Interferon γ-inducible Protein (IFI) 16 Transcriptionally Regulates Type I Interferons and Other Interferon-stimulated Genes and Controls the Interferon Response to both DNA and RNA Viruses

Mikayla R Thompson,Shruti Sharma,Maninjay Atianand,Søren B Jensen,Susan Carpenter,David M Knipe,Katherine A Fitzgerald,Evelyn A Kurt-Jones

doi:10.1074/jbc.m114.554147

Abstract

The interferon γ-inducible protein 16 (IFI16) has recently been linked to the detection of nuclear and cytosolic DNA during infection with herpes simplex virus-1 and HIV. IFI16 binds dsDNA via HIN200 domains and activates stimulator of interferon genes (STING), leading to TANK (TRAF family member-associated NF-κB activator)-binding kinase-1 (TBK1)-dependent phosphorylation of interferon regulatory factor (IRF) 3 and transcription of type I interferons (IFNs) and related genes. To better understand the role of IFI16 in coordinating type I IFN gene regulation, we generated cell lines with stable knockdown of IFI16 and examined responses to DNA and RNA viruses as well as cyclic dinucleotides. As expected, stable knockdown of IFI16 led to a severely attenuated type I IFN response to DNA ligands and viruses. In contrast, expression of the NF-κB-regulated cytokines IL-6 and IL-1β was unaffected in IFI16 knockdown cells, suggesting that the role of IFI16 in sensing these triggers was unique to the type I IFN pathway. Surprisingly, we also found that knockdown of IFI16 led to a severe attenuation of IFN-α and the IFN-stimulated gene retinoic acid-inducible gene I (RIG-I) in response to cyclic GMP-AMP, a second messenger produced by cyclic GMP-AMP synthase (cGAS) as well as RNA ligands and viruses. Analysis of IFI16 knockdown cells revealed compromised occupancy of RNA polymerase II on the IFN-α promoter in these cells, suggesting that transcription of IFN-stimulated genes is dependent on IFI16. These results indicate a broader role for IFI16 in the regulation of the type I IFN response to RNA and DNA viruses in antiviral immunity.

Highlights

interferon ␥-inducible protein 16 (IFI16) binds dsDNA and elicits a type I interferon response
DNA-induced Type I Interferon Production Is Dependent on IFI16 —The type I IFN response induced by DNA in the human THP-1 monocytic cell line or mouse RAW 264.7 cells is dependent on IFI16 or its proposed mouse orthologue IFI204 [25]. siRNA studies have shown that transient knockdown of IFI16 in THP-1 cells leads to a decrease in IFN production and reduced IRF3 and NF-␬B activation in response to herpes simplex virus 1 (HSV-1) as well as to DNA ligands [25]
Because cyclic GMP-AMP synthase (cGAS)/ cGAMP signaling should bypass the need for IFI16 as a DNA sensor and directly activate stimulator of interferon genes (STING), these results provide further evidence that IFI16 can sense DNA leading to STING activation IFI16 regulates IFNstimulated gene (ISG) expression independently of its role in DNA recognition

Summary

Background

IFI16 binds dsDNA and elicits a type I interferon response. Results: IFI16 knockdown cells show a decrease in interferon production and ISG expression in response to DNA and RNA ligands and cyclic dinucleotides. To better understand the role of IFI16 in coordinating type I IFN gene regulation, we generated cell lines with stable knockdown of IFI16 and examined responses to DNA and RNA viruses as well as cyclic dinucleotides. Analysis of IFI16 knockdown cells revealed compromised occupancy of RNA polymerase II on the IFN-␣ promoter in these cells, suggesting that transcription of IFN-stimulated genes is dependent on IFI16 These results indicate a broader role for IFI16 in the regulation of the type I IFN response to RNA and DNA viruses in antiviral immunity. Since the initial discovery of IFI16 and IFI204, compelling recent evidence from both human and mouse cells using RNAi, TALEN (transcription activator-like effector nuclease) knockdown approaches, and gene knock-outs has convincingly demonstrated the importance of a DNA-sensing enzyme called cyclic GMP-AMP synthase (cGAS) in the cytosolic response to dsDNA. These studies describe a regulatory role for IFI16 in the transcriptional regulation of IFN-␣ gene expression and subsequent IFN-inducible gene expression in response to a broader array of IFN inducers than previously anticipated, expanding the function of IFI16 beyond strictly sensing microbial DNA

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION