Influenza virus causes significant illness and mortality in the U.S. and worldwide. Vaccines offer some protection, but must be continuously tailored to mutations of the virus. Some strains of influenza are resistant to one or more of the limited number of available anti-viral drugs. New strategies which attack invariant features of the virus are urgently needed. The viral surface glycoprotein hemagglutinin allows the virus to bind to host cells and enter. Entry depends on high density clusters of hemagglutinin within the viral membrane, but the mechanism of cluster formation is unknown. Assembly depends on hemagglutinin co-clustering with other viral components, including the matrix protein M1, in the host cell plasma membrane. This project will address the fundamental questions of how hemagglutinin forms clusters, how hemagglutinin and M1 co-cluster, and the role of host cell components in these key viral processes. We recently discovered that hemagglutinin and M1 both interact with host cell phosphoinositides, which are able to control important cell signaling and protein trafficking pathways that have been implicated previously in infection. This project will investigate the mechanism of interaction between hemagglutinin, M1, and the phosphoinositides PI4P and PIP2 using super-resolution microscopy, fluorescence spectroscopy, and molecular dynamics simulations, targeting the portions of hemagglutinin that are invariant and therefore less likely to mutate over time. Results will help identify new targets for anti-viral drugs and illuminate how influenza is able to exploit host cell membrane organization for its own life cycle.