Human Genome-Wide RNAi Screen Identifies an Essential Role for Inositol Pyrophosphates in Type-I Interferon Response

The pattern recognition receptor RIG-I is critical for Type-I interferon production. However, the global regulation of RIG-I signaling is only partially understood. Using a human genome-wide RNAi-screen, we identified 226 novel regulatory proteins of RIG-I mediated interferon- production. Furthermore, the screen identified a metabolic pathway that synthesizes the inositol pyrophosphate 1-IP7 as a previously unrecognized positive regulator of interferon production. Detailed genetic and biochemical experiments demonstrated that the kinase activities of IPPK, PPIP5K1 and PPIP5K2 (which convert IP5 to1-IP7) were critical for both interferon induction, and the control of cellular infection by Sendai and influenza A viruses. Conversely, ectopically expressed inositol pyrophosphate-hydrolases DIPPs attenuated interferon transcription. Mechanistic experiments in intact cells revealed that the expression of IPPK, PPIP5K1 and PPIP5K2 was needed for the phosphorylation and activation of IRF3, a transcription factor for interferon. The addition of purified individual inositol pyrophosphates to a cell free reconstituted RIG-I signaling assay further identified 1-IP7 as an essential component required for IRF3 activation. The inositol pyrophosphate may act by -phosphoryl transfer, since its action was not recapitulated by a synthetic phosphonoacetate analogue of 1-IP7. This study thus identified several novel regulators of RIG-I, and a new role for inositol pyrophosphates in augmenting innate immune responses to viral infection that may have therapeutic applications. Author Summary The innate immune system is critical for viral infection control by host organisms. The type I interferons are a family of major antiviral cytokines produced upon the activation of innate immune pattern recognition receptors (PRRs) by viruses. The RIG-I is a major PRR that uniquely detects RNA viruses within the cytoplasm. In this study, we aimed to discover cellular genes and pathways that play regulatory roles in the transcriptional induction of type I interferon- (IFN). Using a human genome wide RNA interference (RNAi) screening, we identified 226 genes whose expression is important for proper IFN production. Through bioinformatics-based mining of the RNAi screen results, we identified that the cellular pathway synthesizing inositol pyrophosphates, a class of inositol phosphates with high-energy diphosphates, is a key positive regulator of RIG-I mediated IFN production. The kinases IPPK, PPIP5K1 and PPIP5K2, that synthesize inositol pyrophosphate 1-IP7, regulated IFN response in a catalytically dependent manner. Mechanistic studies identified that 1-IP7 synthesis pathway was needed for efficient phosphorylation of IRF3. The DIPP family of inositol pyrophosphate hydrolases negatively regulated the IFN response, upon ectopic expression. In summary, this study generated a global view of the regulation of RIG-I signaling, and identified inositol pyrophosphates as important regulators of antiviral response.