Modulation Of MiR-122on Persistently Borna Disease Virus Infected Human Oligodendroglial Cells

Objective: MicroRNAs (miRNAs) are a class of non-coding RNAs with19-24nucleotides, which play important roles in the cleavage and regulating the translationof target genes depending on the extent of matching of complementary sequenceswith the target mRNAs, and implicate in a number of biological processes includingthe host-virus interactions. The abundantly expressed liver-specific miR-122,promotes replication of HCV in cultured human hepatoma Huh7cells stablyexpressing a HCV replicon. To investigate the mechanism of miR-122modulate theantiviral effect in virus infection and host innate immunity, the relationship betweenmiR-122and Borna disease virus (BDV) was studied.Methods: miRNA array in OL and OL/BDV cells was used to ascertain themiRNA which was the target we studied and the level of the miRNA (miR-122).Then we compared the expression level of miR-122in hepatoma Huh7and HepG2,glioma cells of C6and OL by real-time RT-PCR. We next examined the effects ofBDV and the proteins of BDV P and BDV N on the expression of miR-122. Wepredictly examined the most likely binding domains of miR-122with BDV usingRNAhybrid software. BDV N or P gene transfected OL cells were first used to checkthe inhibitory effects of miR-122on BDV translation by western blot. The effects ofmiR-122on BDV protein were further demonstrated in OL/BDV cells treated bymiR-122and AMO-122. On the other hand, to determine whether changes inmiR-122expression have any effect on BDV replication and viral RNA stability,real-time RT-PCR was used to detect the changes of nucleic acids of BDV aftertransfection of miR-122plasmid and AMO-122. To examine further that in addition to its direct antiviral effect, whether miR-122plays any role in innate immunity ofcells against viral infection, we first examined the expression of IFN-alpha andIFN-beta in OL and OL/BDV cells, followed by transfection of miR-122plasmidand treatment of AMO-122to these cells. Meanwhile, to investigate the involvementof miR-122in the IFN induction, the induction abilities of Coxsackievirus B (CVB),polyI:C and exogenous IFN on IFN-alpha and IFN-beta secretion/synthesis werefully detected by AMO-122treatment.Results: The result of miRNA array showed that the significant difference ofmiR-122was found between OL and OL/BDV cells and miR-122is expressed in amoderate level in human oligodendroglial cells (OL), which is much higher than somespecific miRNA, such as miR-124, having a biological effect on brain tissue. Inaddition, real-time RT-PCR showed the expression level of miR-122was lowercompared to Huh7and higher than HepG2, but with a much lower level in BDVpersistent infection (OL/BDV) and cells transfected with BDV gene expressionvectors.Using RNAhybrid software we investigated the antiviral effect of miR-122,on Borna Disease Virus (BDV), and found that the predicted binding sites ofcomplementary sequences between miR-122and the viral mRNAs were of crucialimportance to its functions. Over-expression of miR-122and specific blockingexperiments demonstrated that miR-122was able to specifically inhibit BDV proteinsynthesis, viral gene replication and transcription, and induce the secretion/synthesisof interferon (IFN) in OL and OL/BDV cells. The abolishment of miR-122byAMO-122inhibited the endogenous IFN induction by CVB, polyI:C and exogenousIFN.Conclusion: In summary, our results demonstrated that miR-122might serve asan important endogenous gene regulatory factor, which showed both direct inhibitionof virus replication and translation, and indirect activation of the host innate immunityto exert its antiviral effect. With the result of BDV inhibiting the miR-122expression, the study further suggests that miR-122may plays an important role in the interactionbetween the virus and host cells, what may be involved in the establishment ofpersistent infection of BDV in OL cells.