NS1 Protein of 2009 Pandemic Influenza A Virus Inhibits Porcine NLRP3 Inflammasome-Mediated Interleukin-1 Beta Production by Suppressing ASC Ubiquitination

Abstract

The inflammasome represents a molecular platform for innate immune regulation and controls proinflammatory cytokine production. The NLRP3 inflammasome is comprised of NLRP3, ASC, and procaspase-1. When the NLRP3 inflammasome is activated, it causes ASC speck formation and caspase-1 activation, resulting in the maturation of interleukin-1β (IL-1β). The NLRP3 inflammasome is regulated at multiple levels, with one level being posttranslational modification. Interestingly, ubiquitination of ASC has been reported to be indispensable for the activation of the NLRP3 inflammasome. Influenza A virus (IAV) infection induces NLRP3 inflammasome-dependent IL-1β secretion, which contributes to the host antiviral defense. However, IAVs have evolved multiple antagonizing mechanisms, one of which is executed by viral NS1 protein to suppress the NLRP3 inflammasome. In this study, we compared IL-1β production in porcine alveolar macrophages in response to IAV infection and found that the 2009 pandemic H1N1 induced less IL-1β than swine influenza viruses (SIVs). Further study revealed that the NS1 C terminus of pandemic H1N1 but not that of SIV was able to significantly inhibit NLRP3 inflammasome-mediated IL-1β production. This inhibitory function was attributed to impaired ASC speck formation and suppression of ASC ubiquitination. Moreover, we identified two target lysine residues, K110 and K140, which are essential for both porcine ASC ubiquitination and NLRP3 inflammasome-mediated IL-1β production. These results revealed a novel mechanism by which the NS1 protein of the 2009 pandemic H1N1 suppresses NLRP3 inflammasome activation. Influenza A virus (IAV) infection activates the NLRP3 inflammasome, resulting in the production of IL-1β, which contributes to the host innate immune response. ASC, an adaptor protein of NLRP3, forms specks that are critical for inflammasome activation. Here, we report that the NS1 C terminus of the 2009 pandemic H1N1 has functions to suppress porcine IL-1β production by inhibiting ASC speck formation and ASC ubiquitination. Furthermore, the ubiquitination sites on porcine ASC were identified. The information gained here may contribute to an in-depth understanding of porcine inflammasome activation and regulation in response to different IAVs, helping to further enhance our knowledge of innate immune responses to influenza virus infection in pigs.

The NS1 Protein of Influenza A Virus Participates in Necroptosis by Interacting with MLKL and Increasing Its Oligomerization and Membrane Translocation

“…As expected, our results indicate that the interaction of NS1 with MLKL enhances MLKL-mediated NLRP3 inflammasome activation and IL-1␤ processing. Various RNA viruses, including IAV, vesicular stomatitis virus (VSV), respiratory syncytial virus (RSV), Zika virus, and dengue virus, have been reported to activate the NLRP3 inflammasome (28,34,35). The activated NLRP3 inflammasome can be beneficial to the host, as it can lead to an enhancement of antiviral defense resulting in virus clearance and resolution of tissue damage.…”

“…Since we observed that NS1 increases MLKL oligomerization and membrane translocation, we hypothesized that NS1 would increase MLKL-mediated NLRP3 inflammasome activation and IL-1␤ processing and secretion. To test this, we took advantage of the NLRP3 reconstitution system in HEK293T cells, as we described previously (28). As seen in Fig.…”

“…Influenza A/Puerto Rico/8/34 (PR8) virus was propagated in 11-day-old embryonated chicken eggs as described previously (18). Influenza A/Halifax/210/2009/H1N1 (Hf09) virus was grown in MDCK cells as described previously (28).…”

“…Plasmid expressing wild-type PR8 NS1 (pcDNA-PR8-NS1) and mutant NS1(R38A/K41A) were previously described (23). Similarly, plasmids pcDNA-NLRP3, pCMV-Flag-pro-caspase1, pcDNA-pro-IL-1␤, and pcDNA-ASC were previously described (28). The complete MLKL open reading frame (ORF) was amplified by reverse transcription-PCR (RT-PCR) using the total mRNA from THP1 cells as the template and was cloned into plasmid pCMV-3ϫFlag.…”

NLRP3 inflammasome activation is associated with PM2.5‐induced cardiac functional and pathological injury in mice

“…NLRP3 inflammasome, a main player of inflammatory response, can be activated by diverse stimuli such as pore‐forming toxins, ATP, and viral RNA in macrophages . Once activation is triggered, NLRP3 recruits downstream apoptosis‐associated speck‐like protein contaiining a CARD, cysteine protease caspase‐1 and then assembles NLRP3 inflammasome complex . Subsequently, caspase‐1 precursor (pro‐caspase‐1) is activated into Cleaved caspase‐1 with the formation of p10 and p20 .…”