Peste Des Petits Ruminants Virus-induced MiR-3 Inhibits Type I Interferon Response By Targeting IRAK1

Peste des petits ruminants virus(PPRV),the etiological agent of peste des petits ruminants(PPR),causes an acute or subacute disease in small ruminants,like goat and sheep.PPRV has a strong lymphophilicity,and the main cause of PPRV's pathogenesis is the immunosuppression caused by host infection.As a key factor of the TLR signaling pathway and interferon signaling pathway,Interleukin-1 receptor-associated kinase 1(IRAK1)participates in the hosts' antiviral response.Micro RNAs are a family of small,non-coding RNAs,ranging in length from 18 to 25 nucleotides,which play an important role in innate immune response against viral infection.However,it is unclear what role miRNA play in the immune suppression and pathogenesis of PPRV infection.In this study,goat peripheral blood mononuclear cells(PBMCs)were infected with PPRV.According to the obtained miRNA microarray results,miR-3,which is closely related to the type I interferon pathway.And further explored the role and mechanism of miR-3 in immune suppression caused by PPRV infection.the research contents and results of this project as follow:1: Cells were collected from goat PBMCs infected with different(MOI=0.1,1,10)of PPRV and PPRV(MOI=1)at different time.The kinetic induction of mature miR-3 following PPRV infection indicated that miR-3 was a PPRV infection responsive gene in goat PBMCs,and its induction was accompanied with increased virus replication in a dose-dependent and time-dependent manner.Transfection with plasmids containing the N,H,M,F,C,and V genes of the PPRV into PBMCs,respectively,q RT-PCR assay showed that V protein of PPRV significantly upregulated miR-3 expression.To further determine which transcription factors is responsible for the induction of miR-3,goat PBMCs were treated with inhibitors of the key signal molecules for 1 h and then were infected with PPRV(MOI=1).The q RT-PCR assay showed that PPRV infected goat PBMCs increase miR-3 expression mainly Through NF-?B and P38 pathway.2.To test whether miR-3 can affect PPRV replication,PPRV infection assay were performed in goat PBMCs pre-transfected with miR-3 mimics or miR-3 inhibitor with different concentrations.PPRV infection was examined by Western blot analysis and TCID50 assays,it was proved that miR-3 can facilitate PPRV replication in a dose-dependent manner.3.To explore the mechanism of miR-3 on the regulation of PPRV replication level.According to the Target Scan algorithm,we identified one predict miR-3 targets and found putative miR-3 binding region in the 3'UTRs of goat IRAK1.Double luciferase reporter experiments showed that miR-3 can directly target the 3'UTR of the IRAK1 gene.Furthermore,transfection of miR-3 mimic/inhibitor and its control at different doses,respectively,confirmed that miR-3 negatively regulates IRAK1 expression at the m RNA and protein levels in a dose-dependent manner.Further study showed that,miR-3 mainly inhibits IRAK1-NF-?B pathway-mediated production of IFN-? by targeting IRAK1.4.Transfection of miR-3 inhibitor and its control into goat PBMCs,and the effects of different transfection time on PPRV replication and expression of IFN-? and ISGs were studied.It was proved that miR-3 inhibitor transfection significantly increased IFN-? and ISGs expression compared to IC group at indicated time point,while the level of virus replication and progeny were up downregulated.At the same time,knocking down the expression of IRAK1 rescue the decreased virus replication.In order to further study the effect of IFN-? on PPRV replication,Then,we examined the antiviral effects of IFN-? treatment on virus replication in PPRV infected cells pre-transfected with miR-3 mimic.The results showed that miR-3 mimic transfection enhanced virus replication and progeny,while the enhancement of virus replication by miR-3 was weakened by IFN-? treatment.These results indicate that miR-3 induced upon PPRV infection mainly promotes virus replication by inhibiting the expression of IFN-?.In addition,it is found that PPRV infected PBMCs can induce ISGs in an IRF3-dependent manner.5.To clarify the difference of miR-3 expressed in PBMCs between goats and sheep infected with PPRV,we analyzed the kinetic levels of viral replication,the endogenous expression of miR-3 and IFN-? in goat and sheep PBMCs after infected with PPRV(MOI=1).Compared with sheep,PPRV infected goat PBMCs had higher level of virus titer and miR-3 expression and lower IFN-? expression levels.An inverse correlation was observed between miR-3 expression and IFN-? expression both in goat and sheep PBMCs.Collectively,our findings revealed that PPRV infected PBMCs significantly induce miR-3 expression in NF-?B and p38 dependent manner,which targets IRAK1 to impaire IFN-? production and enhance PPRV replication.