Site-Directed Spin Label EPR Studies of the Interaction Between the Influenza A Proteins (M1 and M2) Involved in Viral Assembly

Influenza A presents a significant concern for public health as it is the cause of seasonal outbreaks and global pandemics. The influenza A proteins, matrix protein 1 (M1) and matrix protein 2 (M2), have been shown to be essential for the propagation of new viruses, especially through their roles in viral assembly and budding. The M2 cytoplasmic tail interacts with the M1 protein, recruiting it to the viral budding site and enabling proper packaging of the viral genome. The Howard lab has previously characterized residues 50-70 in the M2 cytoplasmic tail and the M2 protein’s conformational equilibria by sitedirected spin label electron paramagnetic resonance (SDSL-EPR). This work lays groundwork for the establishment of a system in which to see changes in the M2 protein upon M1 binding. Methods for the overexpression and purification of the M1 protein are presented. Selected M2 sites (43, 57, 68) were studied by SDSL-EPR in the presence of Nterminal M1 (residues 1-165), with M2 sites 43 and 57 acting as indicators of the M2 protein’s conformational dynamics. Binding between M1 and M2 could not be rigorously established, but preliminary results suggest little change in the M2 protein in the presence of the M1 protein

The Distal Cytoplasmic Tail Of The Influenza A M2 Protein Dynamically Extends From The Membrane

The influenza A M2 protein is a multifunctional membrane-associated homotetramer that orchestrates several essential events in the viral infection cycle. The monomeric subunits of the M2 homotetramer consist of an N-terminal ectodomain, a transmembrane domain, and a C-terminal cytoplasmic domain. The transmembrane domain forms a four-helix proton channel that promotes uncoating of virions upon host cell entry. The membrane-proximal region of the C-terminal domain forms a surface-associated amphipathic helix necessary for viral budding. The structure of the remaining ~34 residues of the distal cytoplasmic tail has yet to be fully characterized despite the functional significance of this region for influenza infectivity. Here, we extend structural and dynamic studies of the poorly characterized M2 cytoplasmic tail. We used SDSL-EPR to collect site-specific information on the mobility, solvent accessibility, and conformational properties of residues 61–70 of the full-length, cell-expressed M2 protein reconstituted into liposomes. Our analysis is consistent with the predominant population of the C-terminal tail dynamically extending away from the membranes surface into the aqueous medium. These findings provide insight into the hypothesis that the C-terminal domain serves as a sensor that regulates how M2 protein participates in critical events in the viral infection cycle

Characterization Of The Interaction Between Two Influenza A Proteins (M1 and M2) Involved In Viral Assembly

Two of the proteins required for the efficient assembly of influenza A virus particles are matrix protein 1 (M1) and membrane-bound matrix 2 protein (M2). Our work involves developing a detailed biophysical understanding of the interaction of M1 protein and M2 protein. M2 is a 97-amino acid membrane-bound protein that we have previously characterized using site-directed spin-label electron paramagnetic resonance (SDSL-EPR). M1 is a 252-amino acid protein that associates with M2 protein during viral assembly and budding. Using a multi-pronged biophysical approach, we have probed the interaction of M1 protein with membranes both with and without reconstituted M2 protein. Both M1 and M2 proteins have been previously shown to have multiple conformational states. We hypothesize that the interaction of the two proteins preferentially stabilizes a subset of accessible conformations