Global Bidirectional Transcription of the Epstein-Barr Virus Genome during Reactivation

Abstract

Epstein-Barr virus (EBV) reactivation involves the ordered induction of approximately 90 viral genes that participate in the generation of infectious virions. Using strand-specific RNA-seq to assess the EBV transcriptome during reactivation, we found extensive bidirectional transcription extending across nearly the entire genome. In contrast, only 4% of the EBV genome is currently bidirectionally annotated. Most of the newly identified transcribed regions show little evidence of coding potential, supporting noncoding roles for most of these RNAs. Based on previous cellular long noncoding RNA size calculations, we estimate that there are likely hundreds more EBV genes expressed during reactivation than was previously known. Limited 5= and 3= rapid amplification of cDNA ends (RACE) experiments and findings of novel splicing events by RNA-seq suggest that the complexity of the viral genome during reactivation may be even greater. Further analysis of antisense transcripts at some of the EBV latency gene loci showed that they are "late" genes, they are nuclear, and they tend to localize in areas of the nucleus where others find newly synthesized viral genomes. This raises the possibility that these transcripts perform functions such as new genome processing, stabilization, organization, etc. The finding of a significantly more complex EBV transcriptome during reactivation changes our view of the viral production process from one that is facilitated and regulated almost entirely by previously identified viral proteins to a process that also involves the contribution of a wide array of virus encoded noncoding RNAs. IMPORTANCE Epstein-Barr virus (EBV)is a herpesvirus that infects the majority of the world's population, in rare cases causing serious disease such as lymphoma and gastric carcinoma. Using strand-specific RNA-seq, we have studied viral gene expression during EBV reactivation and have discovered hundreds more viral transcripts than were previously known. The finding of alternative splicing and the prevalence of overlapping transcripts indicate additional complexity. Most newly identified transcribed regions do not encode proteins but instead likely function as noncoding RNA molecules which could participate in regulating gene expression, gene splicing or even activities such as viral genome processing. These findings broaden the scope of what we need to consider to understand the viral manufacturing process. As more detailed studies are undertaken they will likely change the way we view this process as a whole. E pstein-Barr virus (EBV) is a human gammaherpesvirus that is prevalent in all human populations. Although infection is frequently asymptomatic, it has been linked to a number of serious diseases such as Burkitt's lymphoma, nasopharyngeal carcinoma, and posttransplant lymphoproliferative disorder (1). Like other herpesviruses, EBV can exist in both lytic and latent phases, with primary lytic infections often progressing to lifelong latent infections with generally sporadic subsequent episodes o...

Identification of the Physiological Gene Targets of the Essential Lytic Replicative Kaposi's Sarcoma-Associated Herpesvirus ORF57 Protein

“…We correlated these potential polyadenylation sites with the appearance of the RNA-seq profiles to identify sites where the read density decreased abruptly, indicating the possible presence of transcriptional termination. In addition, we searched the RNAseq data for the presence of five or more A residues which did not map to the KSHV genome, in order to identify potential polyadenylation sites, as previously described (39). We also used Northern blot analysis to map the size of each transcript in RNA isolated from cells infected with ⌬ORF57 or WT KSHV and performed 3= RACE to map the polyadenylation and cleavage sites of the mRNAs.…”

New Noncoding Lytic Transcripts Derived from the Epstein-Barr Virus Latency Origin of Replication, oriP , Are Hyperedited, Bind the Paraspeckle Protein, NONO/p54nrb, and Support Viral Lytic Transcription

“…Recent studies have found evidence for previously unknown viral long noncoding RNAs (vlncRNAs) in EBV, Kaposi's sarcoma-associated herpesvirus, MHV68, and herpes simplex virus that are expressed during the lytic cycle (4)(5)(6)(7)(8)(9). For example, evidence from our lab (9,10) and from Dresang et al (4) indicates that EBV likely expresses more than a hundred novel lytic noncoding RNAs. Further, extensive alternative splicing is observed (9,10), illustrating the existence of even greater diversity in the repertoire of expressed coding and noncoding viral transcripts during this process.…”

“…Nevertheless, diverse roles for small noncoding RNAs (ncRNAs) are known to play important roles in EBV infection and have implications to EBV-mediated oncogenesis (2,3). Recent studies have found evidence for previously unknown viral long noncoding RNAs (vlncRNAs) in EBV, Kaposi's sarcoma-associated herpesvirus, MHV68, and herpes simplex virus that are expressed during the lytic cycle (4)(5)(6)(7)(8)(9). For example, evidence from our lab (9,10) and from Dresang et al (4) indicates that EBV likely expresses more than a hundred novel lytic noncoding RNAs.…”

Analysis of EBV Transcription Using High-Throughput RNA Sequencing

“…In order to return both primary and secondary alignments, use the following commands: 19. Note that abundant unannotated transcription has been detected in EBV during reactivation [8].…”