Interactions of the Antiviral Factor Interferon Gamma-Inducible Protein 16 (IFI16) Mediate Immune Signaling and Herpes Simplex Virus-1 Immunosuppression

Abstract

The interferon-inducible protein IFI16 has emerged as a critical antiviral factor and sensor of viral DNA. IFI16 binds nuclear viral DNA, triggering expression of antiviral cytokines during infection with herpesviruses. The knowledge of the mechanisms and protein interactions through which IFI16 exerts its antiviral functions remains limited. Here, we provide the first characterization of endogenous IFI16 interactions following infection with the prominent human pathogen herpes simplex virus 1 (HSV-1). By integrating proteomics and virology approaches, we identified and validated IFI16 interactions with both viral and host proteins that are involved in HSV-1 immunosuppressive mechanisms and host antiviral responses. We discover that during early HSV-1 infection, IFI16 is recruited to sub-nuclear puncta and subsequently targeted for degradation. We observed that the HSV-1 E3 ubiquitin ligase ICP0 is necessary, but not sufficient, for the proteasom e-mediated degradation of IFI16 following infection. We substantiate that this ICP0-mediated mechanism suppresses IFI16-dependent immune responses. Utilizing an HSV-1 strain that lacks ICP0 ubiquitin ligase activity provided a system for studying IFI16-dependent cytokine responses to HSV-1, as IFI16 levels were maintained throughout infection. We next defined temporal IFI16 interactions during this immune signaling response. We discovered and validated interactions with the viral protein ICP8 and cellular ND10 nuclear body components, sites at which HSV-1 DNA is present during infection. These interactions may be critical for IFI16 to bind to nuclear viral DNA. Altogether, our results provide critical insights into both viral inhibition of IFI16 and interactions that can contribute to IFI16 antiviral functions.

Fig. 1.

IFI16 is degraded in a proteasome-dependent manner during HSV-1 infection. A, protein levels of IFI16 and ICP0 at different time points of infection with WT HSV-1 (m.o.i. = 10) by Western blotting. B, protein levels of IFI16 and ICP8 during early periods of infection (m.o.i. = 10) with WT HSV-1, UV-irradiated HSV-1, or no infection in HFFs. Uninf., uninfected. C, mRNA expression levels of ifi16 (circles) and icp0 (triangles) in HFF cells infected with WT HSV-1 (m.o.i. = 10) up to 8 hpi as determined by quantitative PCR analysis in HFF cells. Error bars represent S.E. D, Western blot analysis of protein levels of IFI16, DNA-PK_cs, ICP0, and ICP8 in uninfected or WT HSV-1-infected fibroblasts up to 8 hpi (m.o.i. = 10) in the presence or absence of the proteasome inhibitor MG132. U, uninfected.

Fig. 2.

Endogenous IFI16 has dynamic interactions with viral and host proteins following HSV-1 infection. A, proteomic workflow to map protein interactions with endogenous IFI16 in uninfected and WT HSV-1-infected HFFs. The specificity of interaction was assessed using SAINT and the CRAPome database. B, immunoisolates from IFI16 and control IgG IPs were resolved by SDS-PAGE and stained with Coomassie Blue. IFI16 isoforms are indicated by a bracket. C, filtered interactions with IFI16 were assembled using STRING and visualized in Cytoscape. Gene shapes indicate the detection of an interaction in uninfected (Uninfected; diamond), WT HSV-1 infection (square), or both (circle). Node colors indicate spectral enrichment of an interaction in one of the conditions. D, validation of the IFI16-ICP0 interaction by reciprocal co-immunopurification using α-ICP0 antibody and visualized by Western blotting. HFF cells were infected with WT HSV-1 (m.o.i. = 10), harvested at 3 hpi, and incubated with Protein A/G-agarose beads conjugated to either control α-IgG or α-ICP0 antibodies. Uninf., uninfected.

Fig. 3.

ICP0 contributes to, but is not sufficient for, the degradation of IFI16 during HSV-1 infection. A, schematic of WT HSV-1 genome and the immediate-early gene mutations present in the ICP0-RF and d106 strains. The genome encodes unique long (U_L) and unique short (U_S) sequences (black line) and inverted repeat sequences (IR_L and IR_S) flanked by long (TR_L) and short (TR_L) terminal repeat sequences (white boxes). The immediate-early genes are depicted (black arrows showing direction of transcription). The ICP0-RF strain is deficient in functional ICP0 protein and contains C116G and C156A substitutions in each copy of the ICP0 coding sequence. The d106 strain is deficient in functional ICP4, -22, -27, and -47 proteins and contains a transgene cassette with a GFP reporter gene under the control of the HCMV immediate-early promoter (HCMVIEp) substituted into a partial ICP27 deletion (smaller solid gray box). Deletions (larger solid gray boxes) in ICP4 and the promoter element region of ICP22 and -47, TAATGARAT, result in the loss of these functional immediate-early proteins. B, protein levels of IFI16, ICP0, and PML in HFF cells after infection with WT HSV-1, ICP0-RF, or d106 at various hpi (m.o.i. = 10) were measured by Western blot. PML isoforms are highlighted with a bracket. U, uninfected. C and D, protein levels of IFI16-eGFP, ICP0, and PML in IFI16-eGFP inducible HEK293 cells (IFI16−/+) (C) with and without the transient expression of ICP0. Non-expressing ICP0 cells (ICP0−) were transiently transfected with mCherry (D) and uninfected or infected with WT or ICP0-RF HSV-1 at 4 hpi (m.o.i. = 10). E, protein levels of endogenous IFI16, ICP0, and DNA-PK_cs in HFF cells uninfected or infected with WT or ICP0-RF HSV-1 at 4 hpi and at the indicated m.o.i. Uninf., uninfected.

Fig. 4.

IFI16 co-localizes with ICP0 in discrete nuclear puncta during early stages of HSV-1 infection. A, representative immunofluorescence images of fixed HFF cells stained for IFI16 and ICP0 after infection with WT HSV-1, ICP0-RF, or d106 (m.o.i. = 1). B, cells were pretreated with the proteasome inhibitor MG132 and then analyzed as in A. Uninf., uninfected. Co-localization is indicated with white arrows. Scale bars, 5 μm.

Fig. 5.

IFI16 is required for host immune response after HSV-1 infection and is attenuated by ICP0. A, mRNA levels of cytokines in HFF cells uninfected or infected with WT HSV-1, ICP0-RF, or d106 at 6 hpi (m.o.i. = 10). Results were measured by quantitative PCR and are shown as averages (n = 2), and error bars represent S.E. (p ≤ 0.05 (*) and p < 0.01 (**) by Student's t test). B, mRNA levels measured as in A of ifn-β in HFFs stably knocked down for IFI16 by lentivirus-mediated shRNA transduction (right). IFI16 knockdown was confirmed by Western blotting (left). Ctrl, control.

Fig. 6.

Immunoaffinity purifications of IFI16 during ICP0-RF infection reveal novel interactions, including with PML-containing ND10 bodies. A, immunoisolates of IFI16 and control IgG IPs from HFF cells infected with ICP0-RF at 3 and 8 hpi were resolved by SDS-PAGE and stained with Coomassie Blue. B, SAINT-filtered interactions with IFI16 were assembled using STRING and visualized as a network in Cytoscape. Gene shapes indicate the detection of an interaction uniquely in ICP0-RF HSV-1 infection (diamond) or in common with at least one other condition (uninfected = mock or WT HSV-1 infection; circle). Node colors indicate spectral enrichment of an interaction at 3 or 8 h post ICP0-RF HSV-1 infection. C, validation of the IFI16-ICP8 interaction by reciprocal co-immunopurification using α-ICP8 antibody and visualization by Western blotting. HFF cells were infected with WT HSV-1 (m.o.i. = 10), harvested at 8 hpi, and incubated with Protein A/G-agarose beads conjugated to either control α-IgG or α-ICP8 antibodies. Uninf., uninfected.

Fig. 7.

IFI16 is targeted to PML nuclear bodies following HSV-1 infection. Representative immunofluorescence images of HFF cells stained for IFI16 and PML in uninfected cells (Uninf.) or after infection with either WT HSV-1 or ICP0-RF at 3 and 6 hpi (m.o.i. = 1) are shown. Co-localization is indicated with white arrows. Scale bars, 5 μm.