Putative roles as oncogene or tumour suppressor of the Mid-clustered microRNAs in Gallid alphaherpesvirus 2 (GaHV2) induced Marek’s disease lymphomagenesis

Abstract

In the last decade, numerous microRNAs (miRNAs) have been identified in diverse virus families, particularly in herpesviruses. Gallid alphaherpesvirus 2 (GaHV2) is a representative oncogenic alphaherpesvirus that induces rapid-onset T-cell lymphomas in its natural hosts, namely Marek’s disease (MD). In the GaHV2 genome there are 26 mature miRNAs derived from 14 precursors assembled into three clusters, namely the Meq-cluster, Mid-cluster and LAT-cluster. Several GaHV2 miRNAs, especially those in the Meq-cluster (e.g. miR-M4-5p), have been demonstrated to be critical in MD pathogenesis and/or tumorigenesis. Interestingly the downstream Mid-cluster is regulated and transcribed by the same promoter as the Meq-cluster in the latent phase of the infection, but the role of these Mid-clustered miRNAs in GaHV2 biology remains unclear. We have generated the deletion mutants of the Mid-cluster and of its associated individual miRNAs in GX0101 virus, a very virulent GaHV2 strain, and demonstrated that the Mid-clustered miRNAs are not essential for virus replication. Using GaHV2-infected chickens as an animal model, we found that, compared with parental GX0101 virus, the individual deletion of miR-M31 decreased the mortality and gross tumour incidence of infected chickens while the deletion individually of miR-M1 or miR-M11 unexpectedly increased viral pathogenicity or oncogenicity, similarly to the deletion of the entire Mid-cluster region. More importantly, our data further confirm that miR-M11-5p, the miR-M11-derived mature miRNA, targets the viral oncogene meq and suppresses its expression in GaHV2 infection. We report here that members of the Mid-clustered miRNAs, miR-M31-3p and miR-M11-5p, potentially act either as oncogene or tumour suppressor in MD lymphomagenesis.

Efficient Mutagenesis of Marek’s Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System

“…To further confirm that the observed changes in the virus growth kinetics, especially the improved proliferation of miR-M11-KO virus, were actually caused by the CRISPR/Cas9-mediated miRNA-deletion, we performed a compensatory overexpression of miR-M11 in CEFs in the present study. Our data have shown that the overexpression of miR-M11 successfully suppressed the enhanced growth of RB-1B∆M11 virus, strongly supporting our previous findings [28]. Whether miR-M11 directly regulates MDV genes to affect virus proliferation or through targeting host cellular genes to regulate cell proliferation and/or apoptosis deserves to be studied.…”

“…A previous study [63] has shown that the transcription of the Meq-and the mid-cluster miRNAs gives two distinct transcriptional patterns: during the latent phase of infection the two clusters are driven by the single prmiRM9M4 promoter while during the lytic phase they are transcribed separately using independent promoters, and most of the miRNAs represent two different expression profiles in the different stages of disease progression [64,65]. For the miR-M4-KO mutant, we have observed that once the miR-M4 is omitted from the viral genome, the expression levels of miR-M11 in virus-infected CEFs showed an unusual increase, which greatly strengthened our previous suggestions that the oncogenic or tumor suppressive roles for miR-M4 and miR-M11, respectively, are important to keep a balance during the latency infection and/or MD tumorigenesis [27,28]. Whether miR-M4 participates directly in the regulation of miR-M11 expression in MDV infection deserves to be studied further.…”

“…Interestingly, the in vitro viral growth kinetics of CRISPR/Cas9-edited miR-KO viruses in CEF cells demonstrated that, compared to the parental RB-1B virus, knocking out of the Meq-cluster miRNAs significantly decreased the virus replication while deletion of the mid-cluster miRNAs had an enhancing effect on the virus replication. This was very different to that of the unchanged in vitro replication curves of the similar miR-KO GX0101 mutants produced by BAC mutagenesis, but completely in accordance with their in vivo replication and pathogenicity profiles in virus-challenged birds [27,28]. To further confirm that the observed changes in the virus growth kinetics, especially the improved proliferation of miR-M11-KO virus, were actually caused by the CRISPR/Cas9-mediated miRNA-deletion, we performed a compensatory overexpression of miR-M11 in CEFs in the present study.…”

“…As for the avian herpesviruses, such as MDV-1, MDV-2, and HVT, the BAC mutagenesis has also contributed a lot to editing the viral genomes [59][60][61][62]. In previous studies, we have also produced a series of miRNA-deleted MDV-1 mutants using a GX0101 BAC clone for studying the virus-host interactions mediated by viral miRNAs [21,22,27,28]. However, some of the shortcomings of the BAC engineering approach such as the time-consuming step of cloning of large viral genomes into BAC plasmids, the low frequency of expected homologous recombination, and the insertion of BAC plasmid elements or drug selection markers into the viral genome that sometimes interferes with viral functions, have limited its use.…”

“…Several MDV-1 miRNAs, such as the viral miR-155 ortholog miR-M4-5p [18][19][20][21][22][23][24], miR-M3-5p [25], and miR-M7-5p [26], have been demonstrated to play key roles in MDV life cycle to regulate herpesvirus DNA cleavage/packaging, latency or especially virally-induced tumorigenesis. While it is known that most of the viral miRNA functions are related to the pathogenicity and/or oncogenicity [27,28], underlying molecular regulatory mechanisms mediated by these tiny RNAs in MDV biology still remain unclear.…”