IQGAP2 is a novel interferon-alpha antiviral effector gene acting non-conventionally through the NF-κB pathway

Abstract

Background & aims: Type I interferons (IFN) provide the first line of defense against invading pathogens but its mechanism of action is still not well understood. Using unbiased genome-wide siRNA screens, we recently identified IQ-motif containing GTPase activating protein 2 (IQGAP2), a tumor suppressor predominantly expressed in the liver, as a novel gene putatively required for IFN antiviral response against hepatitis C virus (HCV) infection. Here we sought to characterize IQGAP2 role in IFN response.

Methods: We used transient small interfering RNA knockdown strategy in hepatic cell lines highly permissive to JFH1 strain of HCV infection.

Results: We found that IQGAP2 acts downstream of IFN binding to its receptor, and independently of the JAK-STAT pathway, by physically interacting with RelA (also known as p65), a subunit of the NF-κB transcription factor. Interestingly, our data reveal a mechanism distinct from the well-characterized role of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) in IFN production. Indeed, IFN alone was sufficient to stimulate NF-κB-dependent transcription in the absence of viral infection. Finally, both IQGAP2 and RelA were required for the induction by IFN of a subset of IFN-stimulated genes (ISG) with known antiviral properties.

Conclusions: Our data identify a novel function for IQGAP2 in IFN antiviral response in hepatoma cells. We demonstrate the involvement of IQGAP2 in regulating ISG induction by IFN in an NF-κB-dependent manner. The IQGAP2 pathway may provide new targets for antiviral strategies in the liver, and may have a wider therapeutic implication in other disease pathogeneses driven by NF-κB activation.

Lay summary: In this study, we identify a novel mechanism of action of interferon involving the IQGAP2 protein and the NF-κB pathway that is ultimately protective against hepatitis C virus infection. This newly identified pathway functions independently of the well-known STAT pathway and may therefore provide new targets for antiviral strategies in the liver.

Keywords: Antiviral response; Hepatitis C virus (HCV); IQGAP protein.

Figure 1. IQGAP2 silencing rescues HCV infection from IFN

(A) Huh7.5.1 cells were transfected with Non-targeting siRNA (NT) or IQGAP2 siRNA (#1 to 4 individually or pooled) and IQGAP2 silencing was evaluated 72 hours post-transfection (p.t) on total RNA extracts by qRT-PCR (Left) or on total protein extracts by Western Blotting (Right). (B) Huh7.5.1 cells were transfected with the indicated siRNA and cellular ATP levels were measured 4 days p.t. (C–E) Huh7.5.1 cells were transfected with the indicated siRNA, 2 or 3 days later, cells were treated with 100 IU/ml IFN for 24 hours and then infected with JFH1 at a MOI of 1 TCID₅₀/cell for an extra time as indicated or 48 hours. HCV infection was measured as (C) percentage of infected cells by fluorescence microscopy after HCV Core protein staining, (D) intracellular HCV RNA level in total RNA extracts by qRT-PCR and (E) infectious HCV particle production in the extracellular medium by TCID₅₀ assay.

Figure 2. RelA silencing rescues HCV infection from IFN

(A) Huh7.5.1 cells were transfected with the indicated siRNA and silencing was evaluated 72 hours p.t on total RNA extracts by qRT-PCR (Left) and on total protein extracts by Western Blotting (Right) (B–D) Huh7.5.1 cells were transfected with the indicated siRNA for 2 days, treated with IFN for 24 hours, infected with JFH1 at a MOI of 1 TCID₅₀/cell for an extra 48 hours and HCV infection was measured as (B) percentage of infected cells by fluorescence microscopy after HCV Core protein staining, (C) intracellular HCV RNA level in total RNA extracts by qRT-PCR and (D) infectious HCV particle production in the extracellular medium by TCID₅₀ assay.

Figure 3. IQGAP2 or RelA silencing increases permissiveness to HCV in Huh7/CD81^High cells but not in Huh7.5.1 cells

(A–C) Huh7.5.1 cells or (D–F) Huh7/CD81^High cells were transfected with the indicated siRNA, infected 3 days later with JFH1 at a MOI of 0.5 TCID₅₀/cell for Huh7.5.1 cells or at a MOI of 1 TCID₅₀/cell for Huh7/CD81^High. Two days p.i., HCV infection was measured as (A,D) percentage of infected cells by fluorescence microscopy after HCV Core protein staining, (B,E) intracellular HCV RNA level in total RNA extracts by qRT-PCR and (C,F) infectious HCV particle production in the extracellular medium by TCID₅₀ assay.

Figure 4. IQGAP2 or RelA silencing inhibits NF-κB activation by IFN

(A) Huh7.5.1/pNF-κB-GFP cells were treated with 100 IU/ml IFN, mock-treated or treated with 10 ng/ml of TNFα for 48h and analyzed by fluorescence microscopy after nuclear DAPI staining. (B) Huh7.5.1/pNF-κB-GFP cells were treated for 48 hours with the indicated dose of IFN or 10 ng/ml TNFα or 10 µg/ml PBS-BSA corresponding to the concentration of PBS-BSA in IFN 1000 IU/ml solution (Vehicle) or mock-treated (Mock) and analyzed by Flow Cytometry. (C) Huh7.5.1/pNF-κB-GFP cells were transfected with the indicated siRNA, 72 h later, cells were treated with IFN or mock-treated for 48 h and analyzed by flow cytometry (Left) and representative fluorescent pictures are presented for the IFN treated cells after nuclear DAPI staining (Right). (D) Huh7.5.1 cells were transfected with the indicated siRNA for 72 h, treated with IFN or mock-treated for 20 min. RelA activation by phosphorylation at Ser 536 was assessed on total protein extracts by Western blotting.

Figure 5. IQGAP2 or RelA silencing decreases the induction by IFN of a subset of antiviral ISG

Huh7.5.1 cells were transfected with the indicated siRNA and treated 3 days later with IFN or Mock-treated for 8 hours. The indicated ISGs induction levels were measured on total RNA extracts by qRT-PCR. All values are normalized to Huh7.5.1 cells transfected with NT-siRNA and Mock-treated, set as 1. The dotted line separates previously reported NF-κB target genes on the left, from the genes unrelated to NF-κB on the right.

Figure 6. IQGAP2 and STAT1 have an additive effect on the induction by IFN of a subset of ISG

(A) Huh7.5.1 cells were transfected with the indicated siRNA and silencing was evaluated 72 hours p.t on total RNA extracts by qRT-PCR (Left) and on total protein extracts by Western Blotting (Right). (B) and (C) Huh7.5.1 cells were transfected with the indicated siRNA and treated 3 days later with IFN or Mock-treated. The indicated ISG induction levels were measured 8 hours p.t. unless stated otherwise on total RNA extracts by qRT-PCR. All values are normalized to Huh7.5.1 cells transfected with NT-siRNA (or double NT-siRNA) and Mock-treated, set as 1.