Nuclear IFI16 induction of IRF-3 signaling during herpesviral infection and degradation of IFI16 by the viral ICP0 protein

Abstract

Innate sensing of microbial components is well documented to occur at many cellular sites, including at the cell surface, in the cytosol, and in intracellular vesicles, but there is limited evidence of nuclear innate signaling. In this study we have defined the mechanisms of interferon regulatory factor-3 (IRF-3) signaling in primary human foreskin fibroblasts (HFF) infected with herpes simplex virus 1 (HSV-1) in the absence of viral gene expression. We found that the interferon inducible protein 16 (IFI16) DNA sensor, which is required for induction of IRF-3 signaling in these cells, is nuclear, and its localization does not change detectably upon HSV-1 d109 infection and induction of IRF-3 signaling. Consistent with the IFI16 sensor being nuclear, conditions that block viral DNA release from incoming capsids inhibit IRF-3 signaling. An unknown factor must be exported from the nucleus to activate IRF-3 through cytoplasmic STING, which is required for IRF-3 activation and signaling. However, when the viral ICP0 protein is expressed in the nucleus, it causes the nuclear relocalization and degradation of IFI16, inhibiting IRF-3 signaling. Therefore, HSV-1 infection is sensed in HFF by nuclear IFI16 upon release of encapsidated viral DNA into the nucleus, and the viral nuclear ICP0 protein can inhibit the process by targeting IFI16 for degradation. Together these results define a pathway for nuclear innate sensing of HSV DNA by IFI16 in infected HFF and document a mechanism by which a virus can block this nuclear innate response.

Fig. 1.

Establishment of an HSV-1 infection system to study IRF-3 signaling. (A) Induction of ISG54. HFF were infected with increasing MOIs of d106 or d109 virus, and total cellular RNA was harvested at 6 hpi. ISG54 mRNA levels were normalized to γ-actin levels and further normalized to mock-infected samples. Values are shown as mean ± SEM. (B) Viral genome numbers in infected cells. RAW264.7 cells were infected with d106 or d109 at an MOI of 1. (C) Total cellular DNA was harvested at 2 hpi, and relative viral DNA levels were analyzed by quantitative PCR using primers specific for the ICP8 gene. Levels were normalized to cellular GAPDH gene levels. Shown are ICP8 gene levels from RAW264.7 cells (Left) or HFF (Right) infected with d106 at an MOI of 5 and 50 or with d109 at an MOI of 1 and 10. Values are shown as mean ± SEM (n = 3).

Fig. 2.

HSV-1 induces IRF3-responsive genes in the absence of viral gene expression. RNA was harvested from HFF infected with d106 (MOI 10 or 50) or d109 (MOI 10) at 2, 4, and 6 hpi. RNA levels for IFNβ (A), ISG54 (B), ISG56 (C), and IL-6 (D) were determined by quantitative RT-PCR. Cellular RNA levels were normalized to γ-actin levels and further normalized to control 6-hpi values. Values are shown as mean ± SEM (n = 4). *P ≤ 0.05, compared with d106-infected cells (Student t test).

Fig. 3.

Induction of IRF-3–responsive genes requires release of viral DNA from incoming capsids. HFF were pretreated with DMSO, TPCK, or MG132 for 30 min before and throughout infection with d109 at an MOI of 10. (A and B) Cellular RNA was harvested at 6 hpi, and IFNβ (A) and ISG54 (B) levels were determined by quantitative RT-PCR. RNA levels were normalized to 18S rRNA followed by normalization to corresponding mock-treatment values. *P ≤ 0.05, compared with DMSO-treated cells (Student t test). Values are shown as mean ± SEM (n = 3). (C) Western blot analysis of phospho-TBK1 (pTBK1) and tubulin levels in mock- or d109-infected cells not treated or treated with drugs.

Fig. 4.

IFI16 and STING are required for HSV-1–induced IFN-β induction. HFF were treated with siControl, siIFI16, or siRNA targeting STING (siSTING) for 3 d following infection with d109 (MOI 10) or SeV (100 HAU/8 × 10^5 cells). Cells were harvested for RNA and protein analysis at 6 hpi. (A and B) Western blot analysis of (A) IFI16 and pTBK1 and (B) STING and GAPDH levels in siRNA-treated cells. RNA samples from d109 (C) or SeV (D) infected cells were analyzed by quantitative RT-PCR. IFNβ RNA levels were normalized to 18S rRNA followed by normalization to corresponding mock values. Values are shown as mean ± SEM (n = 3).

Fig. 5.

IFI16 is localized in the nucleus during HSV-1 infection. (A) Fibroblasts infected with d109 virus were fixed and stained with an antibody specific for IFI16 (shown in green) at 4 hpi. Right panel is a higher magnification of a representative cell in the left panel. (Scale bar, 5 μm.) (B) Nuclear and cytoplasmic fractions were prepared from cells infected with d109 and d106 and were analyzed by immunoblot for ICP0, IFI16, and STING localization. Tubulin and lamin A/C represent the fractionation efficiency. (C) Cells were treated with leptomycin B for 30 min before and throughout infection with d109 or SeV for 8 h. Flow cytometry then was used to examine the phosphorylation status of TBK1. Values are shown as mean ± SEM (n = 4).

Fig. 6.

ICP0 inhibits nuclear accumulation of activated IRF-3. HFF were mock-infected or infected with the d109 or d106 viruses. Nuclear and cytoplasmic fractions were prepared at 4, 6, and 8 hpi. Fractions were probed using antibodies specific for ICP0, IRF-3, and phospho-IRF-3 (Ser396). Fractionation efficiency was determined by localization of GAPDH (cytoplasm) and lamin A/C (nucleus).

Fig. 7.

ICP0 sequesters nuclear IRF-3. HFF were infected with d109 (MOI 10) or d106 viruses (MOI 10 or 50) for 6 hpi. Samples were fixed and stained using antibodies specific for ICP0 (shown in green) and IRF-3 (shown in red). (Scale bar, 5 μm.)

Fig. 8.

ICP0 expression promotes IFI16 relocalization and degradation. HFF cells were infected with d109, d106, or WT KOS strain virus. (A) Whole-cell lysates were harvested at 6 hpi and probed for ICP0, IFI16, IRF-3, phospho-IRF-3 (Ser396), STING, and GAPDH using specific antibodies. (B) Infected cells were fixed and stained at 2, 4, and 6 hpi for IFI16 (shown in green) and ICP0 (shown in red). MOIs for d106 are stated in parentheses.

Fig. 9.

ICP0 promotes the degradation of IFI16 in a proteasome- and RING finger-dependent manner. (A) HFF were pretreated with DMSO (control) or MG132 for 30 min before and throughout infection with WT HSV (KOS) virus at an MOI of 10. Whole-cell lysates were harvested at 8 hpi. Western blot analysis of ICP0, IFI16, and tubulin is shown for each of these samples. (B) Cells were infected with the ICP0 RING-finger mutant virus (KOS.RFm) and its rescue (KOS.RFr). Whole-cell lysates were harvested and analyzed as in A.

Fig. 10.

Model of nuclear HSV-1 DNA sensing and inhibition by ICP0. HSV-1 fusion at the plasma membrane or via endosomal compartments deposits viral capsids in the cytoplasm. Capsids traffic to nuclear pores where viral DNA is released into the nucleus. Nuclear IFI16 senses accumulating viral DNA, inducing a nuclear-to-cytoplasmic signaling cascade activating IRF-3, which dimerizes and translocates to the nucleus. Immediate-early expression of ICP0 sequesters IRF-3 from cellular promoters and promotes degradation of IFI16 to inhibit IFN-β expression.

Fig. P1.

Model of nuclear HSV-1 DNA sensing and inhibition by ICP0. HSV-1 fusion at the plasma membrane or via endosomal compartments deposits viral capsids in the cytoplasm. Capsids traffic to nuclear pores where viral DNA is released into the nucleus. Nuclear IFI16 (blue) senses accumulating viral DNA, inducing a nuclear-to-cytoplasmic signaling cascade activating IRF-3 (green), which dimerizes and translocates to the nucleus. Immediate-early expression of ICP0 (red) sequesters IRF-3 from cellular promoters and promotes degradation of IFI16 to inhibit IFN-β expression.