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

Abstract

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...

Epstein‐Barr virus mRNA profiles and viral DNA methylation status in nasopharyngeal brushings from nasopharyngeal carcinoma patients reflect tumor origin

“…Therefore, we conclude that BARF1 mRNA mainly is a latency‐associated gene product in NPC and forms a suitable NPC marker, as suggested before . The high and stable expression of BARTs in NPC biopsies and brushings is consistent with prior data and classifies the BART transcript as EBV housekeeping gene, suitable for internal EBV‐specific normalization of RNA profiles . Relative to BART RNA levels, EBV latency‐II transcripts prevail over immediate early and early lytic transcripts (Fig.…”

Involvement and Roles of Long Noncoding RNAs in the Molecular Mechanisms of Emerging and Reemerging Viral Infections

“…As described by Marquitz et al, the BamHI A rightward transcripts (BARTs) are alternatively spliced and give rise to large wide of viral polyadenylated RNAs in EpsteinÀBarr virusÀrelated epithelial cancers. The BART introns are the origin of 44 miRNAs while the exons are spliced and polyadenylated to form nonprotein-coding RNAs with nuclear localization that regulate infection in a similar mechanism of lncRNAs (Marquitz et al, 2015). In addition, two other nuclear lncRNAs, oriPtLs and oriPtRs, are transcribed during EBV reactivation and contribute to the viral gene expression and evasion of the host immune response (Cao et al, 2015).…”

Long noncoding RNAs: Novel regulators of virus‐host interactions

“…In addition, a 3.91 kb lncRNA M3‐04 generated by gammaherpesviruses regulates viral replication in mice by interacting with antisense miRNAs and the latency gene M2 . Other herpesviruses also encode multiple functional lncRNAs, such as the Epstein‐Barr virus encoding ncRNA, BamH I‐A rightward transcripts, Kaposi's sarcoma‐associated herpesvirus (KSHV) encoding UCA1 , and herpesvirus saimiri encoding U‐rich RNAs . Additionally, the 3′‐untranslated region (UTR) of the flavivirus RNA genome is capable of transcribing an active lncRNA, termed the subgenomic flavivirus RNA (sfRNA), which protects viral RNAs from degradation by the host nuclease Xrn1 and suppresses antiviral RNA interference (RNAi) in infected human cells in culture and, also, interestingly, in mosquitoes by direct interaction with the RNAi machinery .…”