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

Abstract

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 IR 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 miRNA libraries from GaHV-2-infected cells, regardless of the phase of infection.

A phylogenomic analysis of Marek's disease virus reveals independent paths to virulence in Eurasia and North America

“…Several of the listed genes have been shown experimentally to play a role in MDV pathogenicity (Amor et al, 2011;Brown et al, 2006;Jarosinski, Osterrieder, Nair, & Schat, 2005;Kamil et al, 2005;Liu et al, 1999;Lupiani et al, 2004;Nair, 2013;Reddy et al, 2002;Tischer, Schumacher, Messerle, Wagner, & Osterrieder, 2002). While known as an important MDV oncoprotein, Meq is also expressed during lytic replication and has a potential role in the rapid production of virus progeny (Coupeau, Dambrine, & Rasschaert, 2012). Watson, & Nair, 2009), which in the context of MDV virulence evolution should not be overlooked (Zhao et al, 2011).…”

Role of Virus-Encoded microRNAs in Avian Viral Diseases

“…In contrast to the lack of sequence conservation among the different avian herpesviruses, the miRNA sequences are highly conserved among 23 different MDV-1 strains of differing virulence [37,38]. Despite this, the cluster 1 miRNAs are expressed at higher levels in lymphomas produced by vv+MDV than those produced by a vvMDV strain and one polymorphism in the promoter region of these viral miRNAs could be responsible for this differential expression, whereas the LAT-cluster miRNAs expression is equal [37,39], implying that cluster 1 miRNAs may have a more significant role in MD oncogenesis. Indeed, this hypothesis was proved by demonstration that the deletion of the cluster 1 from the viral genome greatly decreased the oncogenicity of the virus [40,41].…”

“…This miRNA was shown to play a key role in MDV-1 induced oncogenesis [40,41]. A recent report has shown that the transcription of cluster 1 and 2 miRNAs is driven by a single promoter, prmiRM9M4, corresponding to the 1300-bp immediately upstream from mdv1-miR-M9, under two distinct transcriptional models during different infection phases [39]. Indeed, this promoter has been shown to be active by both active histone marks and DNA hypomethylation during MDV-1 latency [42], confirming its transcriptional activity.…”

The significance of the individual Meq-clustered miRNAs of Marek’s disease virus in oncogenesis

“…mdv1-miR-M9, mdv1-miR-M5, mdv1-miR-M3, mdv1-miR-M12, mdv1-miR-M2 and mdv1-miR-M4, are sequentially located upstream from the meq oncogene (Burnside et al, 2006;Yao et al, 2008) in the first intron of the transcripts covering the IR L /TR L region, and its transcription is driven by a single promoter, prmiRM9M4, under two distinct transcriptional models during different infection phases (Coupeau et al, 2012). The miRNA mdv1-miR-M4, similar to the KSHV-encoded miR-K12-11 (Gottwein et al, 2007), has been characterized as the second virus-encoded functional orthologue of cellular miR-155 and specifically inhibits the translation of viral proteins involved in the cleavage/ packaging of herpesvirus DNA (Muylkens et al, 2010).…”