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

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.

Toxicity of Influenza A Virus Matrix Protein 2 for Mammalian Cells is Associated with its Intrinsic Proton-Channeling Activity

“…While similar observations have been made in yeast and insect cells cultivated in weakly acidic media, it was assumed that in heterotypic vertebrate expression systems, M2 causes no overt toxicity. 21 Contrary to that, we detected a major effect in 293 HEK cells with 60% of pM2wt-driven cell death occurring within 48 hours after transient transfection. This phenomenon was clearly linked to the well-known proton-channeling ability of M2 with notable changes in mitochondria of pM2wt-transfected cells observed within the same time-period.…”

“…6,19 M2 proton channel activity is known to be cytotoxic in heterologous expression systems, such as E. coli, 20,21 with especially severe effects seen in insect cells and S. cerevisiae, with no overt toxicity observed in vertebrate cells. 21,22 …”

Membrane Organization and Lipid Rafts

“…For instance, proteins that have both a GPI anchor and a trans-membrane domain have been identified, in which the GPI anchor could be raft-associated with the transmembrane domain facing the nonraft bilayer (Kupzig et al 2003). Another protein is the influenza virus M2 protein, which has been postulated to occupy the perimeter of the raft domain, formed when the virus buds out of the plasma membrane (Schroeder et al 2005;Rossman et al 2010). Also, N-Ras has been proposed to act as a linactant in the cytosolic leaflet of a raft (Weise et al 2009).…”

Role of Membrane Rafts in Viral Infection

“…After transportation of the virus to late endosomes, low-pHdependent conformation change of HA induces membrane fusion of the viral envelope with the endosomal membrane. Then viral ribonucleoprotein complexes (RNP) including the viral genome are released to the cytoplasm of host cells by proton influx of viral ion channel M2 protein that requires binding with cholesterol [57,104]. Similar to the entry process of influenza virus, capsid-like core particles of hepatitis B virus (Hepadnaviridae) are internalized through clathrin-dependent and raft-independent endocytosis [105].…”

“…Although these domains of NA are essential for the association with rafts, there is no evidence that NA possesses palmitoylated residues. The mechanism by which NP associates with rafts remains unknown [104,[154][155][156]191]. HA, NA, NP and M2 independently utilize membrane rafts together with apical targeting signal sequence for the apical sorting process, leading to efficient preferential budding and release of progeny viruses from the apical surface membrane.…”