Abstract
Our appreciation for the extent of Epstein Barr virus (EBV) transcriptome complexity continues to grow through findings of EBV encoded microRNAs, new long non-coding RNAs as well as the more recent discovery of over a hundred new polyadenylated lytic transcripts. Here we report an additional layer to the EBV transcriptome through the identification of a repertoire of latent and lytic viral circular RNAs. Utilizing RNase R-sequencing with cell models representing latency types I, II, and III, we identified EBV encoded circular RNAs expressed from the latency Cp promoter involving backsplicing from the W1 and W2 exons to the C1 exon, from the EBNA BamHI U fragment exon, and from the latency long non-coding RPMS1 locus. In addition, we identified circular RNAs expressed during reactivation including backsplicing from exon 8 to exon 2 of the LMP2 gene and a highly expressed circular RNA derived from intra-exonic backsplicing within the BHLF1 gene. While expression of most of these circular RNAs was found to depend on the EBV transcriptional program utilized and the transcription levels of the associated loci, expression of LMP2 exon 8 to exon 2 circular RNA was found to be cell model specific. Altogether we identified over 30 unique EBV circRNAs candidates and we validated and determined the structural features, expression profiles and nuclear/cytoplasmic distributions of several predominant and notable viral circRNAs. Further, we show that two of the EBV circular RNAs derived from the RPMS1 locus are detected in EBV positive clinical stomach cancer specimens. This study increases the known EBV latency and lytic transcriptome repertoires to include viral circular RNAs and it provides an essential foundation and resource for investigations into the functions and roles of this new class of EBV transcripts in EBV biology and diseases.
Highlights
Epstein Barr virus (EBV) is a human oncogenic gamma herpesvirus that is carried by greater than 90% of the world’s population
Our understanding of the extent of EBV transcriptome complexity has recently come to light with discoveries of viral microRNAs, snoRNAs, and a diverse set of transcript
The virus is transmitted to circulating naïve B-cells where a “latency type III” viral gene expression program is utilized (Latent Membrane Proteins (LMPs) -1 and -2, Epstein Barr Nuclear Antigens (EBNAs) -1, -2, -3A, -3B, -3C, and -LP and the non-coding transcripts, EBER1, EBER2, v-sisRNAs, v-snoRNA, RPMS1, and a set of viral miRNAs encoded within the introns of RPMS1) [1,2,3,4]
Summary
Epstein Barr virus (EBV) is a human oncogenic gamma herpesvirus that is carried by greater than 90% of the world’s population. The virus is transmitted to circulating naïve B-cells where a “latency type III” viral gene expression program is utilized (Latent Membrane Proteins (LMPs) -1 and -2, Epstein Barr Nuclear Antigens (EBNAs) -1, -2, -3A, -3B, -3C, and -LP and the non-coding transcripts, EBER1, EBER2, v-sisRNAs, v-snoRNA, RPMS1, and a set of viral miRNAs encoded within the introns of RPMS1) [1,2,3,4] The expression of this full repertoire of latency genes facilitates potent B-cell activation and proliferation, a unique mechanism to expand the infected B-cell population in the host (i.e. independently of new virus production and de novo infection). In immune-competent individuals the viral utilization of non-coding RNAs in addition to low level type I or type II protein coding latency gene expression, perhaps tolerated through local tumor-immune suppression, provides one or more “hits” towards oncogenic progression with minimal impact from the immune system
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