Marek's disease virus serotype 1 (MDV-1) is an oncogenic alphaherpesvirus causing fatal T-cell lymphoma in chickens. MDV latency is characterized by the production of latency-associated transcripts (LATs), a family of non-protein-coding spliced RNAs. A cluster of four microRNAs (cluster mdv1-miR-M8-M10) was identified, but not formally mapped, at the predicted LAT 59 end. We established a LAT cDNA library from latently MDV-infected cell line MSB-1. We identified 22 highly variable LATs, which were due to the extensive alternative splicing of a total of 14 introns. RACE PCR confirmed the predicted 39 end and allowed identification of the 59 end, 400 nt upstream of the previously predicted LAT end. The LATs share their transcription start site with the microRNA-expressing transcript described previously, localizing the microRNAs to the first LAT intron and identifying the LATs as the primary transcripts of the microRNAs. We identified MDV immediate-early (IE) genes ICP4 and ICP27 as putative targets of mdv1-miR-M7-5p, the third microRNA of the cluster mdv1-miR-M8-M10. Endogenously expressed mdv1-miR-M7-5p in MSB-1 cells reduced luciferase activity significantly when microRNA-responsive elements from ICP4 or ICP27 were cloned in the 39 UTR of the firefly luciferase gene. ICP27 protein levels were decreased by 70 % when the mdv1-miR-M7-5p precursor was co-expressed with an ICP27 expression plasmid. Additionally, we showed a negative correlation between the decreased expression of mdv1-miR-M7-5p and an increase in ICP27 expression during virus reactivation. Our results suggest that, by targeting two IE genes, MDV microRNAs produced from LAT transcripts may contribute to establish and/or maintain latency.
INTRODUCTIONMarek's disease virus serotype 1 (MDV-1) is an alphaherpesvirus responsible for T-cell lymphoma in chickens. Its genome organization is similar to that of mardiviruses and simplexviruses: two unique sequences, unique long (U L ) and unique short (U S ), encoding the core genes, are enclosed by the inverted repeat sequences repeat short (R S ) and repeat long (R L ) (Tulman et al., 2000) (Fig. 1a). As in all other herpesviruses, the life cycle of MDV is divided into a lytic and a latent phase, which is associated with oncogenesis in MDV (Schat & Nair, 2008). The first genes expressed during herpesvirus lytic infection are the immediate-early (IE) genes, which control viral gene transcription, triggering a cascade of viral gene expression. In MDV, three genes homologous to the IE genes of other alphaherpesviruses have been identified: ICP4 (located in the R S region), ICP22 and ICP27 (UL54). MDV ICP4 and ICP22 have been described as transactivators (Kato et al., 2002;Pratt et al., 1994), whereas MDV ICP27 has been shown to play a role in post-transcriptional gene regulation, by inhibiting both viral and host-cell splicing (Amor et al., 2011), as also reported for other herpesvirus ICP27 homologues (Smith et al., 2005).As in other herpesviruses, the latency of MDV is characterized by the overexpression of a...