Quantification of Interactions between Influenza Hemagglutinin and Host Cell Phosphoinositides by Super-Resolution Microscopy

The influenza viral membrane protein hemagglutinin (HA) forms dense nanoscale clusters on host cell plasma membranes (PM), but the mechanisms that direct HA clustering are not well understood. Previous studies have observed HA associated with actin rich regions of the PM, but there are no known direct interactions between HA and actin. Phosphatidylinositol 4,5-biphosphate (PIP2) is a signaling lipid in the PM which can regulate the actin cytoskeleton, and actin comets initiated by PIP2 are known to be exploited by HA to reach the PM of infected cells. PIP2 is also used by other viruses, such as HIV and Ebola, to form clusters of viral proteins on the PM. Using diffraction-limited and super-resolution FPALM methods, we observed that HA and PH domain, a protein marker for PIP2, are closely spatially related at the PM. Clusters of PIP2 are also significantly altered in both density and area in the presence of high levels of HA, while HA clusters are significantly altered in the presence of high levels of PIP2, suggestive of an interaction between the two. Although HA mutates rapidly, there are 3 cysteines and 1-2 basic residues in the cytoplasmic tail domain (CTD) which remain highly conserved among HA subtypes. These cysteines are known to undergo palmitoylation in the Golgi, a post-translational modification where hydrophobic palmitic acids are attached. Using HA mutants and super-resolution FPALM, we examined the role of both palmitoylation and charge on the clustering properties of HA and spatial association with PIP2. Mutation of the cysteines or basic residues causes significant reductions to cluster densities (relative to cell average), while mutation of the charges appears to modulate association with PIP2. The greatest changes were observed when both the cysteines and net charge of the HA CTD were changed, causing a maximal 22% ± 6% reduction in the radial distribution functions (RDF) of clusters and a maximal 30% ± 15% increase in associated PH domain RDF amplitude. Cluster properties, density, perimeter, and circularity were also significantly affected. Even though clusters were not eliminated through CTD mutations, these findings suggest that the CTD of HA does play a role in the clustering of HA and spatial association with PIP2