The innate immune system utilizes pattern recognition receptors to detect pathogen-associated molecular patterns

The innate immune system utilizes pattern recognition receptors to detect pathogen-associated molecular patterns, such as viral nucleic acids. While TLR7 and RIG-I detect influenza viral RNA, the NLRP3 senses intracellular ionic fluxes following influenza virus infection. We previously demonstrated that the ion channel activity of viroporins, such as influenza virus M2 or EMCV 2B protein is essential for NLRP3 inflammasome activation32,37. Our findings here have identified a previously unknown mechanism by which influenza virus and EMCV stimulate mtDNA release into the cytosol through their viroporin activity. The cytosolic translocation of mtDNA in response to influenza virus or EMCV infection stimulates cGAS- and DDX41-dependent innate antiviral immune responses. Given that the viroporin-induced disturbance in the intracellular ionic milieu is accompanied by Mn2+ efflux from membrane-enclosed organelles, the ion channel activity of viroporins may be required for increasing the sensitivity of cGAS to dsDNA51. Our data have demonstrated that the infection with ΔNS1 influenza virus enhances cytosolic mtDNA release and the STING-dependent IFN-β gene expression compared with that of WT virus. Several possible mechanisms could explain how the NS1 protein of influenza virus might inhibit mtDNA release into the cytosol and STING-dependent recognition of influenza virus infection. First, the NS1 protein of influenza virus might inhibit cytosolic translocation of mtDNA by inhibiting RIG-I/MAVSdependent signals (Fig. 10). Indeed, we found that influenza virus stimulated cytosolic mtDNA release in a MAVS-dependent manner. In the case of SeV infection, the virus activated-IRF3 associates with Bax to translocate to the mitochondria and cause cytochrome c release29. In addition, previous studies have demonstrated that Bax/Bak play a critical role for mtDNA release into the cytosol27,28. Similarly, we found knockdown of Bax significantly reduced the cytosolic mtDNA release after influenza virus infection. Formation of Bak/Bax macropores elicits inner mitochondrial membrane herniation and stimulate mtDNA release into the cytosol52. These cytosolic mtDNA could be packaged into distinct levels of higher-order structures depending on the ratio of TFAM to mtDNA53 (Fig. 10). Given that cGAS preferentially binds incomplete nucleoid-like structures or U-turn DNA54, cytosolic U-turn DNA bridged by cross-strand binding of TFAM53 could play a major role in the induction of cGAS/ STING-dependent IFN-β gene expression in response to influenza virus infection. Second, because the NS1 protein of influenza virus associated with mtDNA and inhibited detectable levels of cytosolic mtDNA, the NS1 protein may mask mtDNA from recognition by cytosolic DNA sensors (Fig. 10). Indeed, we found that treatment of pure cytosolic extracts of influenza virusinfected cells with proteinase K enhanced detectable levels of cytosolic mtDNA after influenza virus infection. Consequently, transfection of cGAS-293FT cells with proteinase K-treated cytosolic extracts from influenza virus-infected cells significantly enhanced IFN-β gene expression compared with untreated control extracts of influenza virus-infected cells, suggesting that the NS1 protein of influenza virus may associate with mtDNA to evade recognition by cytosolic DNA sensors (Fig. 10). Influenza virus-induces type I IFNs (IFN-α/β) production in a STING-dependent but cGAS-independent manner through a membrane fusion process in human monocyte/macrophage-like cell line THP115. In addition, knockdown of DDX41 in D2SC cells, a mouse myeloid DC line, has no effect on influenza virusinduced IFN-α/β production41. Furthermore, knockdown of STING in MEFs has no effect on influenza virus-induced IFN-β gene expression16. In contrast, our data have demonstrated that influenza virus stimulates cGAS-, DDX41-, and STINGdependent IFN-β gene expression in both mouse (primary lung fibroblasts) and human (HEK293FT and A549) cells. In addition, we found that influenza virus-induced high levels of cGAMP in STING-A549 cells or primary lung fibroblasts within 24 h post infection. Further, treatment of cells with CBX or knockdown of CX43 inhibited the STING-dependent IFN-β gene expression. These data collectively indicate that influenza virus infection stimulates STING-dependent pathways in a cell type-specific manner and that intercellular communication via gap junction plays an important role in spreading STING-dependent antiviral signals to bystander cells (Fig. 10). Although cGAS was required to maximize IFN-β gene expression in the lung after influenza virus infection, cGAS deficiency did not significantly affected the viral titer in the lung compared to WT mice. In contrast, the STING-dependent signals were essential for limiting influenza virus replication in vivo. One possible explanation for this result is that cGAS and other DNA sensors induce redundant signaling pathways required for limiting influenza virus replication in the lung tissue. Another possibility is that STING-dependent translation inhibition could restrict influenza virus replication in vivo, independent of MAVS16. Since cGAS restricts viral replication of flaviviruses including dengue virus and West Nile virus17,55, the antiviral effects of the cGAS could be different for each RNA viruses55. In summary, our finding substantially expand our understanding of how influenza virus and EMCV trigger mtDNA release into the cytosol and stimulate the cGAS- and DDX41- dependent innate antiviral immune responses. Because mitochondrial dsRNA released into the cytosol triggers MDA5- dependent innate antiviral signaling56, our results suggest a possible effect of viroporin-induced mitochondrial dysfunction in the induction of the MAVS-dependent innate antiviral immune responses. Better understanding of crosstalk between RNA and DNA sensing pathways in response to viral infection will aid the development of novel therapeutic strategies to treat viral infections and associated diseases.