Marek's Disease Virus Virulence Genes Encode Circular RNAs.

Circular RNAs (circRNAs) are a recently rediscovered class of functional noncoding RNAs that are involved in gene regulation and cancer development. Next-generation sequencing approaches identified circRNA fragments and sequences underlying circularization events in virus-induced cancers. In the present study, we performed viral circRNA expression analysis and full-length sequencing in infections with Marek's disease virus (MDV), which serves as a model for herpesvirus-induced tumorigenesis. We established inverse PCRs to identify and characterize circRNA expression from the repeat regions of the MDV genome during viral replication, latency, and reactivation. We identified a large variety of viral circRNAs through precise mapping of full-length circular transcripts and detected matching sequences with several viral genes. Hot spots of circRNA expression included the transcriptional unit of the major viral oncogene encoding the Meq protein and the latency-associated transcripts (LATs). Moreover, we performed genome-wide bioinformatic analyses to extract back-splice junctions from lymphoma-derived samples. Using this strategy, we found that circRNAs were abundantly expressed in vivo from the same key virulence genes. Strikingly, the observed back-splice junctions do not follow a unique canonical pattern, compatible with the U2-dependent splicing machinery. Numerous noncanonical junctions were observed in viral circRNA sequences characterized from in vitro and in vivo infections. Given the importance of the genes involved in the transcription of these circRNAs, our study contributes to our understanding and complexity of this deadly pathogen. IMPORTANCE Circular RNAs (circRNAs) were rediscovered in recent years both in physiological and pathological contexts, such as in cancer. Viral circRNAs are encoded by at least two human herpesviruses, the Epstein Barr virus and the Kaposi's Sarcoma-associated herpesvirus, both associated with the development of lymphoma. Marek's disease virus (MDV) is a well-established animal model to study virus-induced lymphoma but circRNA expression has not been reported for MDV yet. Our study provided the first evidence of viral circRNAs that were expressed at key steps of the MDV lifecycle using genome-wide analyses of circRNAs. These circRNAs were primarily found in transcriptional units that corresponded to the major MDV virulence factors. In addition, we established a bioinformatics pipeline that offers a new tool to identify circular RNAs in other herpesviruses. This study on the circRNAs provided important insights into major MDV virulence genes and herpesviruses-mediated gene dysregulation.

Specific transcriptional and post-transcriptional regulation of the major immediate early ICP4 gene of GaHV-2 during the lytic, latent and reactivation phases.

Transcriptional and post-transcriptional mechanisms are involved in the switch between the lytic, latent and reactivation phases of the viral cycle in herpesviruses. During the productive phases, herpesvirus gene expression is characterized by a temporally regulated cascade of immediate early (IE), early (E) and late (L) genes. In alphaherpesviruses, the major product of the IE ICP4 gene is a transcriptional regulator that initiates the cascade of gene expression that is essential for viral replication. In this study, we redefine the infected cell protein 4 (ICP4) gene of the oncogenic Marek's disease virus (MDV or gallid herpesvirus 2) as a 9438 nt gene ended with four alternative poly(A) signals and controlled by two alternative promoters containing essentially ubiquitous functional response elements (GC, TATA and CCAAT boxes). The distal promoter is associated with ICP4 gene expression during the lytic and the latent phases, whereas the proximal promoter is associated with the expression of this gene during the reactivation phase. Both promoters are regulated by DNA methylation during the viral cycle and are hypermethylated during latency. Transcript analyses showed ICP4 to consist of three exons and two introns, the alternative splicing of which is associated with five predicted nested ICP4ORFs. We show that the ICP4 gene is highly and specifically regulated by transcriptional and post-transcriptional mechanisms during the three phases of the GaHV-2 viral cycle, with a clear difference in expression between the lytic phase and reactivation from latency in our model.

Alternative splicing of a viral mirtron differentially affects the expression of other microRNAs from its cluster and of the host transcript.

Interplay between alternative splicing and the Microprocessor may have differential effects on the expression of intronic miRNAs organized into clusters. We used a viral model - the LAT long non-coding RNA (LAT lncRNA) of Marek's disease oncogenic herpesvirus (MDV-1), which has the mdv1-miR-M8-M6-M7-M10 cluster embedded in its first intron - to assess the impact of splicing modifications on the biogenesis of each of the miRNAs from the cluster. Drosha silencing and alternative splicing of an extended exon 2 of the LAT lncRNA from a newly identified 3' splice site (SS) at the end of the second miRNA of the cluster showed that mdv1-miR-M6 was a 5'-tailed mirtron. We have thus identified the first 5'-tailed mirtron within a cluster of miRNAs for which alternative splicing is directly associated with differential expression of the other miRNAs of the cluster, with an increase in intronic mdv1-miR-M8 expression and a decrease in expression of the exonic mdv1-miR-M7, and indirectly associated with regulation of the host transcript. According to the alternative 3SS used for the host intron splicing, the mdv1-miR-M6 is processed as a mirtron by the spliceosome, dispatching the other miRNAs of the cluster into intron and exon, or as a canonical miRNA by the Microprocessor complex. The viral mdv1-miR-M6 mirtron is the first mirtron described that can also follow the canonical pathway.