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

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.

Keywords: DNA; Host-Pathogen Interaction; Innate Immunity; Interferon; Pattern Recognition Receptor (PRR); RIG-I-like Receptor (RLR); RNA; Toll-like Receptor (TLR); Transcription Regulation; Virus.

FIGURE 1.

Stable knockdown of IFI16 by lentiviral transduction. THP-1 (A) or U2OS cells (B and C) were targeted with shRNA against the coding sequence of IFI16 (IFI16 CDS-KD), the 3′-UTR of IFI16 (IFI16 3′UTR-KD), or empty vector control plasmid by lentiviral transduction. Stable clones were selected and monitored for IFI16 expression by immunoblotting and q-RT-PCR. *, p < 0.05 assessed by two-tailed t test compared with empty vector control. Data are represented as mean ± S.E. (error bars). Data represent three biological replicates.

FIGURE 2.

IFI16 knockdown cells display an abrogated type I IFN response to various DNA stimuli. IFI16 knockdown THP-1 cells were challenged with poly(dAdT) (A), HSV 60-mer (B), or HSV-1 virus (C) for 6 h. Levels of IFN-β were measured by q-RT-PCR. IFI16 knockdown U2OS cells were challenged with poly(dAdT) (D) or HSV 60-mer (E) for 6 h, and IFN-β levels were measured by q-RT-PCR. F, IFI16 knockdown U20S cells were challenged with poly(dAdT), and IFN-α levels were measured by q-RT-PCR and normalized to HPRT. *, p < 0.05 assessed by two-tailed t test compared with empty vector control. Data are represented as mean ± S.E. (error bars). Data represent three biological replicates. KD, knockdown.

FIGURE 3.

IFI16-dependent IFN production occurs independently of NF-κB. A, B, and C, IFI16 knockdown THP-1 and U2OS cells were challenged with poly(dAdT), VACV 70-mer, or HSV 60-mer for 12 h. Levels of IFN-α (A and B) and RANTES (C) were measured by ELISA. D and E, IFI16 knockdown THP-1 cells were challenged with poly(dAdT) or HSV 60-mer or infected with HSV-1 for 12 h. Levels of IL-6 (D) and IL-1β (E) were measured by ELISA. F, IFI16 knockdown cells were challenged with poly(dAdT) or infected with HSV-1 for 6 h. Cells were stained with calcein for 1 h, and viability was determined by uptake of calcein stain and FITC fluorescence. *, p < 0.05 assessed by two-tailed t test compared with empty vector control. Data are represented as mean ± S.E. (error bars). Data represent three biological replicates. Bars without * are not significant. KD, knockdown.

FIGURE 4.

IFI16 addback rescues the type I IFN phenotype. IFI16 CDS (A) or 3′-UTR knockdown (B) THP-1 cells were transduced with IFI16 plasmid to create a stable addback cell line. Levels of IFI16 were monitored by q-RT-PCR or by immunoblotting. C and D, cells were transfected with poly(dAdT), HSV 60-mer, VACV 70-mer, or immunostimulatory DNA sequence (ISD) for 6 h for q-RT-PCR or 12 h for ELISA. Levels of IFN-α and IFN-β were measured by ELISA and q-RT-PCR, respectively. *, p < 0.05 assessed by two-tailed t test compared with empty vector control or knockdown versus addback. Data are represented as mean ± S.E. (error bars). Data represent three biological replicates. Bars without * are not significant. KD, knockdown.

FIGURE 5.

IFI16 knockdown cells display an abrogated type I IFN response to various RNA stimuli. A, B, and C, IFI16 stable knockdown THP-1 or U2OS cells were challenged with Sendai virus for 6 (for q-RT-PCR assay of IFN-β levels) or 12 h (for IFN-α levels measured by ELISA). THP-1 cells were stimulated with cyclic-di-AMP (c-di-AMP) or L. monocytogenes (Listeria) (D), 5′-ppp RNA (E), or human metapneumovirus (HMPV) (F) for 6 h, and IFN-β levels were determined by q-RT-PCR. G, THP-1 cells were stimulated with 2′,3′-cGAMP for 12 h, and IFN-α levels were measured by ELISA. H, empty vector (EV) and IFI16 knockdown (KD) cells were challenged with type I IFN or Sendai virus (SeV) for 2 h and monitored for cGAS expression by Western blotting. *, p < 0.05 assessed by two-tailed t test compared with empty vector control. Data are represented as mean ± S.E. (error bars). Data represent three biological replicates. Bars without * are not significant.

FIGURE 6.

IFI16 has a global effect on ISG expression. A, IFI16 knockdown THP-1 cells were challenged with poly(dAdT) or HSV 60-mer for 6 h and monitored for protein expression by immunoblotting. B, IFI16 stable knockdown THP-1 cells were transfected with poly(dAdT) or infected with Sendai virus for 6 h, and RNA was collected. Graphs show selected genes from NanoString analysis. Basal (C) and stimulated (D) levels of RIG-I as determined by NanoString analysis. Data represent one experiment. KD, knockdown; pIRF3, phosphorylated IRF3; pTBK-1, phosphorylated TBK-1.

FIGURE 7.

IFI16 acts at the level of chromatin to modulate IFN-α transcription. A and B, pan-type I IFN was added to cells 2 h before stimulation with Sendai virus (SV) for 2, 8, or 24 h. RIG-I (A) and IFN-α (B) expression was measured by q-RT-PCR. C and D, HEK 293T cells were transfected with a mixture containing 40 ng of TK-Renilla luciferase plus 40 ng of IFN-β or IFN-α4 firefly luciferase and increasing amounts of pRGP-IFI16 as indicated or 80 ng of STING. Luciferase values were monitored and normalized to Renilla values. Values are displayed as -fold change over empty vector control. E, THP-1 cells were stimulated with medium and Sendai virus for 6 h, and nuclear extracts were incubated with biotinylated IFN-α ISRE consensus sequence or biotinylated IFN-α ISRE consensus sequence with increasing amounts of non-biotinylated IFN-α ISRE for 1 h. IFN-α ISRE was immunoprecipitated with streptavidin beads, and the IFI16 protein level was monitored by Western blot analysis. Nuclear extracts were monitored for IFI16 expression by Western blotting. F, cells were stimulated with Sendai virus for 4 h, and a ChIP assay was performed. RNA Pol II recruitment to IFN-α or Il-6 promoter was determined by q-RT-PCR. Data are represented as percent input minus IgG background. Data are representative of three experiments. G, empty vector control and IFI16 knockdown THP-1 cells were stimulated with medium and Sendai virus for 6 h, and nuclear extracts were collected. Nuclear extracts were immunoprecipitated with CBP, and the IRF3 protein level was monitored by Western blotting. Nuclear extracts were monitored for USF2 expression. H, proposed signaling pathway for IFI16. *, p < 0.05 assessed by two-tailed t test compared with empty vector control. Data are represented as mean ± S.E. (error bars). Data represent three biological replicates. Bars without * are not significant. KD, knockdown; RLU, relative luciferase units; IP, immunoprecipitation; IB, immunoblot; Bio, biotinylated; IFNAR, interferon α/β receptor; SeV, Sendai virus.