The Mechanism On The Effect Of Alpha Interferon-Induced Signaling Transduction Pathway By Important Mosquito-borne Flaviviruses

Japanese encephalitis virus(JEV) and Dengue virus(DEN) are the particular important mosquito-borne flavivirus for public health. However, the molecular mechanisms of the pathogenicity for them are not clear. So no new vaccines and effective antiviral drugs exist for the prevention or treatment of infections by them.The clinical trial showed that JEV and DEN may not be sensitive to IFN-α. This research tried to compare and investigate the mechanism of the inhibition of IFN-αinduced signaling transduction pathway by JEV, Beijing-1 strain and DEN2, 43 strain including their non-structural proteins, thus to make some basis for the further understand of the molecular mechanisms of the pathogenicity of the mosquito-borne flavivirus, and provide some advice for the reasonable application of IFN-αin the clinical therapeutics for viral diseases.1. Inhibition of IFN-αinduced signaling transduction pathway by JEV or DEN2 Firstly, we used pISRE-Luc with firefly luciferase reporter gene and measured IFN-αinduced luciferase reporter activity driven by the IFN-stimulated response element (ISRE) to determine ISRE activity level and quantify the suppressive degree of JAK-STAT signaling in infected cells by JEV or DEN2. The results showed that the ISRE activity level significantly decreased in the infected cells by JEV or DEN2, suggesting that these two viruses interfere with the JAK-STAT pathway, and we found that the inhibition of the ISRE activity by JEV was more striking than DEN2.To further investigate that the inhibition of important signaling proteins of JAK-STAT pathway in infected cells by JEV or DEN2. We observed intracellular STAT1 status of JAK-STAT signaling in the infected cells after IFN-αtreatment under a fluorescence microscope. The results showed that the nuclear translocation of the endogenous STAT1 was inhibited by JEV or DEN2, suggesting that these two viruses interfere with the JAK-STAT pathway.These findings tell us that JEV or DEN2 is capable of counteracting IFN-αinduced signaling transduction pathway.To investigate the mechanism of the inhibition of IFN-αinduced JAK-STAT pathway by JEV or DEN2, we then analyzed the STAT1 phosphorylation by Western blotting. The 6results showed that JEV greatly reduced the levels of STAT1 tyrosine phosphorylation (p-STAT1) upon IFN-αstimulation, although the total endogenous STAT1 protein levels were not significantly changed. To further trace the upstream event, we examined whether JAK1 and TYK2, which are responsible for the activation of STATs, were also suppressed by JEV or DEN2 infection. The results showed that the levels of tyrosine-phosphorylated TYK2 (p-TYK2) and JAK1(p-JAK1) in response to IFN-αstimulation were blocked in the infected cells by JEV. However, In the DEN2 infected cells, the levels of p-TYK2,but not p-JAK1 in response to IFN-αstimulation were inhibited. These results indicate that JEV infection blocks the tyrosine phosphorylation of Tyk2 and JAK1, reducing the levels of p-STAT1.And DEN2 infection blocks only the tyrosine phosphorylation of Tyk2, reducing the levels of p-STAT1. Our results suggest that JEV or DEN2 may possess the ability to inhibit IFN-αsignaling in the infected cells through the different mechanism.2. The effect of non-structural proteins in the process of the inhibition of IFN-αinduced signaling transduction pathway by JEV or DEN2To deeply understand the mechanism of the inhibition of IFN-αinduced JAK-STAT pathway by JEV or DEN2, and identify the JEV or DEN2 non-structural proteins responsible for blocking IFN-αsignaling, a series of JEV or DEN2 expression plasmids were generated to individually express V5-tagged NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 and the cellular distribution of JEV or DEN2 non-structural proteins were observed. Then we individually assayed the effect of non-structural proteins of JEV or DEN2 in the IFN-αinduced signaling pathway.Firstly, we used pISRE-Luc to determine ISRE activity level and quantify the suppressive degree of JAK-STAT signaling in infected cells expressing non-structural proteins of JEV or DEN2. To monitor the effect of IFN-αstimulated JAK-STAT signaling by non-structural proteins of JEV or DEN2, we constructed expression plasmids pRed-STAT1, which encodes the human STAT1 fused in frame with a red fluorescent protein, by which the intracellular status of Jak-Stat signaling can be visualized. The cells were cotransfected with plasmids expressing the indicated JEV or DEN2 proteins and pRed-STAT1 and the status of Red-STAT1 upon IFN-αtreatment were observed through the immunofluorescence assay.Then, we analyzed the STAT1 phosphorylation in the cells expressing JEV or DEN2 proteins by Western blotting . The results showed that DEN2 NS5 located in the nuclear, but other JEV or DEN2 proteins all in the cytoplasmic. JEV NS5 clearly blocked nuclear translocation of Red-Stat1 fusion protein, decreased the ISRE activity level, and reduced the levels of p-STAT1 upon IFN-αtreatment. Whereas in cells expressing other viral proteins, the inhibition of IFN-αinduced signaling was not found, suggesting that JEV NS5 is a potent IFN antagonist. However, our findings showed that all the non-structural proteins of DEN2 did not block IFN-αinduced JAK-STAT signaling pathway, suggesting that the mechanism of the inhibition of IFN-αinduced signaling by DEN2 may be different from JEV.3. TypeⅠinterferon response to NS4B of DEN2Recently, the reporter has showed that expression of DEN2-NS4B was capable of blocking IFN signaling. However, this result was not identified in our study. To further investigate the related function of NS4B, we constructed eight expression plasmids which individually express different regions of NS4B according to the predicted topology model of it, finding that present between residues 1 to 54 is important for correct expression of NS4B in the mammalian cells. In addition, our findings showed that different regions of NS4B did not block IFN-αinduced JAK-STAT signaling pathway, suggesting typeⅠinterferon response to NS4B of DEN2 should be further studied. At the same time, we expressed the NS4Bof DEN2 and DEN4 in E. coli and prepared the polyclonal antibody for them. We found the structure of carboxyl end may be important in the immunogenicity of NS4B. These results make some basis for the further understand of the related function of NS4B on typeⅠinterferon system.This research compared and investigated the mechanism of the inhibition of IFN-αinduced signaling transduction pathway by JEV or DEN2 ,including their non-structural proteins, thus to provide some evidence for the further study on the relation between typeⅠinterferon system and the mosquito-borne flavivirus.