Beyond sensing: Retinoic acid inducible gene‐I (RIG‐I) continues to expand its antiviral effector functions
Authors: Amy E.L. Stone, Michael J. Gale
Journal: Hepatology (2017)

Potential conflict of interest: Nothing to report. See Article on Page 1823 Retinoic acid gene‐I (RIG‐I) is a cytosolic RNA helicase and pathogen recognition receptor that recognizes pathogen‐associated molecular patterns (PAMPs) within viral RNA to trigger downstream interferon (IFN) production and innate immune activation during RNA virus infection. Analysis of RIG‐I during hepatitis E virus (HEV) infection now reveals a role in IFN‐independent innate immunity to suppress HEV replication. It has long been accepted that there exists complex signaling and crosstalk among innate immune response pathways to direct Types I and III IFN defenses against microbial pathogens.1 During RNA virus infection IFN is induced as a result of pathogen recognition receptor (PRR) recognition of viral RNA PAMPs. For many RNA viruses PAMP recognition is mediated by RIG‐I1 wherein RIG‐I signaling induces the downstream activation of transcription factors, including IFN regulatory factor 3 (IRF3) and IRF7 as well as nuclear factor kappa B (NF‐κB),2 and drives the expression of virus‐responsive genes, including IFN (reviewed in Loo and Gale1). Secreted IFN then binds to its cognate receptor on the infected cell and on bystander cells to signal through the Janus kinase (JAK)–signal transducer and activator of transcription (STAT) pathway and induce hundreds of IFN‐stimulated genes (ISGs) (Fig. 1). Among their many actions, virus‐responsive genes and ISGs direct antiviral actions to suppress viral replication and spread, and serve to modulate the adaptive immune response to drive antiviral immunity. While RIG‐I can also serve as an antiviral effector gene to bind to viral RNA and block RNA/protein associations,3 it is widely recognized for trigging this innate immune antiviral signaling cascade as a result of PAMP recognition and binding.4 Xu and colleagues5 now reveal a role for RIG‐I in mediating innate immune effector actions against HEV that show roles for RIG‐I in IFN‐independent innate immunity. Remarkably, RIG‐I is observed to drive the expression of a subset of ISGs independently of IRF3, IRF7, and IFN but requiring NF‐κB actions, thus providing evidence for a novel RIG‐I‐directed innate immune effector response pathway in hepatocytes (the target cell of HEV infection) that uncouples IRF and NF‐κB signaling programs to operate outside of the canonical IRF/IFN/ISG innate immune effector pathway (Fig. 1).Figure 1: RIG‐I acts in multiple ways to inhibit virus infection. RIG‐I recognizes PAMP RNA, leading to multiple divergent pathways. (1) MAVS located on the mitochondria‐associated membrane induces IRF3/7 dimerization, leading to the induction of IFN genes. (2) MAVS located on peroxisomes induces NF‐κB activation, leading to the expression of proinflammatory genes. (3) RIG‐I sterically hinders the interaction of viral proteins with viral RNAs. (4) RIG‐I induces STAT1 phosphorylation independent of IFN induction. (5) RIG‐I induces ISG production independent of IRF3/7 and STAT1 signaling, as describes by Xu et al.5 This action may be mediated by MAVS and NF‐κB or potentially other transcription factors.On the surface, this article shows that RIG‐I, an RNA helicase and pathogen recognition receptor, controls HEV infection in human cells.5 While this finding has implications for clinical treatment of patients with HEV by targeting RIG‐I with activator molecules, the authors elucidate that this outcome is mediated independently of IFN.5 This main observation serves as a launching point for a mechanistic study in the downstream signaling of RIG‐I and its role in IFN‐independent ISG induction. Other groups have shown that RIG‐I overexpression, followed by its activation by viral RNA transfection or actual virus infection can induce IRF3 activation and IFN and ISG expression in hepatoma cells and hepatocytes during infection by hepatitis C virus, another liver‐tropic virus (reviewed in Loo and Gale1). Now Xu et al. report that overexpression of RIG‐I and activation of its signaling actions through transfection of cells with PAMP RNA ligand fails to induce IFN production.5 This observation is validated through bioassays that include assessment of a sensitive IFN‐induced promoter‐luciferase reporter gene (ISRE‐Luc)—in this case when RIG‐I was expressed and activated the ISRE‐Luc promoter was highly induced and ISGs were expressed, but no IFN was produced when RIG‐I was overexpressed and activated.5 Examination of tyrosine phosphorylation of STAT1, a marker of STAT activation, showed that in this context STAT1 was phosphorylated/activated, thus linking RIG‐I to IFN‐independent STAT activation and ISG expression.5 RIG‐I signals downstream by forming a complex with the mitochondrial antiviral signaling (MAVS) adaptor protein.3 Of note is that MAVS located on peroxisomes directs signaling independently of IFN and occurring through NF‐κB, AP‐1, IRF1, and IRF3.6 Because Xu et al. also show that cells lacking IRF3 induce ISGs through the RIG‐I/IFN‐independent pathway, NF‐κB could likely serve as the central transcription factor for gene induction in the IFN‐independent RIG‐I signaling pathway. The presence of NF‐κB binding sites in the promoter of many ISGs, including those whose expression is responsive to this novel RIG‐I pathway as shown by Xu et al., support this notion. Unfortunately, Xu et al. do not address the role of NF‐κB or other transcription factors following RIG‐I activation or overexpression in this IFN‐independent pathway. Importantly, tumor necrosis factor‐alpha (TNFalpha), a major inflammatory cytokine, has been shown to suppress HEV infection/replication through NFκB‐dependent ISRE activation,7 such that it could also play a role here to suppress HEV infection. Further, the genes up‐regulated by RIG‐I overexpression independent of IRF3/7 are distinct from those found to be induced by the IFN‐independent peroxisomal MAVS signaling.6 Thus, it is notable that the authors also did not examine the possibility of direct RIG‐I/HEV RNA interaction as an innate immune effector mechanism suppressing HEV infection. We cannot simply conclude that the antiviral effects of RIG‐I against HEV occur exclusively through the ISGs that are induced by IFN‐independent, possibly NF‐κB‐driven, RIG‐I signaling alone, as RIG‐I itself might bind HEV RNA to disrupt viral replication. However, we can conclude that the innate immune action of RIG‐I goes beyond IRF signaling and IFN induction into a more direct role linking RIG‐I and STAT1. How does RIG‐I directly link with the JAK–STAT pathway? Jiang et al. demonstrated that overexpression of RIG‐I in U937 cells leads to increased STAT1 phosphorylation,8 and Hou et al. showed that knocking down RIG‐I reduced the levels of IFN‐α‐induced STAT1 phosphorylation due to increased association of SHP1 and STAT1.9 Here, the STAT1 tyrosine phosphorylation is occurring independently of IFN. While other cytokines were not investigated, it has been shown that STAT1 can be phosphorylated through noncanonical pathways, such as a toll‐like receptor (TLR)–induced, TRAF6‐dependent mechanism.10 RIG‐I has also been associated with TRAF611; but a RIG‐I/TRAF6/STAT1 pathway has yet to be established, and the authors did not examine such an axis, leaving the RIG‐I/STAT1 connection and resulting STAT1 activation undefined. Xu et al. clearly demonstrated the power of activating RIG‐I with ligand RNA to drive antiviral defenses to suppress HEV infection as transfection of cells with PAMP RNA served to trigger RIG‐I activation and signaling through the IFN‐independent RIG‐I pathway. Overall, this work underscores the important role of RIG‐I in innate antiviral immunity and defines RIG‐I as a therapeutic target to control virus infection. Shifting away from IFN‐α as the frontline antiviral is critical to reducing harmful side effects and additional pathogenesis in patients most in need of antiviral treatments, such as transplant recipients. Targeting RIG‐I to engage this novel IFN‐independent pathway therefore could have utility for development of antiviral therapeutics that dispense with IFN altogether. Many groups and biotech companies are developing RIG‐I agonists for clinical applications. Additional studies, like this one by Xu et al., are required to fully define the nature and players within the IFN‐independent pathway for understanding its full application to antiviral immunity. As the authors state, "RIG‐I and its downstream pathways form a large complex web." We need to understand this web and the crosstalk among the various signaling pathways to effectively treat viral diseases. We need new frontline treatments to combat emerging infectious diseases, which are largely RNA viruses. Elucidating the mechanism of action of ISGs will help define these webs and inform us about generating new antiviral treatments. Author names in bold designate shared co‐first authorship.