Role of the amphiphilic helix of M2 for influenza virus budding

Influenza virus assembly is organised by the viral budozone, a raft domain in the plasma membrane, to which the viral glycoprotein hemagglutinin (HA) is targeted. Scission of virus particles is mediated by M2, supposedly governed by a palmitoylated and cholesterol-binding amphiphilic helix in its cytoplasmic tail. We tested several preconditions of this poorly investigated model. Fluorescence resonance energy transfer (FRET) showed that M2 associated with HA, dependent on raft-targeting signals in HA and an intact actin cytoskeleton. Thus, M2 contains intrinsic signals that target the molecule to the budozone.

Using the purified cytoplasmic tail of M2 (M2-CT) we demonstrate that [³H]-photocholesterol is cross-linked to a putative CRAC (cholesterol recognition/interaction amino acid consensus) motif. M2-CT has the capacity to interact with membranes, both in vitro and in vivo, and an influence of palmitoylation and the cholesterol-binding motif on subcellular targeting of M2-CT was observed. In giant plasma membrane vesicles (a cell membrane-derived model system), M2-GFP was enriched in raft-like domains. Raft-association required palmitoylation, but not the CRAC motifs.

However, although palmitoylation and cholesterol binding affect several features of M2 predicted to be required for its membrane scission activity, recombinant viruses revealed only slightly impaired growth when the CRAC motif in M2 in combination with palmitoylation was disrupted. We assume that the process of virus budding is redundant and robust, i.e. other viral or cellular factors take over the function of a (at least partially) defective M2 protein.

Funding was provided by the DFG, SFB 740 (TP C3) and SPP 1175.