Influenza A Virus NS1 Protein Inhibits the NLRP3 Inflammasome

Abstract

The inflammasome is a molecular platform that stimulates the activation of caspase-1 and the processing of pro-interleukin (IL)-1β and pro-IL-18 for secretion. The NOD-like receptor family, pyrin domain containing 3 (NLRP3) protein is activated by diverse molecules and pathogens, leading to the formation of the NLRP3 inflammasome. Recent studies showed that the NLRP3 inflammasome mediates innate immunity against influenza A virus (IAV) infection. In this study, we investigated the function of the IAV non-structural protein 1 (NS1) in the modulation of NLRP3 inflammasome. We found that NS1 proteins derived from both highly pathogenic and low pathogenic strains efficiently decreased secretion of IL-1β and IL-18 from THP-1 cells treated with LPS and ATP. NS1 overexpression significantly impaired the transcription of proinflammatory cytokines by inhibiting transactivation of the nuclear factor-κB (NF-κB), a major transcription activator. Furthermore, NS1 physically interacted with endogenous NLRP3 and activation of the NLRP3 inflammasome was abrogated in NS1-expressing THP-1 cells. These findings suggest that NS1 downregulates NLRP3 inflammasome activation by targeting NLRP3 as well as NF-κB, leading to a reduction in the levels of inflammatory cytokines as a viral immune evasion strategy.

Fig 1. NS1 variants and their characterization.

Plasmids expressing the NS1 variants NS1-HK and NS1-WSN were transiently transfected into HEK293T cells (A and B). (A) Expression of NS1 variants in the cells was examined by western blot analysis using an anti-MYC antibody. Tubulin was used as a loading control. (B) HEK293T cells expressing NS1 variants were fixed at 24 hr post-transfection, immunostained with anti-MYC (red) for NS1 detection, and examined under a confocal laser scanning microscope. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (C) Transfection of HEK293T cells was performed in triplicate by using the reporter construct (IFN-β-luc) and the NS1 expression plasmids (300 and 500 ng); the β-galactosidase expression plasmid served as an internal control. At 24 hr post-transfection, 20 HA of SeV was infected. After 16 hr infection, IFN-β-luc reporter activities were measured and normalized to β-galactosidase activities. Statistical analysis was performed using Student’s t-test (*** denotes a p-value of <0.005.).

Fig 2. Expression of NS1 variants in THP-1 macrophage cells and their inhibitory effects on NLRP3 inflammasome.

(A) NS1 variants were expressed in THP-1 cells by using a lentivirus vector transduction system. After transduction, the expression of each NS1 variant in differentiated THP-1 cells was evaluated by western blot analysis. (B) NS1 variant-transduced THP-1 macrophage cells were immunostained with anti-MYC (red) for NS1 detection. Localization was examined under a confocal laser scanning microscope. Nuclei were stained with DAPI (blue). Scale bar, 20 μm. (C and D) Transduced THP-1 macrophage cells were differentiated with TPA and then treated with LPS (1 μg/mL) for 6 hr, followed by treatment with ATP (2.5 mM) for 15 min. The supernatants were harvested and subjected to ELISA to quantify IL-1β and IL-18. Data represent the mean and standard deviation. Statistical analysis was performed using Student’s t-test to analyze the differences between control and NS1-expressing samples (*** denotes a p-value of <0.005.).

Fig 3. Downregulation of the transcription of proinflammatory cytokines by NS1 variants.

NS1 variants-transduced THP-1 macrophage cells were differentiated with TPA and then treated with LPS (1 μg/mL) for 6 hr and subsequently with ATP (2.5 mM) for 15 min. (A-C) mRNA levels of pro-IL-1β (A), pro-IL-18 (B), and TNF-α (C) were analyzed by quantitative real-time PCR using gene-specific primers. (D) The supernatants were harvested and subjected to ELISA to quantify TNF-α. Data represent the mean and standard deviation. Statistical analysis was performed using Student’s t-test to analyze the differences between control and NS1-expressing samples (* denotes a p-value of <0.05; ** denotes a p-value of <0.01; *** denotes a p-value of <0.005).

Fig 4. Downregulation of NF-κB activation by NS1 variants.

(A) Downregulation of p65-mediated 2xκB-luc activity by NS1 variants. HEK293T cells were transfected with reporter constructs (2xκB-luc) and NS1 expression plasmids (300 and 500 ng) in the presence of p65-expressing plasmid. Each transfection was performed in triplicate, with the β-galactosidase expression plasmid serving as an internal control. After 26 hr transfection, 2xκB-luc reporter gene activities were measured and normalized to β-galactosidase activities. Statistical analysis was performed using Student’s t-test (*** denotes a p-value of <0.005). (B) Inhibition of the NF-κB pathway by NS1 variants in THP-1 macrophage cells. Transduced THP-1 cells were differentiated with TPA treatment, and then treated with LPS (1 μg/mL) for 6 hr, followed by treatment with ATP (2.5 mM) for 15 min. Each indicated proteins were identified by western blot analysis.

Fig 5. Downregulation effect of NS1 variants on the NLRP3 inflammasome and their interaction with NLRP3.

(A) Differentiated THP-1 cells expressing NS1 variants were treated with LPS (1 μg/mL) for 6 hr, followed by treatment with ATP (2.5 mM) for 15 min. The cell lysates were obtained and subjected to western blot analysis using the indicated antibodies. (B and C) NS1 variants interacted with NLRP3, not with ASC or pro-caspase-1. The MYC-NS1-HK (B) or MYC-NS1-WSN (C) construct was co-transfected with a FLAG-tagged NLRP3 inflammasome protein construct (NLRP3, ASC, or pro-caspase-1) into HEK293T cells. The cells were harvested at 48 hr post transfection, and lysates were immunoprecipitated with anti-FLAG-M2 antibody. Protein interaction with NS1 was identified by western blot analysis with an anti-MYC antibody. (D) Differentiated THP-1 cells expressing NS1 variants were treated with LPS (1 μg/mL) for 6 hr, and subsequently with ATP (2.5 mM) for 15 min. The cells were harvested and lysates were immunoprecipitated with anti-MYC antibody. Endogenous NLRP3 protein interaction with NS1 was identified by western blot analysis with anti-NLRP3.