Analyses of non-coding RNAs generated from the Epstein-Barr virus W repeat region

Introduction: The Epstein–Barr virus (EBV) W repeats are transcribed during viral lytic reactivation, a highly oncogenic form of latency known as latency III, and a rare type of latency (Wp-restricted) that is observed in ~15% of endemic Burkitt’s lymphomas. The W repeats encode an EBV protein, EBNA-LP, whose message is produced by splicing out a short (81 nt) and long (2791 nt) intron. We previously discovered a stable intronic sequence (sis)RNA (ebv-sisRNA-1), which is the third most abundant EBV small RNA generated in latency III and comprises all 81 nt of the short intron. We also previously found that regions within the long intron form stable and conserved RNA structures, including a massive (586 nt) hairpin loop. Methods: To identify RNAs that potentially interact with ebv-sisRNA-1, in-silico bimolecular RNA folding and sequence comparison were performed, examining all human and EBV micro (mi)RNAs from the miRBase database. RNA-Seq data from human B cells expressing a latency III program were re-analyzed to identify novel transcripts corresponding to the W repeats. RNA structure modeling was performed using a fold-and-align strategy coupled to manual comparative sequence analysis. Results: ebv-sisRNA-1 is not likely to form conserved stable hybrids with EBV miRNAs. It may, however, interact with several human miRNAs. Analysis of RNASeq data indicates that the entire long W repeat intron is also likely a sisRNA (ebvsisRNA-2). Stable RNA structure, rather than being localized to particular regions, is likely to span the entire ebv-sisRNA-2 sequence. We propose a model for this conserved fold that is supported by consistent and compensatory (structure-preserving) mutations. Conclusions: The EBV W repeats generate two structured latent sisRNAs. EbvsisRNA-1 is predicted to interact with host miRNAs. Ebv-sisRNA-2 is modeled to have wide-spread and exceptionally thermodynamically stable RNA structure that is evolutionarily conserved in related herpesviruses. These results suggest important functions for both sisRNAs in type III latency.