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Structure and coding content of CST (BART) family RNAs of Epstein-Barr virus.

ABSTRACT: CST (BART BARF0) family viral RNAs are expressed in several types of Epstein-Barr virus (EBV) infection, including EBV-associated cancers. Many different spliced forms of these RNAs have been described; here we have clarified the structures of some of the more abundant splicing patterns. We report the first cDNAs representing a full-length CST mRNA from a clone library and further characterize the transcription start. The relative abundance of splicing patterns and genomic analysis of the open reading frames (ORFs) suggest that, in addition to the much studied BARF0 ORF, there may be important products made from some of the upstream ORFs in the CST RNAs. Potential biological functions are identified for two of these. The product of the RPMS1 ORF is shown to be a nuclear protein that can bind to the CBF1 component of Notch signal transduction. RPMS1 can inhibit the transcription activation induced through CBF1 by NotchIC or EBNA-2. The protein product of another CST ORF, A73, is shown to be a cytoplasmic protein which can interact with the cell RACK1 protein. Since RACK1 modulates signaling from protein kinase C and Src tyrosine kinases, the results suggest a possible role for CST products in growth control, perhaps consistent with the abundant transcription of CST RNAs in cancers such as nasopharyngeal carcinoma.

SUBMITTER: Smith PR

PROVIDER: S-EPMC111807 | biostudies-literature | 2000 Apr

REPOSITORIES: biostudies-literature

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Structure and coding content of CST (BART) family RNAs of Epstein-Barr virus.

Smith P R PR de Jesus O O Turner D D Hollyoake M M Karstegl C E CE Griffin B E BE Karran L L Wang Y Y Hayward S D SD Farrell P J PJ

Journal of virology 20000401 7

CST (BART BARF0) family viral RNAs are expressed in several types of Epstein-Barr virus (EBV) infection, including EBV-associated cancers. Many different spliced forms of these RNAs have been described; here we have clarified the structures of some of the more abundant splicing patterns. We report the first cDNAs representing a full-length CST mRNA from a clone library and further characterize the transcription start. The relative abundance of splicing patterns and genomic analysis of the open r ...[more]

PMID: 10708423

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Project description:UnlabelledEpstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high levels of BamHI-A rightward transcripts (BARTs), which include long noncoding RNAs (lncRNAs) and BART microRNAs (miRNAs). It is hypothesized that the mechanism for regulation of BARTs may relate to EBV pathogenesis in NPC. We showed that nuclear factor-κB (NF-κB) activates the BART promoters and modulates the expression of BARTs in EBV-infected NPC cells but that introduction of mutations into the putative NF-κB binding sites abolished activation of BART promoters by NF-κB. Binding of p50 subunits to NF-κB sites in the BART promoters was confirmed in electrophoretic mobility shift assays (EMSA) and further demonstrated in vivo using chromatin immunoprecipitation (ChIP) analysis. Expression of BART miRNAs and lncRNAs correlated with NF-κB activity in EBV-infected epithelial cells, while treatment of EBV-harboring NPC C666-1 cells with aspirin (acetylsalicylic acid [ASA]) and the IκB kinase inhibitor PS-1145 inhibited NF-κB activity, resulting in downregulation of BART expression. Expression of EBV LMP1 activates BART promoters, whereas an LMP1 mutant which cannot induce NF-κB activation does not activate BART promoters, further supporting the idea that expression of BARTs is regulated by NF-κB signaling. Expression of LMP1 is tightly regulated in NPC cells, and this study confirmed that miR-BART5-5p downregulates LMP1 expression, suggesting a feedback loop between BART miRNA and LMP1-mediated NF-κB activation in the NPC setting. These findings provide new insights into the mechanism underlying the deregulation of BARTs in NPC and identify a regulatory loop through which BARTs support EBV latency in NPC.ImportanceNasopharyngeal carcinoma (NPC) cells are ubiquitously infected with Epstein-Barr virus (EBV). Notably, EBV expresses very few viral proteins in NPC cells, presumably to avoid triggering an immune response, but high levels of EBV BART miRNAs and lncRNAs which exhibit complex functions associated with EBV pathogenesis. The mechanism for regulation of BARTs is critical for understanding NPC oncogenesis. This study provides multiple lines of evidence to show that expression of BARTs is subject to regulation by NF-κB signaling. EBV LMP1 is a potent activator of NF-κB signaling, and we demonstrate that LMP1 can upregulate expression of BARTs through NF-κB signaling and that BART miRNAs are also able to downregulate LMP1 expression. It appears that aberrant NF-κB signaling and expression of BARTs form an autoregulatory loop for maintaining EBV latency in NPC cells. Further exploration of how targeting NF-κB signaling interrupts EBV latency in NPC cells may reveal new options for NPC treatment.

Project description:This study assessed the effects of Epstein-Barr virus (EBV) and one form of virally encoded BART long noncoding RNAs (lncRNAs) on cellular expression in epithelial cells grown in vitro and as tumors in vivo determined by high-throughput RNA sequencing of mRNA and small RNAs. Hierarchical clustering based on gene expression distinguished the cell lines from the tumors and distinguished the EBV-positive tumors and the BART tumors from the EBV-negative tumors. EBV and BART expression also induced specific expression changes in cellular microRNAs (miRs) and lncRNAs. Multiple known and predicted targets of the viral miRs, the induced cellular miRs, and lncRNAs were identified in the altered gene set. The changes in expression in vivo indicated that the suppression of growth pathways in vivo reflects increased expression of cellular miRs in all tumors. In the EBV and BART tumors, many of the targets of the induced miRs were not changed and the seed sequences of the nonfunctional miRs were found to have homologous regions within the BART lncRNA. The inhibition of these miR effects on known targets suggests that these induced miRs have reduced function due to sponging by the BART lncRNA. This composite analysis identified the effects of EBV on cellular miRs and lncRNAs with a functional readout through identification of the simultaneous effects on gene expression. Major shifts in gene expression in vivo are likely mediated by effects on cellular noncoding RNAs. Additionally, a predicted property of the BART lncRNA is to functionally inhibit the induced cellular miRs. IMPORTANCE This study identified the total effects of EBV and a viral long noncoding RNA (BART lncRNA) on cellular RNA expression when grown as cells in culture and when grown as tumors in immunodeficient mice. The effects on cellular mRNA expression, lncRNA expression, and cellular and viral miR expression were determined using next-generation sequencing (NGS) and bioinformatics functional analysis. Many cellular growth pathways that are activated during growth in culture are decreased during growth as tumors. This study shows that these changes in expression are accompanied by induction of cellular-growth-inhibitory miRs. However, in the EBV tumors and in tumors expressing the BART lncRNA, many of the known targets of the inhibitory miRs are not affected. Regions of strong homology to the seed sequences of these miRs were identified in the BART lncRNA. These findings suggest that the BART lncRNA functions as a sponge for growth-inhibitory miRs.

Dataset Information

Structure and coding content of CST (BART) family RNAs of Epstein-Barr virus.

Publications

Structure and coding content of CST (BART) family RNAs of Epstein-Barr virus.

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