Influenza A Virus M1 Protein Non-Specifically Deforms Charged Lipid Membranes and Specifically Interacts with the Raft Boundary.

Topological rearrangements of biological membranes, such as fusion and fission, often require a sophisticated interplay between different proteins and cellular membranes. However, in the case of fusion proteins of enveloped viruses, even one molecule can execute membrane restructurings. Growing evidence indicates that matrix proteins of enveloped viruses can solely trigger the membrane bending required for another crucial step in virogenesis, the budding of progeny virions. For the case of the influenza A virus matrix protein M1, different studies report both in favor and against M1 being able to produce virus-like particles without other viral proteins. Here, we investigated the physicochemical mechanisms of M1 membrane activity on giant unilamellar vesicles of different lipid compositions using fluorescent confocal microscopy. We confirmed that M1 predominantly interacts electrostatically with the membrane, and its ability to deform the lipid bilayer is non-specific and typical for membrane-binding proteins and polypeptides. However, in the case of phase-separating membranes, M1 demonstrates a unique ability to induce macro-phase separation, probably due to the high affinity of M1's amphipathic helices to the raft boundary. Thus, we suggest that M1 is tailored to deform charged membranes with a specific activity in the case of phase-separating membranes.

Influenza virus Matrix Protein M1 preserves its conformation with pH, changing multimerization state at the priming stage due to electrostatics.

Influenza A virus matrix protein M1 plays an essential role in the virus lifecycle, but its functional and structural properties are not entirely defined. Here we employed small-angle X-ray scattering, atomic force microscopy and zeta-potential measurements to characterize the overall structure and association behavior of the full-length M1 at different pH conditions. We demonstrate that the protein consists of a globular N-terminal domain and a flexible C-terminal extension. The globular N-terminal domain of M1 monomers appears preserved in the range of pH from 4.0 to 6.8, while the C-terminal domain remains flexible and the tendency to form multimers changes dramatically. We found that the protein multimerization process is reversible, whereby the binding between M1 molecules starts to break around pH 6. A predicted electrostatic model of M1 self-assembly at different pH revealed a good agreement with zeta-potential measurements, allowing one to assess the role of M1 domains in M1-M1 and M1-lipid interactions. Together with the protein sequence analysis, these results provide insights into the mechanism of M1 scaffold formation and the major role of the flexible and disordered C-terminal domain in this process.

Structural analysis of influenza A virus matrix protein M1 and its self-assemblies at low pH.

Influenza A virus matrix protein M1 is one of the most important and abundant proteins in the virus particles broadly involved in essential processes of the viral life cycle. The absence of high-resolution data on the full-length M1 makes the structural investigation of the intact protein particularly important. We employed synchrotron small-angle X-ray scattering (SAXS), analytical ultracentrifugation and atomic force microscopy (AFM) to study the structure of M1 at acidic pH. The low-resolution structural models built from the SAXS data reveal a structurally anisotropic M1 molecule consisting of a compact NM-fragment and an extended and partially flexible C-terminal domain. The M1 monomers co-exist in solution with a small fraction of large clusters that have a layered architecture similar to that observed in the authentic influenza virions. AFM analysis on a lipid-like negatively charged surface reveals that M1 forms ordered stripes correlating well with the clusters observed by SAXS. The free NM-domain is monomeric in acidic solution with the overall structure similar to that observed in previously determined crystal structures. The NM-domain does not spontaneously self assemble supporting the key role of the C-terminus of M1 in the formation of supramolecular structures. Our results suggest that the flexibility of the C-terminus is an essential feature, which may be responsible for the multi-functionality of the entire protein. In particular, this flexibility could allow M1 to structurally organise the viral membrane to maintain the integrity and the shape of the intact influenza virus.