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

Eleonora V Shtykova,Alexander L Ksenofontov,Cy M Jeffries,Natalia V Fedorova,Alexey A Dolgov,Victor A Radyukhin,Dmitri I Svergun,Lyudmila A Baratova,Liudmila A Shilova,Alexander V Shishkov,Vladimir V Volkov,Oleg V Batishchev,Petri Kursula

doi:10.1371/journal.pone.0082431

Abstract

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.

Highlights

Influenza A viruses are important pathogens that still rank among the major global health problems
The M1 protein layer is seen on electron microphotographs as a regularly arranged lattice of rods with the size about 4.064.066.0 nm, which are densely packed perpendicular to the viral membrane [7]
Overall characteristics of M1 protein and its NM-domain The overall characteristics of the full-length M1 protein and its NM-domain in solution were first assessed with Analytical ultracentrifugation (AUC)

Summary

Introduction

Influenza A viruses are important pathogens that still rank among the major global health problems. The M1 protein is membrane associates and forms a rigid matrix layer under the viral envelope utilizing multiple protein-lipid and protein-protein interactions (lipid-M1, M1- haemagglutinin, M1- neuraminidase, M1- ribonucleoprotein particles, M1-M1) [4,5,6]. The rigid matrix layer of M1 maintains the integrity and shape of the intact influenza virus and structurally organises the virus membrane [2,3,4,5,6,8,9]. Acting as an endoskeleton, M1 provides the basement and an anchor point for the HA fusion machinery [5,6] These studies revealed an important inherent tendency of the protein to self-associate. In a detailed investigation into the self-assembly of recombinant M1 from E. coli and of its sub-fragments, the pH-dependent oligomerization of M1 in vitro was shown to be determined by both the N- and C-terminal domains of the protein [10]

Methods

Results

Conclusion