for a trans-acting noncoding RNA.
High-Throughput RNA sequencing based virome analysis of 50 lymphoma cell lines from the Cancer Cell Line Encyclopedia project
Using high-throughput RNA sequencing (RNA-seq) data from 50 common lymphoma cell culture models from the Cancer Cell Line Encyclopedia project, we performed an unbiased global interrogation for the presence of a panel of 740 viruses and strains known to infect human and other mammalian cells. This led to the findings of previously identified infections by Epstein-Barr virus (EBV), Kaposi’s sarcoma herpesvirus (KSHV), and human T-lymphotropic virus 1 (HTLV-1). In addition, we also found a previously unreported infection of one cell line (DEL) with a murine leukemia virus. High expression of MuLV transcripts was observed in DEL cells and we identified 4 transcriptionally active integration sites, one being in the TNFRSF6B gene. We also found low levels of MuLV reads in a number of other cell lines and provided evidence suggesting cross-contamination during sequencing. Analysis of HTLV-1 integrations in two cell lines, HuT 102 and MJ, identified 14 and 66 transcriptionally active integration sites with potentially activating integrations in immune regulatory genes including IL15, IL6ST, STAT5B, HIVEP1, and IL9R. While KSHV and EBV do not typically integrate into the genome, we investigated a previously identified integration of EBV into the BACH2 locus in Raji cells. This analysis identified a BACH2 disruption mechanism involving splice donor sequestration. Through viral gene expression analysis, we detected expression of stable intronic RNAs from the EBV BamHI W repeats that may be part of long transcripts spanning the repeat region. We also observed transcripts at the EBV vIL10 locus exclusively in the Hodgkin’s lymphoma cell line, Hs 611.T, the expression of which were uncoupled from other lytic genes. Assessment of the KSHV viral transcriptome in BCP-1 cells showed expression of the viral immune regulators, K2/vIL6, K4/vIL8-like vCCL1, and K5/E2-ubiquitin ligase 1 that was significantly higher than expression of the latency-associated nuclear antigen, LANA. Together, this investigation sheds light into the virus composition across these lymphoma model systems and provides insights into common viral mechanistic principles.
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Influenza A virus utiizes RNA throughout infection. Little is known, however, about the roles of RNA structures in these processes. A previous bioinformatics survey predicted multiple regions of influenza A virus that are likely to generate evolutionarily conserved and stable RNA structures. One predicted conserved structure is in the pre-mRNA coding for essential proteins, M1 and M2. This structure is 39 nucleotides downstream of the M2 mRNA 5′ splice site. Here a combination of biochemical structural mapping, mutagenesis, and NMR confirms the predicted three-way multi-branch structure of this RNA. Imino proton spectra reveal no change in secondary structure when 80 mM KCl is supplemented with 4 mM MgCl2. Optical melting curves in 1 M NaCl and in 100 mM KCl with 10 mM MgCl2 are very similar, with melting temperatures ~14 °C higher than for 100 mM KCl alone. These results provide a firm basis for designing experiments and potential therapeutics to test for function in cell culture.
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