IFI16 functions as an antiviral factor during influenza virus infection by binding viral RNA and enhancing the RIG-I signaling (RIP-seq)

RIG-I is thought to be the most important sensor of influenza virus infection and plays critical roles in the recognition of cytoplasmic dsRNA and activation of type I IFNs and initiates the innate antiviral immune responses. How the binding of viral RNA to and activation of RIG-I are regulated remains enigmatic. Here, by an affinity proteomics approach with viral RNA as the bait, we found that IFI16, previously identified as a DNA sensor, was significantly induced both in vitro and in vivo during influenza virus infection. Using an IFI16 knockout cells and p204-deficient mice model, we demonstrated that IFI16 enhanced RIG-I-mediated production of type I IFNs and thereby inhibited viral replication during influenza virus infection. Furthermore, we showed that IFI16 regulated the RIG-I signaling by enhancing its transcriptional expression through recruitment of RNA Pol II to the RIG-I promoter. We also verified that IFI16 directly interacted with both viral RNA by HINa domain and associated with RIG-I through its PYRIN domain as well as promoted influenza virus-induced K63-linked polyubiquitination of RIG-I. In addition, we found that IFI16 lost its ability to inhibit viral replication in the absence of RIG-I in virus-infected cells. These results indicate that IFI16 is a key regulator of the RIG-I signaling during antiviral innate immune responses, which highlights a novel mechanism of IFI16 in IAV and other RNA viruses infection, expands our knowledge in antiviral innate immunity, and suggests its possible use as a new strategies to manipulate antiviral responses. Overall design: To test if IFI16 bound IAV RNA, we eluted RNA co-precipitating with FLAG-tagged IFI16 from IAV-infected RIG-I knockout cells and examined eluates for the presence of IAV RNA. In parallel, we evaluated RNA co-precipitating with FLAG-RIG-I, a known sensor of IAV RNA, as a positive control. And the Flag empty vector as the negative control. We readily detected IAV-specific vRNA mapping to all eight IAV gene segments in IFI16 or RIG-I immunoprecipitates.