IFITM3 inhibits virus-triggered induction of type I interferon by mediating autophagosome-dependent degradation of IRF3

Abstract

Interferon-induced transmembrane protein 3 (IFITM3) is a restriction factor that can be induced by viral infection and interferons (IFNs). It inhibits the entry and replication of many viruses, which are independent of receptor usage but dependent on processes that occur in endosomes. In this study, we demonstrate that IFITM3 plays important roles in regulating the RNA-virus-triggered production of IFN-β in a negative-feedback manner. Overexpression of IFITM3 inhibited Sendai virus-triggered induction of IFN-β, whereas knockdown of IFITM3 had the opposite effect. We also showed that IFITM3 was constitutively associated with IRF3 and regulated the homeostasis of IRF3 by mediating the autophagic degradation of IRF3. These findings suggest a novel inhibitory function of IFITM3 on the RNA-virus-triggered production of type I IFNs and cellular antiviral responses.

Keywords: IFITM3; IFNs; IRF3; autophagy.

Figure 1

Overexpression of IFITM3 inhibits virus-triggered induction of IFN-β. (a and c) HEK293 cells were transfected with the indicated reporters and IFITM3 (a) or IFITM1/2 (c) expression plasmids (0, 25, 50 ng per 5 × 10⁵ cells). Twenty-four hours after transfection, the cells were infected with SeV or left untreated for 12 h before luciferase assays were performed. (b) HEK293 cells were transfected with the IRF1 promoter reporter and the indicated plasmids. The cells were treated with IFN-γ or left untreated before performing the luciferase assays. (d–g) HEK293 cells were transfected with control or Flag-IFITM3 (100 ng per 10⁶ cells) plasmid for 24 h. The cells were infected with SeV (d and f) for 10 h or transfected with poly(I:C) (e and g) followed by real-time PCR (d and e) or ELISA analysis (f and g). (a–f) Graphs show the means±s.d., n=3, *P<0.05, **P<0.01.

Figure 2

Knockdown of IFITM3 potentiates virus-triggered induction of IFN-β and inhibits viral replication. (a) HeLa cells were transfected with the control or indicated RNAi constructs. Cell lysates were analyzed on immunoblots with the indicated antibodies. Graphs show the means±s.d., n=3, *P<0.05, **P<0.01. (b and c) HEK293 cells were transfected with the indicated RNAi constructs together with the indicated reporter plasmids. The cells were then infected with SeV (b) or IFN-γ (c) for 12 h before luciferase assays were performed. (d–g) HEK293 cells were transfected with the indicated RNAi constructs. Cells were then infected with SeV (d and f) for 10 h or transfected with poly(I:C) (e and g) before real-time PCR (d and e) or ELISA (f and g) was performed. (h) HEK293 cells were transfected with control or IFITM3-RNAi construct for 36 h and then transfected with poly(I:C) for 24 h. The supernatants were collected and applied to Vero cells for 24 h. These Vero cells were infected with GFP-VSV or GFP-NDV (MOI=0.1) for 24 h and visualized by microscopy or analyzed on immunoblots with the indicated antibodies.

Figure 3

IFITM3 regulates virus-triggered signaling at the level of IRF3. (a) HEK293 cells were transfected with the indicated expression plasmids together with control or IFITM3 plasmids for 24 h. Real-time PCR was performed 24 h later. (b) Stable IFITM3-knockdown HEK293 cells were transfected with the indicated expression plasmids and then analyzed by real-time PCR. Graphs show means±s.d., n=3, *P<0.05, **P<0.01. (c and d) Stable IFITM3-knockdown HEK293 cells were infected with SeV or left uninfected for 8 h. The cell lysates were separated by native (top) or SDS (bottom) PAGE and analyzed on immunoblots with the indicated antibodies (c). The ratio of the IRF3 dimer to total IRF3 was calculated by measuring the grayscale values of the bands (d). (e) IRF3 mRNA levels in stable IFITM3-knockdown HEK293 cells after SeV stimulation were measured by real-time PCR. (f and g) HEK293 cells were transfected with IFITM3 plasmid (f) or IFITM3-RNAi construct (g). The cells were then infected with SeV and analyzed on immunoblots with the indicated antibodies. The ratio of IRF3 or TBK1 to actin was calculated by measuring the grayscale values of the bands and is shown in the lower panels. (h) HEK293 cells were transfected with increased amounts of IFITM1 or IFITM2 plasmids. The endogenous level of IRF3 was then detected by immunoblotting.

Figure 4

IFITM3 Associates with IRF3. (a) HEK293 cells were transfected with Flag-IFITM3 plasmid and infected with SeV. The cell lysates were tested by coimmunoprecipitation and analyzed on immunoblots with the indicated antibodies. (b) HEK293 cells were transfected with HA-IFITM3 and Flag-IRF3 or Flag-IRF3-5A plasmid. The cell lysates were coimmunoprecipitated and analyzed by immunoblots with the indicated antibodies. (c) Glutathione-Sepharose beads coupled to GST-IFITM3 were incubated with HEK293 cell extracts expressing Flag-IRF3 or Flag-IRF3-5A. Proteins bound to the beads were analyzed by western blotting. The experiment was repeated twice with comparable results. (d) Mapping of the minimal interaction domains between IRF3 and IFITM3. HEK293 cells (1 × 10⁷) were transfected with the indicated plasmids. Coimmunoprecipitation and immunoblots were performed. (e) HeLa cells were treated with IFNα (upper panel) or transfected with poly(I:C) (lower panel) for the indicated times. Cell lysates were then applied to the immunoblot.

Figure 5

IFITM3 mediates autophagic degradation of IRF3. (a) Stable IFITM3-knockdown HEK293 cells were transfected with the indicated plasmids and treated with 3-MA or MG132 for 6 h before an immunoblot analysis was performed. (b) HEK293 cells were transfected with increasing amounts of Flag-IFITM3 and analyzed on immunoblots with the indicated antibodies. (c and d) HeLa cells transfected with the indicated plasmids were infected with SeV for 12 h or left untreated and then analyzed under immunofluorescence microscopy. (e) HEK293 cells were transfected with the indicated plasmids and applied to the coimmunoprecipitation and immunoblot experiments. (f) HeLa cells were transfected with Flag-IFITM3 and LC3-GFP plasmids, or infected with SeV. Immunofluorescence assays were performed using anti-IRF3 antibody as the primary antibody. (g) Stable IFITM3-knockdown HEK293 cells were infected with SeV and treated with CHX for the indicated times before immunoblot analysis was performed (left panel). The expression levels of IRF3 and actin were semi-quantified by measuring the grayscales of the bands on the western blots. The normalized expression of IRF3 is shown in the line chart (right panel).