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The influenza virus ion channel and maturation cofactor M2 is a cholesterol-binding protein

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Abstract

The influenza-virus M2 protein has proton channel activity required for virus uncoating and maturation of hemagglutinin (HA) through low-pH compartments. The proton channel is cytotoxic in heterologous expression systems and can be blocked with rimantadine. In an independent, rimantadine-resistant function, M2, interacting with the M1 protein, controls the shape of virus particles. These bud from cholesterol-rich membrane rafts where viral glycoproteins and matrix (M1)/RNP complexes assemble. We demonstrate that M2 preparations from influenza virus-infected cells and from a baculovirus expression system contain 0.5–0.9 molecules of cholesterol per monomer. Sequence analyses of the membrane-proximal M2 endodomain reveal interfacial hydrophobicity, a cholesterol-binding motif first identified in peripheral benzodiazepine receptor and human immunodeficiency virus gp41, and an overlapping phosphatidylinositol 4,5-bisphosphate-binding motif. M2 induced rimantadine-reversible cytotoxicity in intrinsically cholesterol-free E. coli, and purified E. coli-expressed M2 functionally reconstituted into cholesterol-free liposomes supported rimantadine-sensitive proton translocation. Therefore, cholesterol was nonessential for M2 ion-channel function and cytotoxicity and for the effect of rimantadine. Only about 5–8% of both M2 preparations, regardless of cholesterol content, associated with detergent-resistant membranes. Cholesterol affinity and palmitoylation, in combination with a short transmembrane segment suggest M2 is a peripheral raft protein. Preference for the raft/non-raft interface may determine colocalization with HA during apical transport, the low level of M2 incorporated into the viral envelope and its undisclosed role in virus budding for which a model is presented. M2 may promote clustering and merger of rafts and the pinching-off (fission) of virus particles.

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Abbreviations

CRAC:

Cholesterol recognition/interaction amino acid consensus

DMPC:

l-α-dimyristoylphosphatidylcholine

DRM:

Detergent-resistant membrane

HA:

Hemagglutinin

HDL:

High-density lipoprotein

KPS:

Potassium phosphate buffer with K2SO4

LDL:

Low-density lipoprotein

MDCK:

Madin-Darby canine kidney

NA:

Neuraminidase

NaPS:

Sodium phosphate buffer with Na2SO4

Ni-NTA:

Nickel-nitrilotriacetic acid

OG:

N-octyl-β-d-glucopyranoside

PM:

Plasma membrane

PS:

Phosphatidylserine

RNP:

Ribonucleoprotein

Sf9 :

Spodoptera frugiperda

TDC:

Taurodeoxycholate

TGN:

trans-Golgi network

TM:

Transmembrane

T. ni:

Trichoplusia ni

TX-100:

Triton X-100

Φ:

Hydrophobic amino acid

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Acknowledgements

C.S. expresses her gratitude to Prof. Nikolaus Müller-Lantzsch and Prof. Friedrich Graesser at the Department of Virology of the University of the Saarland in Homburg. The authors are grateful for advice from and critical discussion with Drs. Barry Ely and Stephen A. Wharton, and thank Alan J. Hay (National Institute for Medical Research, London, U.K.) for the gift of antisera. We are indebted to Drs. Bernd Rüstow (Surfactant Laboratory of the Charité) for his support of lipid analysis and Brigitte Brux (Protein Analytics Laboratory of the Charité) for advice on cholesterol assays and native gels. Prof. Andreas Herrmann generously granted access to fluorimeters at the Institute of Biophysics, Humboldt University. Nadine Hardel helped with the cloning and Kathlen Schröder is thanked for reliable technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (grant No. Schr 554/2) and the Charité Berlin (project No. 98-273).

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  1. Cornelia Schroeder

    Present address: Jadolabs GmbH, Pfotenhauerstr. 108, 01307, Dresden, Germany

  2. Tse-I Lin

    Present address: Tibotec BVDV, 2800, Mechelen, Belgium

Authors and Affiliations

  1. Abteilung Virologie, Institut für Mikrobiologie und Hygiene, Universität des Saarlandes, Homburg/Saar, 66421, Homburg, Germany

    Cornelia Schroeder

  2. Institut für Virologie, Universitätsklinikum Charité, Humboldt-Universität zu Berlin, 10098, Berlin, Germany

    Harald Heider, Elisabeth Möncke-Buchner & Tse-I Lin

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  1. Cornelia Schroeder
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  2. Harald Heider
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Correspondence to Cornelia Schroeder.

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Schroeder, C., Heider, H., Möncke-Buchner, E. et al. The influenza virus ion channel and maturation cofactor M2 is a cholesterol-binding protein. Eur Biophys J 34, 52–66 (2005). https://doi.org/10.1007/s00249-004-0424-1

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  • DOI: https://doi.org/10.1007/s00249-004-0424-1

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