Date of Award
Summer 8-1-2025
Level of Access Assigned by Author
Open-Access Thesis
Degree Name
Doctor of Philosophy (PhD)
Department
Physics
First Committee Advisor
Samuel T. Hess
Second Committee Member
MacKenzie R. Stetzer
Third Committee Member
Ioan-Augustin Chioar
Additional Committee Members
Melissa Maginnis
Karissa Tilbury
Abstract
Influenza A virus is a recurring problem every year that causes significant morbidity and mortality in the human population. The vaccine is chosen to optimize protection against all strains, but the effectiveness varies due to the high mutation rate of the virus. On average, about 8% of the world’s population is infected annually. The need for a universal vaccine is crucial to fight all strains of the flu, as it has already developed resistance to some of the existing drugs. Investigating the viral life cycle to gain knowledge about how viral proteins interact with host cell components such as lipids and proteins will lead to the development to new antiviral drug targets. The influenza virus A spike protein hemagglutinin (HA) is crucial for viral attachment, entry, and fusion. HA forms high density clusters on the plasma membrane (PM) of infected cells, which lead to clusters in the viral envelope, and which are necessary for viral replication. HA also contains a cytoplasmic tail domain (CTD) which contains certain features that are highly conserved across all known HA subtypes. There are 3 cysteines known to be palmitoylated and 1-2 basic amino acids that remain conserved despite a high mutation rate in HA overall. Phosphatidylinositol 4,5-bisphosphate (PIP2) is a minor lipid found in the PM which plays an important role in numerous cellular functions. It has been shown that HA and PIP2 co-localize at the PM, suggesting that they are interacting, but the mechanism of interaction is unknown. Using
molecular dynamics (MD) simulations and super-resolution microscopy, we observe an increase in the density of PIP2 around wild-type HA (HAwt) compared to various HA mutants that change the charge of the CTD, presence of palmitoylation, or both, suggesting that the mechanism of interaction is both electrostatic and hydrophobic. The greatest changes were observed between HAwt and RREQ, which changes the net charge of the TCD from positive to negative. In the MD simulations there was a depletion of PIP2 around the CTD of HA and in the super-resolution FPALM images there is a decrease in the amount of free PIP2 around HA clusters. In addition, we show that there are other highly conserved amino acid motifs like in the CTD of HA in other viral spike proteins. These motifs could mediate PIP2 interactions with other viral spike proteins in a similar way. From MD simulations we show that PIP2 clusters more strongly around the CTD of influenza B HA compared to influenza A HA. We also find that PIP2 may not be the only phospholipid interacting with the CTD of HA. Phosphatidylinositol 4-phosphate (PI4P) is a minor lipid component found mainly in the Golgi and the PM, but FPALM results show that the PI4P-binding protein P4C co-localizes with HA inside of the cell, and MD simulations reveal that PI4P is found around the CTD of HA at a higher density than PIP2. This could lead to new antiviral targets to interrupt the viral life cycle.
Recommended Citation
Winski, David, "Determining the Mechanisms of Interaction Between Influenza Spike Protein Hemagglutinin and Host Cell Phosphoinositides by Super-Resolution Imaging and Molecular Dynamics Simulations" (2025). Electronic Theses and Dissertations. 4247.
https://digitalcommons.library.umaine.edu/etd/4247