Axin1: A novel scaffold protein joins the antiviral network of interferon

Abstract

Acute respiratory infection by influenza virus is a persistent and pervasive public health problem. Antiviral innate immunity initiated by type I interferon (IFN) is the first responder to pathogen invasion and provides the first line of defense. We discovered that Axin1, a scaffold protein, was reduced during influenza virus infection. We also found that overexpression of Axin1 and the chemical stabilizer of Axin1, XAV939, reduced influenza virus replication in lung epithelial cells. This effect was also observed with respiratory syncytial virus and vesicular stomatitis virus. Axin1 boosted type I IFN response to influenza virus infection and activated JNK/c-Jun and Smad3 signaling. XAV939 protected mice from influenza virus infection. Thus, our studies provide new mechanistic insights into the regulation of the type I IFN response and present a new potential therapeutic of targeting Axin1 against influenza virus infection.

Keywords: Axin1; JNK/c-Jun; Smad3; influenza virus; interferon.

Figure 1.. Axin1 is reduced during influenza pneumonia.

Mice were intranasally inoculated with H1N1 influenza A/PR/8/34 virus (250 pfu/mouse). The lung tissue and bronchoalveolar lavage (BAL) were collected at 1 to 7 days post infection (dpi). (A) H&E staining of paraffin sections of lungs. Scale bar = 100 μm. (B) Differential immune cell counts in BAL. n=3. (C, D) Relative mRNA expression of viral genes (NP and HA) and IFNβ1 and MX1 in whole lung tissues were measured by real-time PCR and normalized to 18S rRNA. n=3. (E) Axin1 protein levels in the lung tissues were measured by Western blot. (F, G) Human bronchial/tracheal epithelial cells (HBTEC) and THP1-derived macrophages (MФ) were infected with H1N1 influenza A/PR/8/34 virus at an MOI of 0.1 for 24 h. Axin1, Axin2, viral NP and β-actin were determined by Western blot. Axin1 and Axin2 protein levels were quantitated to β-actin. Control: CON. n=3. *p < 0.05, ****p < 0.0001 vs CON.

Figure 2.. Axin1 inhibits virus replication.

(A, B) HEK293 cells were transfected with GFP, Axin1, or Axin2 plasmid (OE-GFP, OE-Axin1 or OE-Axin2) for 24 h (A) and then infected with H1N1 influenza A/PR/8/34 virus (MOI=2) (B). The cells were collected at 8 to 24 h post infection (hpi). Axin1 and Axin2 protein levels were determined by Western blot and mRNA expression of viral genes (NP, M1 and HA) was analyzed by real-time PCR and normalized to β-actin. n=2. (C, D) A549 cells were transfected with 10 μM of siRNA control or siRNA Axin1 (Dharmacon) for 48 h using Lipfectamine RNAimax (C). The cells were then infected with H1N1 influenza A/PR/8/34 virus (MOI=0.01) for 48 h (D). Axin1, Axin2, β-actin and viral NP levels were determined by Western blot and quantified. n=3. *p < 0.05, ***p < 0.001 v.s. siCon. (E) HEK293 cells were transfected with GFP, Axin1 or Axin2 plasmid and infected with 1 MOI of IAV-Gluc, or VSV-Luc for 16 h. The luciferase activity was determined. n=3. *p <0.05, **P<0.01 v.s. OE-GFP. (F, G) HEK293 cells were transfected with GFP, Axin1, or Axin2 plasmid for 24 h and then infected with GFP-labeled RSV (MOI=1) for indicated times. Viral G and M protein levels were measured by Western blot in lysed cells with β-actin as a loading control. Myc-tagged Axin1, Axin2, and GFP protein levels were also determined using anti-myc antibody (F). GFP images of infected cells were taken (G). Scale bar = 100 μm.

Figure 2.. Axin1 inhibits virus replication.

(A, B) HEK293 cells were transfected with GFP, Axin1, or Axin2 plasmid (OE-GFP, OE-Axin1 or OE-Axin2) for 24 h (A) and then infected with H1N1 influenza A/PR/8/34 virus (MOI=2) (B). The cells were collected at 8 to 24 h post infection (hpi). Axin1 and Axin2 protein levels were determined by Western blot and mRNA expression of viral genes (NP, M1 and HA) was analyzed by real-time PCR and normalized to β-actin. n=2. (C, D) A549 cells were transfected with 10 μM of siRNA control or siRNA Axin1 (Dharmacon) for 48 h using Lipfectamine RNAimax (C). The cells were then infected with H1N1 influenza A/PR/8/34 virus (MOI=0.01) for 48 h (D). Axin1, Axin2, β-actin and viral NP levels were determined by Western blot and quantified. n=3. *p < 0.05, ***p < 0.001 v.s. siCon. (E) HEK293 cells were transfected with GFP, Axin1 or Axin2 plasmid and infected with 1 MOI of IAV-Gluc, or VSV-Luc for 16 h. The luciferase activity was determined. n=3. *p <0.05, **P<0.01 v.s. OE-GFP. (F, G) HEK293 cells were transfected with GFP, Axin1, or Axin2 plasmid for 24 h and then infected with GFP-labeled RSV (MOI=1) for indicated times. Viral G and M protein levels were measured by Western blot in lysed cells with β-actin as a loading control. Myc-tagged Axin1, Axin2, and GFP protein levels were also determined using anti-myc antibody (F). GFP images of infected cells were taken (G). Scale bar = 100 μm.

Figure 3.. Axin1 enhances type I IFN response during influenza virus infection.

(A, B) HEK293 cells were transfected with GFP, Axin1, or Axin2 plasmid (OE-GFP, OE-Axin1 or OE-Axin2) for 24 h and then infected with H1N1 influenza A/PR/8/34 virus (MOI=2) for 8 h. mRNA expression of IFNβ1 and OAS1 were measured by real-time PCR and were normalized to β-actin. n=3. ***p < 0.001 v.s. OE-GFP. (C) Western blot was carried out to determine the protein expression of p-STAT1 (Tyr 701) and total STAT1 with and without virus infection (2 hpi). (D) HEK293 cells were transfected with GFP or Axin1 and then infected with H1N1 influenza A/PR/8/34 virus (MOI=2) in the presence of vehicle (DMSO), 30 μM fludarabine or 4 μM ruxolinitib for 16 h. NP mRNA levels were determined by real-time PCR and normalized to β-actin. n=3. *p < 0.05 v.s. vehicle-OE-GFP, †p < 0.05 v.s vehicle-Axin1.

Figure 4.. Axin1 activates JNK/c-Jun pathway and Smad3 signaling.

(A, B) HEK293 cells were transfected with GFP, Axin1, or Axin2 plasmid (OE-GFP, OE-Axin1 or OE-Axin2) for 24 h and infected without (Control) or with (PR/8) H1N1 influenza A/PR/8/34 virus (MOI=1) for 2 h. The protein levels of phosphorylated JNK (p-JNK), total JNK (JNK), phosphorylated c-Jun (p-c-Jun), total c-Jun (Jun), phosphorylated ATF2 (p-ATF2), and total ATF2 (ATF2) were determined by Western blot. Relative band intensities of phosphorylated proteins were quantified and normalized to respective total proteins. n=4. *p < 0.05, **p < 0.01 v.s. OE-GFP. (C) Smad3 signaling reporter and pRL-TK plasmids were transfected together with GFP, Axin1, or Axin2 plasmid into HEK293 cells for 24 h and then infected without (Control) or with (PR/8) H1N1 influenza A/PR/8/34 virus (MOI=1) for 8 h. Dual-luciferase activities was performed. The results were expressed as a ratio of Smad3 signaling reporter Firefly luciferase activity to pRL-TK Renilla luciferase activity. n=3. **p < 0.05, ****p < 0.0001 v.s. OE-GFP. (D) HEK293 cells pretreated with 10 μM SP600125 (JNK inhibitor), 10 μM SIS3 (Smad3 inhibitor), or 0.05% DMSO (Vehicle) for 6 h and were transfected with ISRE reporter together with Axin1 plasmid for 24 h. Cells were then infected with H1N1 influenza A/PR/8/34 virus (MOI=2) for 8 h. Dual-luciferase activities were determined and the ISRE_Luc Firefly luciferase activity was normalized to pRL-TK Renilla luciferase activity. n=3. *p < 0.05 v.s. Control. †p < 0.05, ††p < 0.01 v.s. vehicle.

Figure 5.. XAV939 inhibits virus replication in lung epithelial cells.

(A-C) A549 cells were pretreated with 10 μM XAV939 or 0.05% DMSO (vehicle) for 24 h. Cells were then infected with H1N1 influenza A/PR/8/34 virus (MOI=0.01) for 48 h. Protein levels of NP, NS1 and Axin1 were determined by western blots and quantitated. n=3. *p < 0.05 v.s vehicle. (D) A549 cells were pretreated with 20 μM XAV939 or 0.05% DMSO (vehicle) for 24 h. Cells were then infected with H1N1 influenza A/PR/8/34 virus (MOI=2) for indicated times. Virus titers in culture media from XAV939-treated A549 cells were determined by TCID₅₀ assay. n=3. *p < 0.05 v.s. vehicle. (E, F) Human bronchial/tracheal epithelial cells were pretreated with 10 μM XAV939 or 0.05% DMSO (vehicle) for 24 h and infected with H1N1 influenza A/PR/8/34 virus (MOI=1) for 8 h. The cells were immunostaining with NP antibodies and NP-positive cells were counted. n=3. *p < 0.05 v.s vehicle. (G, H) Hep2 cells were pretreated with 20 μM XAV939 or 0.05% DMSO (vehicle) for 24 h. Cells were then infected with GFP-labeled RSV (MOI=0.1 or 1) for 48 h. Viral G and M protein expression was measured by Western blot in lysed cells with GAPDH as a loading control. GFP images of infected cells were also taken. Scale bar = 100 μm. (I) A549 cells were treated 10 μM XAV939 or 0.05% DMSO (vehicle) for 24 h and then infected with H1N1 influenza A/PR/8/34 virus (MOI=0.01) in the presence of vehicle (DMSO), 30 μM fludarabine or 4 μM ruxolinitib for 24 h. NP mRNA levels were determined by real-time PCR and normalized to β-actin. n=3. *p < 0.05 v.s. respective control.

Figure 6.. XAV939 protects mice from a lethal influenza virus challenge.

Mice were challenged intranasally with a lethal dose of H1N1 influenza A/PR/8/34 virus. XAV939 treatment was given orally and daily, beginning with one day before infection (−1 dpi) and continued until 2 [XAV939 (I)] or 4 [XAV939 (II)] days post infection. Control animals received vehicle alone. (A) The illumination of experiment design. (B) Virus titer in the homogenized infected lungs at 2 dpi was measured by TCID₅₀ assay in MDCK cells and normalized to total protein amount. n=5. *p < 0.05 v.s. vehicle. (C) Survival rate was monitored throughout the course of the study (10-11 mice/group). Mantel-Cox χ² test on Kaplan-Meier survival data was used to compare the survival rate between groups.