Date of Award

2020

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pathology

First Advisor

Vikas Anathy

Abstract

The influenza A virus (IAV) causes severe respiratory illness in humans. Current treatments are rapidly becoming ineffective due to the emergence of viral resistance to available therapies such as oseltamivir and zanamivir. Given the impact of this virus there is an urgent need to explore novel targets for new treatments less susceptible to viral mutation; targeting host proteins utilized by the virus may avoid these limitations. It has been shown in vitro that interactions with host ER based protein disulfide isomerases (PDIs) are required for specific IAV proteins to reach their functional conformations. The viral proteins hemagglutinin (HA) and neuraminidase (NA) both contain numerous disulfide bonds necessary for their functionality. Our results demonstrate both HA and NA interact with host PDIA3; a chaperone responsible for the catalysis of disulfide bonds in newly formed glycoproteins. However, it is unknown whether these host-viral protein interactions are required during active infection and whether they represent a putative therapeutic target for the treatment of influenza infection.

In this dissertation I investigated the role of host PDIA3 in the folding of IAV proteins and development of subsequent immunopathology. The impact of PDIA3 during both H1N1 and H3N2 influenza infection was examined using lung epithelial specific PDIA3 knockout mice and inhibitors of PDIs in primary human bronchial epithelial cells and isolated mouse tracheal epithelial cells. Among PDI inhibitors tested, we found LOC14 inhibits PDIA3 at an IC50 of 5μM. Treatment with LOC14 inhibits PDIA3 activity in lung epithelial cells and subsequently decreases disulfide bonds and oligomerization of HA in both H1N1 and H3N2 infected primary lung epithelial cells. Biotin switch assays indicated that LOC14 also decreased disulfide bonds in NA and these differences in oxidative folding correspond to a subsequent decrease in NA activity. Furthermore, following LOC14 treatment we observed a decrease in detectable viral protein in supernatants from infected cells, suggesting potential deficiencies in viral release. Moreover, these decreases in disulfide bonds significantly decrease viral load, and pro-inflammatory responses from primary lung epithelial cells. Lung epithelial specific deletion of PDIA3 in mice results in a significant decrease in viral burden and levels of inflammatory-immune markers in mouse lung, as well as significantly improved airway mechanics. Additionally, in vivo administration of LOC14 partially mirrored these results, yielding significant decreases in overall viral burden. Taken together, these data suggest that lung epithelial PDIA3 plays a critical role in the maturation of IAV proteins and propagation of the virus and illustrate the potential of utilizing host PDIs as a target for anti-viral therapies.

Language

en

Number of Pages

188 p.

Available for download on Tuesday, October 12, 2021

Share

COinS