Screening And Functional Identification Of Host MicroRNA Targeting PRRSV Receptor CD163

Porcine reproductive and respiratory syndrome(PRRS)is a viral infectious disease which caused by porcine reproductive and respiratory syndrome virus(PRRSV).PRRSV causes reproductive problems in sows and respiratory distress in growing pigs.PRRSV targets cells of the monocyte or macrophage lineage,causing severe cell death,immunosuppression and persistent infections.It can inhibit or escape the innate immune system of the host.However,the commercial PRRSV vaccine developed in the market cannot provide effective protection for pigs.Therefore,to develop the antiviral strategies that can apply to different PRRSV strains and provide broad protection for pigs has been a major challenge for PRRSV prevention and control.MicroRNA(miRNA)is a class of single-strand non-coding RNA with length of 18-23 nt.It is an important regulatory factor,which play roles on the expression of protein-coding gene in transcriptional or post-transcriptional levels.Many studies have confirmed that miRNA plays an important role in the interaction between PRRSV and the host during the process of PRRSV infection.Previous studies demonstrated that CD163 is the critical receptor in the process of PRRSV infection.In this study,bioinformatics was used to explore the role and mechanism of microRNAs which can regulate the expression of CD163 in the process of PRRSV infection,which will provide effective strategies for the prevention and control of PRRSV.The research results were as following:1.Prediction and identification of the miRNAs which regulate the CD163 geneFirstly,the 3’UTR sequences of CD163 mRNA were downloaded from NCBI database.Four mature microRNAs: miR-124 a,miR-143-3p,miR-338 and miR-450b-3p,which can target CD163 3’UTR were predicted by bioinformatics software(UCSC,miRbase,RNA22 V2,RNAhybrid).Then,the 3’UTR of monkey and pig CD163 were cloned to the reporter vector with double luciferase recognition sites of psiCHECK-2.Using the double Luciferase Report System,results showed that miR-124 a could bind to the 3’UTR of CD163,the luciferase activity was effectively inhibited(up to 61%)when co-transfected with miR-124 a mimics and psiCHECK-2-[CD163-3’UTR].Then,the recombinant vector with mutation sequence of miR-124 a and CD163 binding sites was constructed.microRNA mimic and the double luciferase reporter plasmid was transfected into HEK293 T cells.Results showed that the binding capacity of miR-124 a and the mutant recombinant plasmid decreased,which indicated that miR-124 a can specifically combine to the site of 138~144 of 3’UTR region of porcine CD163 and site of 31~36 of 3’UTR region of monkey CD163.2.Regulatory mechanism of miR-124 a targeting CD163In order to study the regulatory mechanism of miR-124 a on CD163,the expression of mRNA and protein of CD163 were detected after overexpressing the miR-124 a with microRNA mimics in PAMs.The results showed that miR-124 a could inhibit the expression of CD163 at mRNA and protein levels.Moreover,further study proved that miR-124 a inhibited the expression of CD163 in a dose-dependent manner after overexpressing the different concentrations(20 nm,50 nm,100 nm)of miR-124 a mimics.3.Functional roles of miR-124 a in PRRSV infectionIn order to study the function of miR-124 a in PRRS virus infection,the expression levels of miR-124 a and CD163 of PAMs infected by different PRRSV strains were detected using qRT-PCR.the results showed that when the highly pathogenic PRRSV strain(HP-PRRSV HNP5(0.01 MOI))and the low pathogenic PRRSV strain(N-PRRSV CH-1R(0.01 MOI))infected PAMs,the expression of CD163 increased and the expression of miR-124 a decreased.Then,the strategy of anti PRRSV infection by over expression of miR-124 a was detected by qRT-PCR,TCID50 and Western blot.It was found that overexpression of miR-124 a could effectively inhibit the replication of different PRRSV strains in host cells.Overexpressing the miR-124 a mimics in the natural host cells(PAM)of PRRSVcan effectively inhibit the replication of high pathogenic PRRSV strain(HP-PRRSV HN-P5)and low pathogenic PRRSV strain(N-PRRSV CH-1R).In conclusion,this study elucidated the regulatory roles of miR-124 a on the receptor CD163 of PRRSV during the virus infection,overexpression of miR-124 a can inhibited the replication of PRRSVin the host.The results of this study not only contribute to a better understanding of miRNA mediating PRRSV and host cells interaction mechanism,but also provide a new clue for PRRSV prevention and control.