In Vivo Dynamic Expression Profiles Of MDV MiRNAs And Their Self-regulatory Founctions On The Viral Protein-coding Genes

Marek’s disease virus serotype 1(MDV-1) is an oncogenic α-herpesvirus that induces Marek’s disease characterized by fatal T cells lymphomas in chickens. Although MD could be controlled by vaccination, wild-type isolates of MDV-1 have tended to increase in virulence and cause MD even in vaccinated chickens. MDV-1 cause serious economic losses to poultry industry. As a typically oncogenic virus, MDV-1 represents an ideal model for investigating the biology, genetics, and immunology of herpesvirus-induced tumors. Micro RNAs(mi RNAs) are new regulators of gene expression, which play important regulation roles at post-transcriptional level in most biological processes.In MDV-1 genomes, a total of 26 mi RNAs are encoded in 14 precursors and concentrated in three gene clusters, namely the Meq-cluster, the Mid-cluster and LAT-cluster. Up to now, only a few of the MDV-1 mi RNAs, such as mi R-M4-5p, mi R-M3-5p and mi R-M7-5p, have been shown to play critical roles in MDV replication, latency, pathogenesis and/or oncogenes. However, for most of the viral mi RNAs, their potential regulatory role in MDV biology remains unknown.Firstly, the study systematically analyzed in vivo dynamic expression profiles of MDV-1 mi RNAs during the virus life cycle and development of Marek’s disease lymphomas. And then further research on self-regulation of the MDV-1 mi RNAs on viral genes were carried out. The experiments and results of the study were as follows:1 Firstly, quantitative real-time RT-PCR(q RT-PCR) methods detecting 4 viral protein-coding genes including g B, ICP4, pp38 and meq and all of the 26 MDV mi RNAs were developed. And then dynamic gene expression in chickens infected with very virulent MDV strain GX0101 was analyzed at 3 dpi, 7 dpi, 14 dpi, 21 dpi, 30 dpi, 45 dpi and 60 dpi, respectively. The results showed that the first peak of g B, ICP4, pp38 and meq expression levels appered at the early cytolytic phase(7 dpi), decreased at the latent phase(14 dpi), and then peaked at the late cytolytic phase(21 dpi). At the transformation and proliferative phase(the late time period of 30-60 dpi), the gene expression level of g B, ICP4, and pp38 were gradually reduced, but meq remained expressed at a higher level, which is in accord with the subsequent pathogensis of MD described based on the‘Cornell Model’, which indicated that animal experiments GX0101 infected chickens achieved success.However, distinct from the viral protein-coding genes, the MDV-1 26 mi RNAs could be divided into three groups with different temporal expression profiles during the progress of the disease. 12 viral mi RNAs such as mi R-M4-5p, mi R-M2-5p, mi R-M2-3p in the first group increased after infection and reached a first peak at 14–21 dpi, decreased by 30 dpi, and were then highly expressed again at 45–60 dpi.These mi RNAs displayed an expression pattern that may be closely correlated to the latent, late cytolytic, and/or the proliferative phases in MD pathogenesis.The other 12 MDV-1 mi RNAs such as mi R-M4-3p, mi R-M7-5p and mi R-M11-3p showing a different expression pattern constitute the second group.Most of these mi RNAs were expressed at higher levels during the first 1–2 weeks post-challenge with subsequent low levels of expression, although some display a slight increase at 45 or 60 dpi. Based on these results, it seems that the mi RNAs in group II are more closely correlated to the early cytolytic and/or the latent phases in MDV’s life cycle. Two other mi RNAs in group III display no identical expression features.2 we have presently predicted and analyzed the MDV genome using bioinformatics software and found 357 binding sites of the viral mi RNAs in 75 protein-coding genes of MDV. The functions of candidate target genes are involved in a lot of aspects including gene regulation, transformation, apoptosis, control of cell cycle, immune regulation, virus replication and transmission, DNA processing and repair. According to founctions of related viral genes and in vivo dynamic expression profiles of MDV-1 mi RNAs, we gave preference to 10 candidate target genes including 30 binding sites associated with protein phosphorylation, DNA replication, gene regulation and transformation, constructed 11 expression vectors of mi RNA precursor and 26 recombinant psi CHECKTM-2 of candidate targets, and then cotransfectied 293 T cells. Finally, interactions between candidate target and mi RNA were analyzed by Dual Luciferase Reporter Assay(DLRA) and the results showed that 5 pairs of target and mi RNA(MDV025 and mi R-M4-5p, meq and mi R-M5-3p, meq and mi R-M7-5p, meq and mi R-M11-5p, meq and mi R-M12-5p) were found that mi RNA could interact with 3’-UTR of their corresponding target. Further analyzing by q RT-PCR proved that the oncogene meq of MDV-1 may be self-regulated by viral mi R-M5-3p, mi R-M7-5p, mi R-M11-5p and mi R-M12-3p.As mentioned above, in vivo dynamic expression profiles of MDV-1 mi RNAs during pathogenesis and development of lymphomas were systematically uncoverd in the study, which may provide important clues in the evaluation and search for further potential functions of viral mi RNAs in MDV-1 biology. Simultaneously, it is confirmed that mi R-M5-3p, mi R-M7-5p, mi R-M11-5p and mi R-M12-3p could regulate the most important oncogene meq in MDV, which indicated that the viral mi RNA may play important roles during contributing to MDV oncogenesis. It layed an important foundation for uncovering the molecular mechanisms on MDV-1 infection and tumorigenesis mediated by mi R-M5-3p, mi R-M7-5p, mi R-M11-5p and mi R-M12-3p that trigger the development of MD lymphoma need further studies.