Duck Plague Virus Encoded MicroRNA Dev-miR-D28-3p Inhibits Virus Proliferation By Targeting UL30

Duck plague virus(DPV),also known as Duck enteritis virus(DEV),is a herpesvirus that causes high morbidity and mortality in waterfowl.Previous research of our group showed that the DPV CHv strain encoded eight unique miRNAs compared to DPV VAC strain.In this paper,we first predicted the targets of these 8 unique miRNAs,and then selected dev-miR-D28-3p as the target to investigate the mechanism of DPV proliferation.The results are as follows:1.Construction of a regulatory network of the 8 miRNAs targeting DPV itselfBy bioinformatics software,the targets of eight miRNAs targeted by DPV CHv strain were predicted.The results showed that eight miRNAs might potentially regulate 26 gene transcripts,including the immediate early gene ICP4,early gene UL30,and late gene UL27.Among them,dev-miR-D28-3p might target UL24,UL27 and UL30.2.Study on the mechanism of dev-miR-D28-3p targeting UL30 to inhibit viral proliferationThe effects of dev-miR-D28-3p on DPV proliferation were detected by q RT-PCR,Western blot and TCID50.The results showed that,after overexpressing dev-miR-D28-3p,the viral copy numbers,virus titers and viral protein expressions were all inhibited(P<0.05)in DEF.However,the opposite results were obtained(P<0.05)after knockdowning dev-miR-D28-3p.These results indicated that: dev-miR-D28-3p inhibited DPV proliferation.It was further verified by dual luciferase reporter gene assay,q RT-PCR,and Western blot that dev-miR-D28-3p could bind to UL24,UL27,and UL30,but only suppressed the m RNA transcript levels and protein expression levels of UL27 and UL30(P<0.05),indicating that UL27 and UL30 were the dev-miR-D28-3p target sites.Subsequently,the effects of target UL27 and UL30 on DPV proliferation were detected by q RT-PCR,Western blot,and TCID50.The results showed that the viral copy numbers,virus titers and viral protein expressions were increased after overexpression of UL27 and UL30 in DEF(P<0.05),while the opposite results were obtained after knockdown(P<0.01),indicating that dev-miR-D28-3p could inhibit DPV proliferation by suppressing UL27 or UL30.Finally,the effects of dev-miR-D28-3p on the expression of UL30-UL42 complex were investigated.The ability of UL30 to form a heterodimeric complex with UL42 was confirmed by IFA,Co-IP,and then after adding gradient concentrations of UL30-UL42 complex to the DNA polymerase activity reaction system,it was found that the substrate of30 nt size would gradient decrease to constant,while the product of 50 nt size would increase from no gradient to constant,indicating that the UL30-UL42 complex was DNA polymerase.Co-IP assay revealed that overexpression of dev-miR-D28-3p decreased the protein expression of both U30 and UL42 in the IP group,indicating that overexpression of dev-miR-D28-3p inhibited the expression of the UL30-UL42 complex(viral DNA polymerase).In summary,our study found that dev-miR-D28-3p,encoded by the DPV CHv strain,inhibited the expression of the UL30-UL42 complex(viral DNA polymerase)by targeting and inhibiting UL30,which in turn inhibited DPV proliferation.