Influenza a virus NS2 suppresses NFKB/NF-κB signaling to facilitate viral replication by mediating the autophagic-degradation of IKBKG/NEMO

Influenza A virus (IAV) is an important zoonotic pathogen responsible for substantial respiratory morbidity and mortality. Elucidating the mechanisms by which IAV evades host innate immunity is critical for developing novel antiviral strategies. Although the IAV non-structural protein 2 (NS2) is well-characterized for the export of viral ribonucleoproteins (vRNPs) from the host cell nucleus, the function of NS2 in evading host innate immunity, especially the NFKB/NF-κB (nuclear factor kappa B) signaling pathway, remains poorly understood. The present study uncovered that NS2 is a novel viral inhibitor of the NFKB pathway. Mechanistically, NS2 interacted with and mediated the degradation of the NFKB essential modulator (IKBKG/NEMO), thereby suppressing downstream signal transduction. The macroautophagy/autophagy receptor OPTN (optineurin) was exploited by NS2 to mediate the selective autophagic degradation. Furthermore, the K72 residue was critical for the NS2-mediated degradation of IKBKG/NEMO, as the K72R substitution in NS2 disrupted the IKBKG/NEMO-NS2 interaction and abrogated the autophagic degradation. In addition, NS2K72R mutant virus displayed less viral load and milder pathogenicity in mice. In conclusion, these findings highlighted the novel biological function of IAV NS2 in exploiting selective autophagy to evade host defenses, and offered a potential target for controlling IAV infections.Abbreviations: 3-MA: 3-methyladenine; AIV: avian influenza virus; ATG7: autophagy related 7; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CHX: cycloheximide; co-IP: co-immunoprecipitation; CHUK/IKKα: component of inhibitor of nuclear factor kappa B kinase complex; DAPI: 4ʹ, 6-diamidino-2-phenylindole, dihydrochloride; dsRNA: double-stranded RNA; dpi: days post-infection; EID50: 50% egg infective dose; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; hpi: hours post-infection; IAV: influenza A virus; IFN: interferon; IKBKB/IKKβ: inhibitor of nuclear factor kappa B kinase subunit beta; IFNG: interferon gamma; IKBKG/NEMO: inhibitor of nuclear factor kappa B kinase subunit gamma; IKK: IκB kinase; IP: immunoprecipitation; IRF3: interferon regulatory factor 3; IRF7: interferon regulatory factor 7; LAMP1: lysosome associated membrane protein 1; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MAP3K14/NIK: mitogen-activated protein kinase kinase kinase 14; MAVS: mitochondrial antiviral signaling protein; MLD50: 50% mouse lethal dose; MOI: multiplicity of infection; MRV/Sendai virus: murine respirovirus; NBR1: NBR1 autophagy cargo receptor; NEP: nuclear export protein; NFKB/NF-κB: nuclear factor kappa B; NFKB2/p100: nuclear factor kappa B subunit 2; NFKBIA/IκBα: NFK inhibitor alpha; NP: nucleoprotein; NS1: non-structural protein 1; OPTN: optineurin; PB1: basic polymerase 1; PBS: phosphate-buffered saline; poly(I:C): polyriboinosinic polyribocytidylic acid; PRRs: pattern recognition receptors; RELA/p65: RELA proto-oncogene, NF-kB subunit; RELB: RELB proto-oncogene, NF-kB subunit; RIGI: RNA sensor RIG-I; RIGI-IN: RIGI-CARD; RLR: RIGI-like-receptor; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SIM: SUMO-interacting motif; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TBK1: TANK binding kinase 1; TNF: tumor necrosis factor; TRAF6: TNF receptor associated factor 6; TOLLIP: toll interacting protein; Vec: empty vector; vRNP: viral ribonucleoprotein.