Screening Of MicroRNA That Regulate Swine Influenza A Virus Replication And Molecular Mechanism Study On Host Protein PCNA Regulating Influenza A Virus Replication

Influenza A viruses can infect many mammal and birds,such as human,pigs,chicken and ducks.Swine influenza,which is an acute and highly infectious respiratory disease caused by swine influenza A virus,makes the infected pigs to lose productivity,causing great production economic losses.In addition,pigs are the ’mixing vessel’ in which different influenza viruses reassort,for they are naturally infected with different sources of influenza viruses.The pandemic H1N1/2009 influenza A virus,which caused the first influenza pandemic of the 21st century,probably derives from the reassortment of North American H3N2 and H1N2 swine viruses with Eurasian avian-like swine viruses,posing a potential threat to public health.Therefore,it is very important for economics,health of animals and human to strengthen the prevention and control of swine A viruses.microRNAs(miRN As),which are single-stranded,endogenous and noncoding RN A of approximately 22 nucleotides,can regulate gene expression at post-transcriptional levels through an RNA interference pathway,playing important roles in a broad spectrum of host biological processes.In recent years,the involvement of miRNAs in influenza A virus infection causes more and more attention.However,much work focused on human,mice or chicken miRNAs which regulate human or avian influenza viruses rather than swine miRNAs which play roles in regulating swine influenza A virus infection.Therefore,the first part of this work aims to screen swine miRNAs which are involved in SIV-H1N1/2009 infection,and to figure out the biological functions and mechanisms of action of these miRNAs.Host proteins also play vital roles in influenza A virus infection,for example,the viral ribonucleoprotein(vRNP)complex need to interact with some host proteins to mediate the viral RNA transcription and replication.A research group screens PCNA as a potential host protein to interact with RNP complex,indicating that PCNA may be involvement in regulating influenza A virus replication.In addition,it is reported that PCNA-interacting peptides can reduce Akt phosphorylation and TLR-mediated cytokine secretion,suggesting that PCNA may also play a role in cellular signaling.Therefore,the second part of this work preliminarily investigate the molecular mechanism of PCNA regulating influenza A virus replication by affecting viral polymerase activity and host innate immunity response.Detailed contents are as follows:1.The bioinformatics analysis of swine miRNA microarray and gene microarraySwine miRN A microarray and gene microarray were carried out after SIV-H1N1/2009 infection in NPTr cells.A total of 66 miRNAs were identified to be differentially expressed,among which 9 miRNAs were upregulated and the other 57 were downregulated.Meanwhile,there were 166 differentially expressed transcripts which could be functionally annotated,among which 89 genes were upregulated and 77 were downregulated.According to reported studies and bio informatics analysis,14 miRN A-gene pairs,whose expressions were negatively correlated,were identified to be involved in influenza A virus replication.2.miRNAs indirectly regulate the replication of SIV-H1N1/2009 by targeting host genesAfter a comprehensive analysis of these 14 miRN A-gene pairs,we focused on the pair of ssc-miR-361-3p and PLK3.Firstly,ssc-miR-361-3p was validated to target the 3’UTR of PLK3 by a dual-luciferase reporter assay,and to repress the mRNA and portein levels of PLK3.SIV-H1N1/2009 infection could upregulate PLK3 levels by downregulating ssc-miR-361-3p.Then PLK3 was validated to promote SIV-H1N1/2009 replication,whereas ssc-miR-361-3p was found to inhibit the virus replication.Furthermore,ssc-miR-361-3p was verified to target neither another predicted host gene HMGA1,nor the viral genomic RNA,which further indicated that ssc-miR-361-3p negatively regulated SIV-H1N1/2009 replication by targeting PLK3 in some extent.In addition,some other predicted miRNA-gene pairs were preliminary validated.As a result,ssc-miR-339-5p was found to probably negatively regulate SIV-H1N1/2009 replication by targeting host gene MYBL2,which needed further investigation.3.miRNAs directly regulate the replication of SIV-H1N1/2009 by targeting viral genomic RNAWe screened miRNAs that targeted the genomic RNA of SIV-H1N1/2009,and found that ssc-miR-204 and ssc-miR-4331 targeted viral HA and NS respectively by dual-luciferase reporter assays,and that the target sites were located in HA1 and NS1 in detail.In addition,ssc-miR-204 and ssc-miR-4331 were validated to negatively regulate the replication of SIV-H1N1/2009 by repressing the expression of HA1 and NS1 respectively,and the antiviral mechanism of them might be virus sequence-specific.Furthermore,the virus infection downregulated ssc-miR-204 and ssc-miR-4331 levels reversely,which might weaken their inhibition effect on virus replicaton and further facilitate the virus to replicate in host.4.Host protein PCNA affects influenza A virus replication by regulating viral polymeraseFirstly,PCNA was validated to interact with polymerase subunits PB2 and PB1 using co-immunoprecipitation.Next,PCNA was found to compete with importin-α5 for binding the C terminal of PB2,which resulted in nuclear import of PB2 by importin-α5.Furthermore,the assembly of influenza A virus ribonucleoprotein complex was impaired because of less PB2 in nucleus,which decreased the polymerase activity,further inhibited the influenza A virus replication.In turn,virus infection downregulated the expression of PCNA,which might be related with PB2 and PB1.5.Host protein PCNA regulates innate immunityIn addition,PCNA was found to promote the IRF3 phosphorylation induced by SeV infection and further potentiate IFNβ production,indicating that PCNA could also affect influenza A virus replication by regulating host innate immunity.In turn,IFNβ induced by SeV infection increased the expression of PCNA.Next,PCNA was verified to interact with MAVS,TRAF3,TRAF6 and IKKε using co-immunoprecipitation,and the TRAF domain of TRAF3 is specifically required for interacting with PCNA.However,PCNA neither affect the expression of MAVS,TRAF3,TRAF6 and IKKε,nor influence interactions between different components of the RLR pathway.The mechanism of PCNA positively regulating innate immunity needs to be investigate further.This work investigated how swine miRNAs to regulate swine influenza virus on porcine cell lines for the first time,and successfully screened the ssc-miR-361-3p,ssc-miR-339-5p,ssc-miR-204 and ssc-miR-4331 which negatively regulated the replication of SIV-H1N1/2009,and preliminarily illuminated their mechanisms of action.In addition,this work studied the roles of host protein PCNA in regulating influenza A virus polymerase activity and host innate immunity.Taken together,this work provided candidate targets and theoretical basis for developing anti-influenza drugs.