The Molecular Mechanism Of Japanese Encephalitis Virus NS1' In Inhibiting Type ? Interferon

Japanese encephalitis virus(JEV)is a kind of infectious disease common to human beings and animals which is transmitted by mosquitoes.It mainly causes Japanese encephalitis(JE)after infection.JEV was first discovered in Japan in 1871 and is susceptible to various animals.The infection of JEV on humans and horses will develope into an obvious encephalitis.JEV also caused serious veterinary problems,from which the pig industry suffered serious losses because of the reproduce failure.JEV targets the central nervous system(CNS),which causes morbidity and mortality in humans and animals by causing excessive inflammatory responses to the CNS,but the specific pathogenic mechanism remains to be elucidated.Recent studies have shown that the non-structural protein NS1' of JEV which produces due to ribosome frameshifting can significantly increase the invasiveness of JEV According to this,NS1' may play an important role in the invasion of the central nervous system through the blood brain barrier(BBB).The specific reason for this phenomenon is not clear.As an early defense mechanism for host anti-virus infection,type ? interferon(IFN)signaling pathway plays an important role in host antiviral immunity.After JEV infects the host,it can reach a high virus titer in the peripheral blood by inhibiting the host interferon response and downstream signal transduction,which facilitates the virus to invade the CNS.Therefore,the study about relationship between JEV NS1' protein and host type ? interferon signaling pathway has important scientific significance for revealing the neuroinvasiveness and pathogenic mechanism of JEV.Based on this,this paper aims to explore the effect of NS1' protein on the replication and invasion of the central nervous system by using the NS1' protein of JEV as a research object.The details are as follows:1.Loss of NS1' attenuates neuroinvasivenesss of JEVAfter successfully constructing the NS1' deletion type virus(rG66A),the Ba1B/C mouse model was used to assess the neurovirulence and neuroinvasiveness of rG66A and its parental virus(pSA14)by intracerebral and intraperitoneal inoculation.The results of intracerebral inoculation showed that there was no significant difference in clinical signs and mortality of mice caused by the two virus,indicating that the absence of NS1' protein did not significantly affect the neurovirulence of JEV.The result of intraperitoneal inoculation showed that:The morbidity and mortality caused by rG66A were significantly lower than its parental virus,pSA14,and the viral load in the brain and blood of rG66A-infected mice was also significantly lower than that of the pSA14 group,indicating that the absence of NS1' protein significantly affected the invasion of the CNS by JEV.Basis on these result,we evaluated the function of NS1' on lethality in mice by intravenous injection of NS1' protein in the rG66A-infected BalB/C mouse.The results showed that rG66A-infected mouse treated with NS1' protein caused a significant increase in clinical signs and mortality of mice,which was consistent with the pSA14 virus.The viral load in the blood and brain tissues of mice in NS1' treated group was significantly increased.This shows that NS1' protein has the function of enhancing JEV neuroinvasiveness.2.NS1' protein inhibits the expression of host type ? interferonTo explore the mechanism by which NS1' protein enhances neuroinvasiveness of JEV,we used plaque methods to determine the viral growth curve of pSA14 and rG66A virus in different cell lines.We found that the two virus had the same viral growth curve in vero cells,while in A549 cells and TM4 cells,the viral growth curve of NS1' deletion virus(rG66A)was significantly lower than that of the parental pSA14.However,when the cells were treated with the type ? interferon receptor(IFNAR2)pAb,the viral growth curve of the two virus were similar.Further studies revealed that the rG66A strain can induce higher levels of IFN-(3 in host cells,indicating that the NS1' protein has the function of inhibiting host type ? interferon.On this basis,NS1,NS1',and ?NS1'52aa proteins were over-expressed by transfection and then infected with Sendai virus.The results confirmed that NS1' protein can directly inhibit the expression of host IFN-P,thereby inhibiting the expression of ISGs.The inhibitory effect of NS 1' was significantly stronger than NS1 protein,but ?NS1'52aa protein did not inhibit the expression of type ? interferon,indicating that NS1' protein has stronger ability to inhibit type ? interferon than NS1 protein,but the integrity structure is the key to its function.To further demonstrate the effect of NS1' protein on the inhibition of type ?interferon production,in vivo experiments,we uesd type ? interferon receptor-deficient C57BL/6J mice and wild-type C57BL/6J mice.The lethality of intraperitoneal inoculated with wild-type C57BL/6J mice was significantly lower than that of the pSA14 virus.This result was consistent with the infection of the BalB/C mouse model.While there was no significant difference in the lethality and morbidity of infection between type ? interferon receptor-deficient C57BL/6J mice by the two virus.The results showed that NS1' protein can increase the viremia in mice by inhibiting the expression of host type ? interferon,and enhance the neuroinvasiveness of JEV.3.NS1' protein inhibits the expression of host MAVS and type ? interferon by targeting miR-22To explore the molecular mechanism by which NS1' protein inhibits type ? interferon expression,we found that NS1' protein inhibits the activity of the IFN-P promoter by the Dual-Luciferase Reporter assay,thereby reducing the transcription and expression of IFN-?.The key proteins(RIG-I,MAVS,IKKs,TBK1 and IRF3)in the RLR signaling pathway were co-expressed with NS1' protein in HEK293T cells,respectively.The results showed that overexpressing MAVS could not be contacted with inhibition,while overexpression of IKK? and TBK1 could abolish the inhibitory effect Further studies have found that NS 1' protein can inhibit the expression of MAVS mRNA and protein and inhibit the activity of IFN-?-luc,thus determining that MAVS may be a target of NS1'protein.According to previous research in the laboratory,JEV can up-regulate miR-22 to inhibit the mRNA and protein expression of MAVS.Based on this,this experiment demonstrates that NS1' protein can up-regulate miR-22 to regulate the expression of MAVS.This result indicates that NS1' protein can inhibit the expression of endogenous MAVS by up-regulating miR-22,thereby inhibiting the transcription and expression of IFN-P,promoting the natural immunity of the virus escape host,and facilitating virus replication.This study found that NS1;protein plays an important role in JEV evasion of host antiviral response,and initially demonstrated that NS1;protein regulates endogenous MAVS expression by miR-22,inhibits type ? interferon expression,promotes virus proliferation,and enhances JEV neurological invasion.Force provides important clues for further clarifying the pathogenesis of JEV.