MiRNA-A6 Encoded By Hepatitis E Virus Induced The Expression Of Signal Regulatory Protein Alpha To Promote Viral Replication

Hepatitis E virus(HEV)is a major pathogen of acute hepatitis worldwide.However,its pathogenic mechanism and replication mechanism is still unclear.Virus encoded miRNA(VmiRNA)can regulate the expression of viral proteins to escape the host innate immune system to promote its replication.Signal regulator protein alpha(SIRP-a)is one of the target protein of miR-A6 encoded by HEV,which play a key role in regulation of innate immunity.Moreover,SIRP-a may play a critical role in escape the immune system when virus infection.The aim of this research is to explore the mechanism of HEV encoded miRNA-A6 regulates the expression of SIRP-a to promote viral replication as follows:1.miRNA-A6 induce the expression of SIRP-aSIRP-a is the target protein of miR-A6 encoded by HEV,in order to explore how HEV regulate the expression of SIRP-a by its VmiRNA,A549 cells were inoculated with HEV(ultraviolet inactive HEV,UV-HEV,serve as control),results indicated that HEV infection induces SIRP-α expression.To further identify the relationship between miR-A6 and SIRP-a,miR-A6 eukaryotic expression vector was constructed and transfected into A549 cells,and SIRP-a was induced abundantly,which reveal that miR-A6 encoded by HEV induces the expression of SIRP-a.To analyze the specificity of miR-A6 regulation of SIRP-a,miR-A6 mutants eukaryotic expression vector(the seed sequence were mutated)were constructed and transfected into A549 cells.Results showed that miR-A6 specifically regulates the expression of SIRP-α.The mutation of miR-A6 seed sequences decrease the expression of SIRP-a.More importantly,miR-A6 can effectively promote HEV replication.Meanwhile,over-expression of SIRP-a can also promote the replication of HEV,in contrast,knockdown the expression of SIRP-a inhibited HEV replication.2.The interaction between HEV and SIRP-aThe interaction between virus and host is a hotspot for researching the virus pathogenic mechanism.In order to explore the open reading frame of HEV that interacted with SIRP-a,HEV three ORFs(ORF1,ORF2 and ORF3)eukaryotic expression vector was separately constructed and transfected into A549 cells.Results showed that the HEV ORF3 protein induced the expression of SIRP-a.Further researches showed that the D1 domain of ORF3 plays a critical role in inducing the expression of SIRP-α.Meanwhile,co-immunoprecipitation experiments indicated that the HEV ORF3 protein interacted with SIRP-a.3.miRNA-A6 inhibits IFN-β through regulation of SIRP-a expressionInnate immune system is the first line of host to defense against invading pathogens.Virus dsRNA was recognized by host pattern recognition receptors(PRRs),which activate IRF3 phosphorylation,and then result the production of type I interferons(IFNs)to eradicate the pathogen.However,whether or not HEV could escape host innate immune system is still unclear.In order to analysis the effect of HEV on IFN-β expression,the cells were stiimulated with dsRNA analogue,poly(I:C),to produce a large amounts of IFN-β,and then inoculated with live HEV or UV-HEV.Results indicated that HEV inhibited IRF3 phosphorylation and IFN-β expression.To further identify the effect of miR-A6 encoded by HEV on the expression of IFN-β,the miR-A6 eukaryotic expression vector was transfected into cells.Results showed that miRNA-A6 effectively inhibited IRF3 phosphorylation and the expression of IFN-β.More importantly,the ability of miR-A6 inhibits IRF3 phosphorylation and IFN-β was decreased significantly when the expression of SIRP-a was knockdown.In addition,the HEV ORF3 protein induced the expression of SIRP-a and interacted with each other.In order to analyze the effect of HEV ORF3 on the expression of IFN-β,ORF3 eukaryotic expression vector was transfected into cells,which revealed that HEV ORF3 could effectively inhibit IRF3 phosphorylation and IFN-β expression.Meanwhile,the D1 domain of ORF3 plays a critical role.In summary,HEV encoded miRNA-A6 induces SIRP-a expression to inhibit IRF3 phosphorylation and then inhibit the expression of IFN-β,and results in increase of viral replication.