The Influenza Hemagglutinin Cytoplasmic Tail Domain Interacts with Phosphatidylinositol 4,5-bisphosphate

During the life cycle of the influenza virus, hemagglutinin (HA) plays a crucial role in binding, entry, and fusion of the virus. HA forms densely packed clusters at the host cell plasma membrane (PM), which are necessary for generating infectious virions. However, the process behind HA clustering remains unclear. Previous research revealed that HA co-clusters with phosphatidylinositol 4,5-bisphosphate (PIP2) in PM, but the nature of this interaction is not well understood.

In this study, molecular dynamics simulations and fluorescence photoactivation localization microscopy (FPALM) were employed to better understand the relationship between HA and PIP2.

The findings indicate that the interaction primarily occurs between the PIP2 head group and the cytoplasmic tail domain (CTD) of HA. By introducing specific mutations in the CTD, researchers were able to alter the charge, palmitoylation sites, or a combination of both. Molecular dynamics simulations demonstrated that the mutations HARREQ and RREQMAY had the most significant impact on HA-PIP2 interactions.

These mutations caused a depletion in PIP2's radial distribution function around HA at distances of 2.5 nm or less. Additionally, FPALM revealed that CTD mutations resulted in a reduced HA cluster density at the PM. Notably, the mutation that altered both charge and acylation components (HAREMAY) exhibited the most substantial reduction in HA cluster density. Furthermore, these clusters showed structural changes in terms of circularity and perimeter.

Mutations in the HA transmembrane domain also led to minor modifications in the HA cluster properties and its co-clustering with PIP2. Importantly, FPALM highlighted that PIP2 co-clustering with HA was influenced by HA mutations, with more free PIP2 being localized under HAREMAY clusters. The study developed a chemical model that allows for the interpretation of HA-PIP2 interactions and uncovers quantitative differences between PIP2 binding by HA CTD mutants.

Ultimately, the researchers concluded that the mechanism of HA-PIP2 interaction involves both electrostatic and hydrophobic components. This insight into HA-PIP2 interaction, coupled with the prevalence of putative PIP2-interacting domains in various viral spike proteins, suggests that understanding and potentially disrupting these interactions could lead to effective therapeutic methods for combating viral infections.