Date of Award

Fall 12-18-2020

Level of Access Assigned by Author

Open-Access Thesis

Degree Name

Master of Science (MS)




Samuel T. Hess

Second Committee Member

Robert W. Meulenberg

Third Committee Member

MacKenzie R. Stetzer


Influenza virus, colloquially known as the flu, is an acute respiratory disease that infects several millions of individuals each year in the U.S. and kills tens of thousands of those infected. Yearly viral vaccines are widely available, however, due to the virus’s high mutation rate, their efficacy varies greatly. Due to the variability in vaccine efficiency against seasonal influenza, and the potential for even more pathogenic versions of influenza to emerge at any time, there is a high demand for a universal treatment option.

Influenza virus hijacks a variety of host cell components in order to replicate. The glycoprotein hemagglutinin (HA), which is found in the envelope of influenza virions and assembles in the plasma membrane (PM) of host cells, is involved in the attachment, entry, and assembly stages of the viral life cycle. To perform its membrane fusion function, HA must cluster at high densities, although the mechanism for HA clustering remains unknown. Previous work has observed an association between the cytosolic protein actin and HA at the PM of cells. Actin has been shown to affect the motion of HA within clusters, but their connection is not understood. Phosphoinositides, such as phosphatidylinositol 4,5-bisphosphate (PIP2), are implicated in actin polymerization, remodeling, and depolymerization and are theorized to be the mediator between the HA-actin connection through their direct interactions with actin binding proteins (ABPs). Disruption of factors that induce clustering of HA may lead to novel treatment methods for influenza infection.

To elucidate the mechanism of HA clustering, we used Fluorescence Photoactivation Localization Microscopy (FPALM) to study HA and PIP2 in living cells at the PM. We found that HA and PIP2 colocalize at the PM in living cells and that HA modulates the mobilities of PIP2 molecules. Further analysis of PIP2 and HA revealed a time-dependent correlation in their dynamics, indicating the existence of a direct connection between the molecules. In addition, we found that HA and PIP2 are delivered together to the PM at high frequencies, suggesting that HA is delivered to the PM already clustered. These HA clusters persist long enough at the PM that HA and PIP2 recycling events are observed at similar frequencies. Our observations strengthen the hypothesis that HA and PIP2 interact at the PM and suggest that PIP2 plays a role in HA clustering mechanisms.