The Regulation Of Bovine Herpesvirus5Encoded MiR-10on Its Target Gene Us3

Bovine herpesvirus5(Bovine herpesvirus5, BoHV5) belongs to subfamily of alphaherpesvirus, which has the characteristics of neurotropic nature and latent infection.BoHV-5could enter the Central Nervous System(CNS), causing severe fatal calf meningoencephalitis and great economic losses of cattle industry. Our previous reports indicated that BoHV-5had11miRNA genes which encode16mature miRNA in epithelial cells. Among the11miRNAs of BoHV5, miR-10is most abundant, and the pre-miRNAs encoded two different mature miRNAs, of which one is on the5’end and another on the3’end. But its biological function is not known.In this study we found several potential target genes regulated by miR-10through bioinformatic analysis. Luciferase assay report system was employed to validate the putative targets. We found that miR-10could target Us3and UL39genes. And the further validated regulation of Us3was proved in transcription and translation levels. The main work contains:1. The prediction and validation of miR-10target genesThrough bioinformatics prediction we got multiple potential target genes of miR-10. And UL23、BICP22、UL28、UL37、UL39and Us3were chosed through the analysis and comparison.The3’UTRs of these target genes were cloned and connected to the reported plasmid psiCHECK-2. The dual luciferase report system was employed to validate the putative targets. The results showed that miR-10could target on UL39and Us3genes, miR-10-5p could target on Us3gene and miR-10-3p could target on UL39gene.Meanwhile, miR-10-5p mutanted its seed sequence, failing to up-regulate the target genes.2. The regulation of miR-10-5p on Us3On the basis of the above work, we chosed the Us3gene which play an important role in the interaction between virus and host for the further research.Real-time PCR and Western Blot demonstrated miR-10-5p could regulate the US3. The results showed the regulation of Us3mainly occurred in the translation phase of protein expression and could upregulated the expression of this protein.3. The influence of overexpreed miR-10-5p on virus propagationIn order to verify the effect of miR-10-5p target on Us3gene on virus propagation. The miR-10-5p was transfected into MDBK cells, changed the medium4hours later and incubrated with5MOI BoHV-5wild virus. We chosed several different times to poison and then measured the virus titer. The study indicated that the virus titer had no different. It showed that over expression of miR-10-5p had little influence on the replication and proliferation of virus.4. The influence of overexpressed miR-10-5p on epithelial cells apoptosisAs Us3gene play an important role in the process of virus induced apoptosis.The miR-10-5p was transfected into MDBK cells, and then inoculated with BoHV-5wide virus. The sample was collected and extractied total protein, detecting the expression of caspase-3. Experimental results showed that the overexpression of miR-10-5p could improve the expression of caspase-3, Indicating that miR-10-5p could promote virus-induced apoptosis.The flow cytometry was aslo used to detect cell apoptosis. The results showed that the apoptotic of overexpression miR-10-5p cells was improved. It indiciated that miR-10-5p overexpression did promote apoptosis virus-induced in consistent with detection of apoptotic protein caspase-3results.5The influence of overexpressed miR-10-5p on nerve cells apoptosisAs BoHV-5has the characteristics of neurotropic nature, The miR-10-5p was transfected into N2a cells, and then inoculated with BoHV-5wide virus. The sample was collected and extracted total protein to detect the expression of caspase-3. Experimental results show that overexpression miR-10-5p could improve the expression of caspase-3. Indicated that overexpression of miR-10-5p could promote virus-induced apoptosis.The flow cytometry assey also demonstrated that the apoptotic cells of overexpression miR-10-5p was improved. It indiciated that miR-10-5p overexpression did promote virus-induced nerve cells apoptosis.In this study, dual luciferase experiment, Real-time PCR, western-blot, viral proliferation and apoptosis detection were performed to verify the reaction of miR-10and its target gene Us3.Andit will provides referencefor further revealed BoHV-5nerve pathogenic Andit also provides important material for the pathogenic difference with BoHV-1and BoHV-5,at the same time provides reference for developing better vaccines and drugs.