Phase Partitioning of Influenza Virus Neuraminidase in a Model Membrane

Today, more than ever, understanding the biophysics of viruses, specially enveloped viruses with spike transmembrane glycoproteins like SARS-CoV-2 and influenza seems imperative. From three transmembrane proteins of influenza virus i.e. neuraminidase (NA), hemagglutinin (HA), and the proton channel protein M2, the former is of most interest as it contains a bulky head which nominates it as a key player in the virus budding process due to its ability in generating lateral pressure. Recent reports suggest that localized protein-protein crowding can bend membranes and drive curvature and tubulation. Such crowding can only happen through a clustering mechanism like e.g. in the membrane raft domains (characterized by high contents of cholesterol, sphingolipids and phospholipids with saturated fatty acids). Here, we utilize thermoplasmonic-induced fusion and investigate how the virus NA proteins partition into lipid ordered (lo) and disordered (ld) phases of a model membrane. Using phase segregated Giant Unilamellar Vesicles (GUVs) as mimics of raft and non-raft domains of cell plasma membranes, we successfully fuse these vesicles to Giant Plasma Membrane Vesicles (GPMVs) derived from transfected cells. The fusion event, in a condition that is close to the physiological condition, leads to formation of hybrid vesicles with micron-size ordered and disordered domains with properly oriented NA proteins on their membrane. The NA proteins, then, surprisingly start to populate in the disordered region of the fused structure and as time evolves, they remain there. This can be interpreted as NA preference for the non-raft domains of the membrane. The same behavior is also observed for an engineered variant of the NA protein that lacks the bulky head. Our method provides a novel platform for studying peripheral and transmembrane proteins in a model membrane in a physiologically relevant condition.