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Influenza M2 protein regulates MAVS-mediated signaling pathway through interacting with MAVS and increasing ROS production.

Autophagy (2019-02-12)
Ruifang Wang, Yinxing Zhu, Xian Lin, Chenwei Ren, Jiachang Zhao, Fangfang Wang, Xiaochen Gao, Rong Xiao, Lianzhong Zhao, Huanchun Chen, Meilin Jin, Wenjun Ma, Hongbo Zhou
ABSTRACT

Influenza A virus can evade host innate immune response that is involved in several viral proteins with complicated mechanisms. To date, how influenza A M2 protein modulates the host innate immunity remains unclear. Herein, we showed that M2 protein colocalized and interacted with MAVS (mitochondrial antiviral signaling protein) on mitochondria, and positively regulated MAVS-mediated innate immunity. Further studies revealed that M2 induced reactive oxygen species (ROS) production that was required for activation of macroautophagy/autophagy and enhancement of MAVS signaling pathway. Importantly, the proton channel activity of M2 protein was demonstrated to be essential for ROS production and antagonizing the autophagy pathway to control MAVS aggregation, thereby enhancing MAVS signal activity. In conclusion, our studies provided novel insights into mechanisms of M2 protein in modulating host antiviral immunity and uncovered a new mechanism into biology and pathogenicity of influenza A virus. Abbreviations: AKT/PKB: AKT serine/threonine kinase; Apo: apocynin; ATG5: autophagy related 5; BAPTA-AM: 1,2-Bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis; BECN1: beclin 1; CARD: caspase recruitment domain; CCCP: carbonyl cyanide m-chlorophenylhydrazone; CQ: chloroquine; DCF: dichlorodihyd-rofluorescein; DPI: diphenyleneiodonium; DDX58: DExD/H-box helicase 58; eGFP: enhanced green fluorescent protein; EGTA: ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid; ER: endoplasmic reticulum; hpi: hours post infection; IAV: influenza A virus; IFN: interferon; IP: immunoprecipitation; IRF3: interferon regulatory factor 3; ISRE: IFN-stimulated response elements; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; MMP: mitochondrial membrane potential; MOI, multiplicity of infection; mRFP: monomeric red fluorescent protein; MTOR: mechanistic target of rapamycin kinase; NC: negative control; NFKB/NF-κB: nuclear factor kappa B; PI3K: class I phosphoinositide 3-kinase; RLR: RIG-I-like-receptor; ROS: reactive oxygen species; SEV: sendai virus; TM: transmembrane; TMRM: tetramethylrhodamine methylester; VSV: vesicular stomatitis virus.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Monoclonal ANTI-FLAG® M2 antibody produced in mouse, 1 mg/mL, clone M2, affinity isolated antibody, buffered aqueous solution (50% glycerol, 10 mM sodium phosphate, and 150 mM NaCl, pH 7.4)
Sigma-Aldrich
Sodium dodecyl sulfate, BioReagent, suitable for electrophoresis, for molecular biology, ≥98.5% (GC)
Sigma-Aldrich
Dimethyl sulfoxide, ≥99.5% (GC), suitable for plant cell culture
Sigma-Aldrich
Chloroquine diphosphate salt, powder or crystals, 98.5-101.0% (EP)
Sigma-Aldrich
Carbonyl cyanide 3-chlorophenylhydrazone, ≥97% (TLC), powder
Sigma-Aldrich
LY-294,002 hydrochloride, solid, ≥98% (HPLC)
Sigma-Aldrich
Glycerin, meets USP testing specifications
Sigma-Aldrich
Diphenyleneiodonium chloride, ≥98%
Sigma-Aldrich
1-Adamantylamine, 97%
Sigma-Aldrich
Bovine Serum Albumin, chromatographically purified, New Zealand origin, low endotoxin, suitable for cell culture, pH 7, ≥98%
Sigma-Aldrich
BAPTA-AM, ≥95% (HPLC)
Sigma-Aldrich
Amantadine hydrochloride
Sigma-Aldrich
Ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid, BioXtra, ≥97 .0%
Sigma-Aldrich
4′-Hydroxy-3′-methoxyacetophenone, 98%
Sigma-Aldrich
p3XFLAG-CMV-14 Expression Vector, shuttle vector for transient or stable intracellular expression of C-terminal 3xFLAG
Sigma-Aldrich
Gly-Pro