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.
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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)