EBV BART MicroRNAs Target Multiple Pro-apoptotic Cellular Genes to Promote Epithelial Cell Survival

Kang, Dong Woo; Skalsky, Rebecca L.; Cullen, Bryan R.

doi:10.1371/journal.ppat.1004979

Cited by 100 publications

(91 citation statements)

References 46 publications

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“…In the time since the publication of our high-throughput experiment that proposed CASP3 as a target of EBV miRNAs, several groups have published conflicting evidence of this interaction [11, 18, 22, 24, 25]. Here we confirmed that human CASP3 is a direct target of nine EBV BART miRNAs in luciferase assays by testing all canonical, predicted sites.…”

Section: Discussionsupporting

confidence: 75%

Epstein-Barr viral microRNAs target caspase 3

Harold

Cox

Riley

2016

Virol J

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The Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that transforms B cells and causes several malignancies including Burkitt’s lymphoma. EBV differentially expresses at least 49 mature microRNAs (miRNAs) during latency in various infected epithelial and B cells. Recent high-throughput studies and functional assays have begun to reveal the function of the EBV miRNAs suggesting roles in latency, cell cycle control, and apoptosis. In particular, the central executioner of apoptosis, Caspase 3 (CASP3), was proposed as a target of select EBV miRNAs. However, whether CASP3 is truly a target of EBV miRNAs, and if so, which specific miRNAs target CASP3 is still under debate. Based on previously published high-throughput biochemical data and a bioinformatic analysis of the entire CASP3 3′-UTR, we identified 12 EBV miRNAs that have one or more seed binding sites in the CASP3 3′-UTR. We individually tested all 12 miRNAs for repression of CASP3 in luciferase reporter assays, and nine showed statistically significant (P < 0.001) repression of a full-length CASP3 reporter. Further, three EBV miRNAs, including BART22, exhibited repression of endogenous CASP3 protein. These data confirm that CASP3 is a direct target of specific EBV BART miRNAs.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0602-7) contains supplementary material, which is available to authorized users.

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Section: Discussionsupporting

confidence: 75%

Epstein-Barr viral microRNAs target caspase 3

Harold

Cox

Riley

2016

Virol J

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“…AGS cells infected with EBV express high levels of the BART miRNAs (15), and previous profiling of one AGS line by microarray suggested that a substantial subset of the downregulated genes after latent infection were direct targets of the BART miRNAs (4). To confirm that the BART miRNAs contribute to the changes in gene expression profiled in the multiple infected cell lines in this report, the genes changed in expression were overlapped with the most extensive lists of BART miRNA targets published to date, where the BART miRNA targets were identified by PAR-CLIP in a peripheral effusion lymphoma cell line dually infected with EBV and KSHV, as well as the NPC cell line C666 (25,26). A higher percentage of the downregulated genes and a lower number of the upregulated genes overlapped with the PAR-CLIP data sets compared to the null hypothesis, which would be the percentage of all genes expressed in AGS cells (based on the RNA-seq data) that overlap the PAR-CLIP data set ( Fig.…”

Section: Resultsmentioning

confidence: 89%

Host Gene Expression Is Regulated by Two Types of Noncoding RNAs Transcribed from the Epstein-Barr Virus BamHI A Rightward Transcript Region

Marquitz

Mathur

Edwards

et al. 2015

J Virol

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In Epstein-Barr virus-infected epithelial cancers, the alternatively spliced BamHI A rightward transcripts (BARTs) are the most abundant viral polyadenylated RNA. The BART introns form the template for the production of 44 microRNAs (miRNAs), and the spliced and polyadenylated exons form nuclear non-protein-coding RNAs. Analysis of host cell transcription by RNA-seq during latency in AGS cells identified a large number of reproducibly changed genes. Genes that were downregulated were enriched for BART miRNA targets. Bioinformatics analysis predicted activation of the myc pathway and downregulation of XBP1 as likely mediators of the host transcriptional changes. Effects on XBP1 activity were not detected in these cells; however, myc activation was confirmed through use of a myc-responsive luciferase reporter. To identify potential regulatory properties of the spliced, polyadenylated BART RNAs, a full-length cDNA clone of one of the BART isoforms was obtained and expressed in the Epstein-Barr virus (EBV)-negative AGS cells. The BART cDNA transcript remained primarily nuclear yet induced considerable and consistent changes in cellular transcription, as profiled by RNA-seq. These transcriptional changes significantly overlapped the transcriptional changes induced during latent EBV infection of these same cells, where the BARTs are exclusively nuclear and do not encode proteins. These data suggest that the nuclear BART RNAs are functional long noncoding RNAs (lncRNAs). The abundant expression of multiple forms of noncoding RNAs that contribute to growth regulation without expression of immunogenic proteins would be an important mechanism for viral oncogenesis in the presence of a functional immune system. IMPORTANCEInfection with Epstein-Barr virus (EBV) is nearly ubiquitous in the human population; however, it does contribute to the formation of multiple types of cancer. In immunocompromised patients, EBV causes multiple types of lymphomas by expressing viral oncogenes that promote growth and survival of infected B lymphocytes. EBV-positive gastric carcinoma does not require immune suppression, and the viral oncoproteins that are frequent targets for an immunological response are not expressed. This study demonstrates using transcriptional analysis that the expression of various classes of viral non-protein-coding RNAs likely contribute to the considerable changes in the host transcriptional profile in the AGS gastric cancer cell line. This is the first report to show that the highly expressed polyadenylated BamHI A rightward transcripts (BART) viral transcript in gastric carcinoma is in fact a functional viral long noncoding RNA. These studies provide new insight into how EBV can promote transformation in the absence of viral protein expression. L atent infection with Epstein-Barr virus (EBV), a nearly ubiquitous human herpesvirus, is responsible for multiple malignancies in the two types of cells that it infects: B lymphocytes and epithelial cells (1). The mechanism by which EBV leads to transformation dep...

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“…So far, EBV-encoded miRNAs have been shown to play a role in immune evasion (Albanese et al, 2016; Tagawa et al, 2016; Xia et al, 2008), apoptosis (Kang et al, 2015; Lei et al, 2013), and inhibition of tumor suppressors (Bernhardt et al, 2016). Additionally, inactivation of the miR-BHRF1 miRNA cluster results in a reproducible and robust reduction in B cell transformation and LCL growth (Feederle et al, 2011a, 2011b; Haar et al, 2015; Seto et al, 2010), which suggested that the miR-BHRF1 miRNA cluster was promoting EBV transformation by downregulating the expression in trans of cellular mRNAs that inhibit this process.…”

Section: Introductionmentioning

confidence: 99%

The Epstein-Barr virus miR-BHRF1 microRNAs regulate viral gene expression in cis

et al. 2017

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The Epstein-Barr virus (EBV) miR-BHRF1 microRNA (miRNA) cluster has been shown to facilitate B-cell transformation and promote the rapid growth of the resultant lymphoblastoid cell lines (LCLs). However, we find that expression of physiological levels of the miR-BHRF1 miRNAs in LCLs transformed with a miR-BHRF1 null mutant (Δ123) fails to increase their growth rate. We demonstrate that the pri-miR-BHRF1-2 and 1-3 stem-loops are present in the 3′UTR of transcripts encoding EBNA-LP and that excision of pre-miR-BHRF1-2 and 1-3 by Drosha destabilizes these mRNAs and reduces expression of the encoded protein. Therefore, mutational inactivation of pri-miR-BHRF1-2 and 1-3 in the Δ123 mutant upregulates the expression of not only EBNA-LP but also EBNA-LP-regulated mRNAs and proteins, including LMP1. We hypothesize that this overexpression causes the reduced transformation capacity of the Δ123 EBV mutant. Thus, in addition to regulating cellular mRNAs in trans, miR-BHRF1-2 and 1-3 also regulate EBNA-LP mRNA expression in cis.

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EBV BART MicroRNAs Target Multiple Pro-apoptotic Cellular Genes to Promote Epithelial Cell Survival

Cited by 100 publications

References 46 publications

Epstein-Barr viral microRNAs target caspase 3

Epstein-Barr viral microRNAs target caspase 3

Host Gene Expression Is Regulated by Two Types of Noncoding RNAs Transcribed from the Epstein-Barr Virus BamHI A Rightward Transcript Region

The Epstein-Barr virus miR-BHRF1 microRNAs regulate viral gene expression in cis

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