Kinetic expression analysis of the cluster mdv1-mir-M9-M4, genes meq and viL-8 differs between the lytic and latent phases of marek's disease virus infection

Marek's disease virus (GaHV-2) is an alphaherpesvirus that induces T-cell lymphoma in chickens. The infection includes both lytic and latent stages. GaHV-2 encodes three clusters of microRNAs (miRNAs) located in the internal (I)/terminal (T) repeat (R) regions. We characterized transcripts encompassing the mdv1-mir-M9-M4 and mir-M11-M1 clusters located in the _L/TR _L region, upstream and downstream from the meq oncogene, respectively. By 59- and 39-RACE-PCR and targeted RT-PCR, we showed that mdv1-mir-M9-M4 could be transcribed from an unspliced transcript or from at least 15 alternatively spliced transcripts covering the IR _L/TR _L region, encompassing the meq and vIL-8 genes and localizing the mdv1-mir-M9-M4 cluster to the first intron at the 59-end. However, all these transcripts, whether spliced or unspliced, seemed to start at the same transcriptional start site, their transcription being driven by a single promoter, prmiRM9M4. We demonstrated alternative promoter usage for the meq and vIL-8 genes, depending on the phase of GaHV-2 infection. During the latent phase, the prmiRM9M4 promoter drove transcription of the meq and vIL-8 genes and the mdv1-mir-M9-M4 cluster in the first intron of the corresponding transcripts. By contrast, during the lytic phase, this promoter drove the transcription only of the mdv1-mir-M9-M4 cluster to generate unspliced mRNA, the meq and vIL-8 genes being transcribed principally from their own promoters. Despite the expression of meq and the mdv1-mir- M9-M4 cluster under two different transcriptional processes during the latent and lytic phases, our data provide an explanation for meq expression and mdv1-mir-M4-5P overexpression in miRNAlibraries from GaHV-2-infected cells, regardless of the phase of infection.