Conformation and Directionality of the Influenza A M2 Protein Domain Essential to Viral Replication

Influenza A causes seasonal epidemics, and the possibility of another worldwide influenza A pandemic remains a public health concern. The influenza A M2 protein is implicated in the virus’s ability to bud and create new infectious virus particles. Specifically, the C-terminal region of the virus is required for viral budding, and this protein domain binds another influenza protein, M1. M2-M1 binding is critical to packaging the viral genome into new virions. To probe the region of M2 involved in viral budding and genome packing, this thesis studied the conformation of residues 60-70 of the C-terminus via site-directed spin label electron paramagnetic resonance (SDSL-EPR) spectroscopy. SDSL-EPR spectroscopy provided structural information about M2 reconstituted into 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine : 1-palmitoyl-2- oleoyl-sn-glycero-3-phospho-1-rac-glycerol (POPC:POPG) 4:1 lipid bilayers, a physiologically relevant environment. Mobility and membrane accessibility data revealed that residues 60-64 are associated with the membrane and are less mobile than residues 65-70. Residues 65-70 form a region with some periodic secondary structure, extending into the aqueous phase. A unique contribution of this thesis was the determination of the directionality of M2 insertion into the membrane via trypsin fragmentation and matrixassisted laser desorption ionization-mass spectrometry (MALDI-MS). According to current reconstitution protocols, M2 inserts into the membrane with no directionality, and both the N- and C-termini lie at the liposome exterior

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