Date of Award

Summer 8-20-2021

Level of Access Assigned by Author

Open-Access Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Physics

Advisor

Samuel T. Hess

Second Committee Member

R. Dean Astumian

Third Committee Member

James P. McClymer

Additional Committee Members

Julie A. Gosse

Melissa Maginnis

Abstract

Influenza A is a highly contagious and a pathogenic virus that causes serious respiratory illness, the complications from which can be fatal even to young and healthy adults. On average, approximately 250,000 to 500,000 people die each year from complications due to Influenza A (IAV) worldwide. Frequently occurring mutations can evade the vaccine-developed immune response, so the vaccines need to be updated continuously. Drug resistance to some of the existing drugs has already been established. Hence new antiviral therapeutics need to be explored in order to prevent further morbidity and mortality. To develop such a novel antiviral therapeutic, knowledge of the viral life cycle and interaction between the viral proteins and host cell lipids and proteins is crucial, as influenza viruses hijack these cellular components during infection. IAV Hemagglutinin (HA) is the most abundant viral glycoprotein responsible for viral binding and entry. HA clusters at the host cell plasma membrane, and these clusters need to be high in density to catalyze membrane fusion for viral entry. However, the mechanism by which HA forms clusters remains unknown. We recently showed clustering of HA is modulated by phosphatidylinositol (4,5) bisphosphate (PIP2). Targeting this interaction could lead to possible alternative antiviral therapeutics. CPC (Cetylpyridinium Chloride) is a positively charged quaternary ammonium compound used in mouthwashes and personal care items. CPC has been previously shown to have antibacterial and antiviral properties. While both the antibacterial and antiviral properties of CPC are well understood at high concentrations (millimolar), the effect of CPC on cell function at relatively low (micromolar) concentrations is not well understood. In this study, we use the super-resolution microscopy technique FPALM to study the effect of CPC on PIP2-binding proteins, and to illuminate the mechanism of the antiviral properties of CPC at these much lower, non-cytotoxic micromolar concentrations in the cell model. Results show that CPC at these concentrations significantly modulates PIP2 clustering and HA clustering, and more importantly, reduces the HA density and the co-clustering of HA and PIP2. CPC also disrupts the assembly of HA and the IAV Matrix Protein 1 (M1). These results are important because dense HA clusters correlate with efficient viral entry and infectivity, and modulating PIP2 clusters reduces HA clustering and in turn disrupts the assembly of HA and M1. In addition, we also show CPC at micromolar concentrations can improve the survival of zebrafish infected with IAV. In addition, we also show for the first time that M1 colocalizes with PIP2 and demonstrate the influence of HA in M1 clustering at the plasma membrane, which might have an impactful role in the viral life cycle.

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