Expression and purification of native and functional influenza A virus matrix 2 proton selective ion channel.
Influenza A virus displays one of the highest infection rates of all human viruses and therefore represents a severe human health threat associated with an important economical challenge. Influenza matrix protein 2 (M2) is a membrane protein of the viral envelope that forms a proton selective ion channel. Here we report the expression and native isolation of full length active M2 without mutations or fusions. The ability of the influenza virus to efficiently infect MDCK cells was used to express native M2 protein. Using a Calixarene detergents/surfactants based approach; we were able to solubilize most of M2 from the plasma membrane and purify it. The tetrameric form of native M2 was maintained during the protein preparation. Mass spectrometry shows that M2 was phosphorylated in its cytoplasmic tail (serine 64) and newly identifies an acetylation of the highly conserved Lysine 60. ELISA shows that solubilized and purified M2 was specifically recognized by M2 antibody MAB65 and was able to displace the antibody from M2 MDCK membranes. Using a bilayer voltage clamp measurement assay, we demonstrate a pH dependent proton selective ion channel activity. The addition of the M2 ion channel blocker amantadine allows a total inhibition of the channel activity, illustrating therefore the specificity of purified M2 activity. Taken together, this work shows the production and isolation of a tetrameric and functional native M2 ion channel that will pave the way to structural and functional characterization of native M2, conformational antibody development, small molecules compounds screening towards vaccine treatment.
CALIXAR, 60 Avenue Rockefeller, 69008 Lyon, France.
Elodie
E
Desuzinges Mandon
Laboratoire de Virologie et Pathologie Humaine (VirPath), Centre International de Recherche en Infectiologie (CIRI), U1111 INSERM, UMR 5308 CNRS, ENS Lyon, Université Claude Bernard Lyon1 (UCBL1), Lyon, France.
Aurélien
A
Traversier
CALIXAR, 60 Avenue Rockefeller, 69008 Lyon, France; CNRS, Institut de Chimie et Biologie de Protéines, 69007 Lyon, France.
Anne
A
Champagne
CALIXAR, 60 Avenue Rockefeller, 69008 Lyon, France.
Lorraine
L
Benier
Aix Marseille Univ, CNRS, INSERM, Institut Paoli-Calmettes, CRCM, Marseille Protéomique, Marseille, France.
Stéphane
S
Audebert
Institut Européen des Membranes, UMR5635, Université de Montpellier CNRS ENSCM, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France.
Sébastien
S
Balme
CALIXAR, 60 Avenue Rockefeller, 69008 Lyon, France.
Emmanuel
E
Dejean
Laboratoire de Virologie et Pathologie Humaine (VirPath), Centre International de Recherche en Infectiologie (CIRI), U1111 INSERM, UMR 5308 CNRS, ENS Lyon, Université Claude Bernard Lyon1 (UCBL1), Lyon, France; VirNext, Faculté de Médecine RTH Laennec, EZUS, Lyon, France.
Manuel
M
Rosa Calatrava
CALIXAR, 60 Avenue Rockefeller, 69008 Lyon, France. Electronic address: ajawhari@calixar.com.
Anass
A
Jawhari
D000818:Animals; D002462:Cell Membrane / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004285:Dogs; D015870:Gene Expression; D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000737:chemistry / Q000235:genetics; D007473:Ion Channels / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D061985:Madin Darby Canine Kidney Cells; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000086082:Viroporin Proteins
D007473:Ion Channels; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins
Current/voltage; Influenza infection; Lipid bilayer; MDCK expression; Matrix 2 influenza A; Native solubilization; Oligomeric state; Proton selective ion channel; Purification