Role Of RIG-I-like Receptors During Type I Interferon Response Induced By Dengue Virus Infection

Dengue viruses (DENV) type 1-4, together with West Nile virus (WNV), Japanese Encephalitis virus (JEV), etc, belong to the genus Flavivirus family Flavivirida, containing single-stranded, positive-sense genome RNAs of approximately 11 kb in length. DENV infections can result in mild dengue fever (DF), severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS). In terms of the morbidity and mortality rates, DENV is becoming one of the most important publich health problems in tropical and subtropical countries. The pathogenesis from DF to DHF/DSS has not yet been elucidated, and no vaccine or antiviral drugs are currently available.At the initial stage of virus infection, host pattern recognition recepotors (PRR), including Toll-like receptor (TLRs) and RIG-I-like receptors (RLRs), are responsible for sensing viral RNA and initiates innate antiviral responses, including the activation of proinflammatory cytokines and type I interferon (IFN). Toll-like receptors are abundant on monocytes/macrophages and DCs, and play a key role in regulating the inflammatory response against infectious viruses. RLRs, including RIG-I and MDA5, are supposed to sense cytoplasmic viral RNA mainly in non-immune cells. DENV is transmintted by mosquitoes feed, skin epithelial cells have been supposed to be one of the primary target cells. However, the role of RLRs in human epithelial cells during the induction of IFN by DENV is still unknown. In this study, human lung epithelial cell lines A549 were chosen to observe the IFN response to DENV infection, and then the role of RIG-I and MDA5 during DENV-induced IFN response were analyzed by RNAi and over-expression assays. What's more, the antiviral effects and mechanism of RIG-I against DENV and West Nlie virus were determined. The main results are described as following in three parts:1. DENV induced the expression of RIG-I and IFN-βin A549 cells. Firstly, to study the IFN response to DENV infection in human epithelial cells, A549 cells were infected with DENV and heat-inactivated DENV, respectively, and IFN-βwere found secreted in culture supertants since 12 h post-infection by a commercial human IFN-βELISA kit, while no IFN-βwas detected in A549 cells treated with heat-inactivated virus. Also, transfection of a DENV replicon was found to be able to induce the secretion of IFN-βin A549 cells. These results suggested that the production of IFN-βwere due to virtual virus infection or viral RNA replication. Further, RT-PCR and Western blotting resuluts showed that the expression of RIG-I and it's adaptor, IPS-1, were upregulated upon DENV infection in A549 cells. CAT reportor plasmids that waer under the control of the IRF-3 and IFN-βpromoter were used to analyze the effects of DENV infection, and the results indicated that IRF-3 and IFN-βpromoter were activated 24 hrs post infection.Then, different isolates of DENV that differed in serotypes and virulence clearly were used to infect A549 cells, and IFN-βlevels were compared and assayed. While no significant difference was found among those isolates. What's more, similar RIG-I activation and IFN-βproduction were observed in A549 cells infected with WNV. Together, these results suggested that DENV infection can activate the IRG-I signal pathway molecules, including RIG-I, IPS-1 and IRF-3, finally induce the expression of IFN-βin human epithelial cells. Serotype and virulence of DENV have no effect on the IFN-βresponse to DENV infection in A549 cells.2. RIG-I and MDA5 are both involved in IFN-βresponse to DENV infection.In order to demonstrate the role of RIG-I in the IFN-βresponse to DENV infection, full-length RIG-I and its dominant negative muatant, RIG-IC, were transiently expressed in A549 cells followed by DENV infection. ELISA and CAT assays results showed that IFN-βlevel and promoter activity was induced by RIG-I and further enhanced by DENV infection, and that RIG-IC caused a reduction in IFN-βlevel and promoter activity. Further, siRNAs were used to knockdown the endogenous expression of RIG-I in A549 cells, while no significant difference was found between the DENV-induced IFN-βresponse in RIG-I-konckout cells and control cells. Next, the role of MDA5 in the IFN-βresponse to DENV infection was analyzed by RNAi and over-expression. The expression of MDA5 was up-regulated upon DENV infection, and over-expression of MDA5 in A549 cells also induced the secretion of IFN-β. While MDA5 knockout and over-expression had no significant effects on the IFN-βresponse to DENV infection in A549 cells.Furthermore, stable knockdown cell lines of human RIG-I were developed and MDA5 were subsequently knocked out by siRNA. The IFN-βresponse to DENV infection was almost blocked in this cell line. Together, these results suggest that both RIG-I and MDA5 are involved, and either of the two is essential for the induction of IFN-βresponse to DENV infection, and RIG-I plays a principle role in the IFN-βresponse to DENV infection.3. Antivial effects and mechanism of RIG-I against DENV and WNV.Previous experiments based on the stable RIG-I-knockout cells showed that RIG-I knockout enhanced the cytopathic effects, and increased the yield of progeny DENV, suggesting a potential antiviral effects of RIG-I. To test whether RIG-I? can inhibit replication of DENV and WNV, RIG-I was transiently over-expressed in A549 cells followed by infection with DENV or WNV, and the progeny virus yield decreased sharply in a dose-dependent manner. Further, treatment of A549 cells with human IFN-βantibodies had no effects on the antiviral activity, demonstrating that the IFN-β-provoked JAK-STAT pathways are not essential for the antiviral effect of RIG-I. Next, over-expression of RIG-I and RIG-IC, not RIG-IN, showed similar antiviral effects, suggesting the C-terminal of RIG-I was responsible for the antiviral activity. What's more, the DENV E protein level decreased in RIG-I and RIG-IC expressing A549 cells compared with that in RIG-IN expressing and control cells. So at the early stage of DENV infection, the activated RIG-I not only induces IFN-β, but also inhibits the synthesis of viral protein through its C-terminal region.In conclusion, our results demonstrate that DENV infection can activate both RIG-I and MDA5, and either of the two is essential for the induction of IFN-βresponse to DENV infection. In addition, the C teiminal region of RIG-I can inhibit the synthsis of viral protein, possessing antiviral activity against multiple flaviviruses. Our findings indicat the critical role of RIG-I and MDA5 during DENV infection, and describe the possible process of type I interferon production in DENV-infected human lung cells for the first time, provide insights for the understanding of virus-host interaction and development of intervention strategy for viral infections.