Host Protein AGO2 Inhibiting IFN-? Expression And Promoting Influenza A Virus Proliferation

Influenza A virus is an acute zoonosis and raises high awareness worldwide.Host-virus interactions in various types and forms are the hot spots.Influenza virus infection mainly activates RIG-I pathway.AGO2 is a multifunctional protein,and the study on this protein focused more on the tumor and developmental biology,but the role in antiviral innate immunity is still unclear.This study found that AGO2 inhibited the production of IFN-? by competitive binding to IRF3 with CBP/p300.Preliminary study has shown that influenza virus can make use of AGO2 to regulate the distribution of v RNA and to promote proliferation.The main contents and results are as follows:1 Construction and expression of AGO2 eukaryotic expression plasmidAGO2 gene was amplified and the eukaryotic expression plasmid was successfully constructed.HEK293 T cells and A549 cells were transfected with the plasmids.Western Blotting results showed that AGO2 could express in cells and the size was consistent with what we know.The A549 cells were transfected with the constructed AGO2 eukaryotic expression plasmid or si RNA to change the expression level of AGO2 and then infected with H5N1.The results showed that overexpression of AGO2 enhanced the proliferation of H5N1 in A549 cells,but the opposite result occurred while silencing AGO2.These indicated that AGO2 affected the virus proliferation.At the same time,preliminary studies also showed that AGO2 affected the expression of IFN-? as well as downstream genes.2 Study on protein AGO2 inhibiting IFN-? expressionThis study first examined the effect of AGO2 on the expression of IFN-? in HEK293 T cells.The results showed that AGO2 inhibited the expression of IFN-?.On this basis,we subsequently explored the specific mechanisms of AGO2 inhibiting IFN-?expressionRNA viruses including Influenza virus mainly activate RIG-I signaling pathway.Therefore,we investigated the various adaptor-mediated IFN-?promoter activation when AGO2 was overexpressed by dual luciferase report system.The results showed that overexpression of AGO2 markedly inhibited the upstream of IRF3-mediated IFN-?induction.Further research suggested that AGO2 did not affect the phosphorylation,nucleation and DNA binding ability of IRF3.All of above indicated that AGO2 inhibited the expression of interferon in the downstream of IRF3.We found that AGO2 interacted with IRF3 by immunoprecipitation experiments.Then the N-terminal region and C-terminal region of IRF3 were constructed.It was found that AGO2 interacted with the C-terminal region of IRF3,which hinted that AGO2 affected the interaction between IRF3 and downstream molecules.The expression plasmid of IBi D domain of CBP/p300 was constructed,which interacts with IRF3.The competitive binding experiments showed that AGO2 inhibited the interaction between IRF3 and IBi D.Further research showed that AGO2 was an inhibitor of the binding of IRF3 to CBP which plays a crucial role in the formation of transcriptional initiation complex and the expression of interferon.The eukaryotic expression plasmids of different functional regions of AGO2 were constructed,and the results showed that the MID region of AGO2 interacted with IRF3.To elucidate the accuracy of the results,the MID-deletion mutant of AGO2 expression plasmid was constructed.The competitive binding experiments showed that the deletion mutant could not compete with CBP in combination with IRF3.The above results concluded that AGO2 competitively bound to IRF3 with CBP/p300 and thus affected the formation of transcriptional complexes and the expression of IFN-?.3 The relationship between the host protein AGO2 and H5N1 influenza virusThis study explored the relationship between the host protein AGO2 and H5N1 influenza virus replication.The distribution of AGO2 were examined in H5N1 infected A549 cells.The results showed that the content of AGO2 in nucleus was increased at 24 hours post infection with H5N1 and decreased at 48 hpi.Then,the effects of AGO2 on the replication and distribution of influenza virus v RNA were detected.It was found that AGO2 promoted the replication of v RNA and affect the distribution of v RNA.Therefore,it could be presumed that,in favor of virus replication,AGO2 can be promoting the v RNA distribution in the nucleus during the early stage of H5N1 infection.However,at the late stage,the decreased number of AGO2 in the nucleus could benefit v RNA to release.AGO2 is a multifunctional protein that participates in regulating a variety of physiological functions of the organisms.Whether AGO2 protein has a direct effect on the replication of virus particles need to be further explored.