Influenza Virus A M2 Protein Generates Negative Gaussian Membrane Curvature Necessary for Budding and Scission

Abstract

The M2 protein is a multi-functional protein, which plays several roles in the replication cycle of the influenza A virus. Here we focus on its ability to promote budding of the mature virus from the cell surface. Using high resolution small angle X-ray scattering we show that M2 can restructure lipid membranes into bicontinuous cubic phases which are rich in negative Gaussian curvature (NGC). The active generation of negative Gaussian membrane curvature by M2 is essential to influenza virus budding. M2 has been observed to colocalize with the region of high NGC at the neck of a bud. The structural requirements for scission are even more stringent than those for budding, as the neck must be considerably smaller than the virus during ‘pinch off’. Consistent with this, the amount of NGC in the induced cubic phases suggests that M2 proteins can generate high curvatures comparable to those on a neck with size 10x smaller than a spherical influenza virus. Similar experiments on variant proteins containing different M2 domains show that the cytoplasmic amphipathic helix is necessary and sufficient for NGC generation. Mutations to the helix which reduce its amphiphilicity and are known to diminish budding attenuated NGC generation. An M2 construct comprising the membrane interactive domains, the transmembrane helix and the cytoplasmic helix, displayed enhanced ability to generate NGC, suggesting that other domains cooperatively promote membrane curvature. These studies establish the importance of M2-induced negative Gaussian curvature during budding and suggest that antagonizing this curvature is a viable anti-influenza strategy.

The Amphipathic Helix of Influenza A Virus M2 Protein Is Required for Filamentous Bud Formation and Scission of Filamentous and Spherical Particles

“…Other recent in silico data support the ability of M2 to cause membrane curvature as well as demonstrate its sensitivity to penta-alanine mutations within the amphipathic helix (53). It was found that the M2 amphipathic helix is sufficient to cause negative Gaussian curvature (NGC), which is a prerequisite alteration of membrane shape for budding and scission mechanisms, similar to that proposed for influenza virus virions (28).…”

Helical antimicrobial polypeptides with radial amphiphilicity

“…For example, this is the amount of NGC found in a transmembrane pore with a size of ∼40 nm, if we ignore all other effects. If on the other hand, the destabilization mechanism is that of a budding event followed by scission, then we can estimate the size of the defect using a catenoid surface, which is an approximate representation of the surface of a scission pore (56,57). The Gaussian curvature of a minimal catenoid surface with a neck radius of c along its z axis is defined by K(z) = −[sech 4 (z∕c)]/c 2 .…”

Viral fusion protein transmembrane domain adopts β-strand structure to facilitate membrane topological changes for virus–cell fusion

“…NGC generation is often facilitated by negative-curvature lipids such as PE (41)(42)(43), which commonly enhance fusogenicity (44). Our measured hKi can be visualized using a catenoid surface, which is an approximation of a scission neck or a fusion pore (45,46). The Gaussian curvature of a minimal catenoid surface with a neck radius of c along its z axis is KðzÞ = −½sech 4 ðz=cÞ =c 2 .…”