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

PLoS Pathogens, Feb 2014

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-β 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 β-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.

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

et al. (2014) Human Genome-Wide RNAi Screen Identifies an Essential Role for Inositol Pyrophosphates in Type-I Interferon Response. PLoS Pathog 10(2): e1003981. doi:10.1371/journal.ppat.1003981 Human Genome-Wide RNAi Screen Identifies an Essential Role for Inositol Pyrophosphates in Type-I Interferon Response Niyas Kudukkil Pulloor Sajith Nair Aleksandar D. Kostic Pradeep Bist Jeremy D. Weaver Andrew M. Riley Richa Tyagi Pradeep D. Uchil John D. York Solomon H. Snyder Adolfo Garca- Sastre Barry V. L. Potter Rongtuan Lin Stephen B. Shears Ramnik J. Xavier Manoj N. Krishnan Karen L. Mossman, McMaster University, Canada 0 www.plospathogens.org 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-b 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 to1IP7) 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 b-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. - Funding: MNK was funded by ASTAR and Ministry of Education. SHS was funded by NIH grant MH-18501. RL was funded by the Canadian Institutes of Health Research grant MOP42562. AGS was funded by NIAID grant U19AI083025 and by CRIP (Center for Research on Influenza Pathogenesis), a NIAD-funded Center of Excellence for Influenza Research and Surveillance (CEIRS, contract # HHSN266200700010C). SBS was supported by the Intramural Research Program of the NIH, National Institute of Environmental Health Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. The innate immune system, a primordial yet highly organized defense mechanism, plays critical roles in the host response against RNA viruses. The first step in the innate immune response involves recognition of pathogen-associated molecular patterns by several host encoded pattern recognition receptors (PRR). A key mediator of antiviral immunity is the type-I interferon family of cytokines, which are transcribed upon detection of RNA viruses by the pattern recognition receptors [13]. Cells have developed PRRs that are specialized for detecting pathogens in the cytosol, the site where many RNA viruses replicate. One such PRR is the retinoic acid inducible gene - I (RIG-I) [13]. RIG-I recruits the adaptor protein MAVS to activate a signaling pathway that causes TBK1 to phosphorylate the latent transcription factor IRF3 [47]. Signaling cascades triggered by multiple PRRs indeed converge to activate IRF3. Once phosphorylated, IRF3 dimerizes and translocates to the nucleus, where it forms a complex with the transcriptional coactivators CBP/p300, which together stimulate the expression of type-I interferon [1]. This initiates the antiviral immune responses [810]. An optimal interferon response is essential to control viral infections; however, excessive interferon exposure is detrimental to the human body [11]. Significant research effort has been invested in determining the nature of the positive and negative signaling pathways which regulate the responses that RIG-1 elicits following 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-b (IFNb). Using a (...truncated)


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Niyas Kudukkil Pulloor, Sajith Nair, Aleksandar D. Kostic, Pradeep Bist, Jeremy D. Weaver, Andrew M. Riley, Richa Tyagi, Pradeep D. Uchil, John D. York, Solomon H. Snyder, Adolfo García-Sastre, Barry V. L. Potter, Rongtuan Lin, Stephen B. Shears, Ramnik J. Xavier, Manoj N. Krishnan. Human Genome-Wide RNAi Screen Identifies an Essential Role for Inositol Pyrophosphates in Type-I Interferon Response, PLoS Pathogens, 2014, Volume 10, Issue 2, DOI: 10.1371/journal.ppat.1003981