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.

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 0 splice site (SS) at the end of the second miRNA of the cluster showed that mdv1-miR-M6 was a 5 0 -tailed mirtron. We have thus identified the first 5 0 -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.

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.

Herpesviruses encode a unique serine protease essential for viral capsid maturation. This protease undergoes autoprocessing at two sites, R and M, at the consensus sequence (V, L, I) P3 -X P2 -A P1 /S P1 (where X is a polar amino acid). We observed complete autoprocessing at the R and M sites of Marek's disease virus (MDV) protease following production of the polyprotein in Escherichia coli. Site-directed mutagenesis confirmed the predicted sequence of the R and M sites, with the M site sequence being nonconsensual: M P3 -N P2 -A P1 /S P1 . Mutagenesis and expression kinetics studies suggested that the atypical MDV M site was cleaved exclusively by the processed short protease, a feature making MDV unique among herpesviruses.