Student Theses and Dissertations

Date of Award

2018

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Hang Lab

Abstract

To rapidly detect early stage infections the innate immune system maintains an assortment of pathogen recognition mechanisms interspersed throughout both the extracellular and intracellular environments. These sensors recognize key components of viral, bacterial and fungal pathogens, and stimulate an inflammatory response which leads to the expression of an extensive network of host defense proteins. One such canonical network is regulated by type I interferon. This pathway responds to viral infections by upregulating hundreds of interferon stimulated genes (ISG) critical for host immunity. One of the more pivotal proteins for viral control is interferon-induced transmembrane protein 3 (IFITM3). IFITM3 is a host protein known to play a key role in inhibiting numerous virus infections, including influenza, Dengue, West Nile, HIV and Ebola. It is active in the early stages of infection and interferes with viral fusion and content delivery to the cell cytoplasm. Despite this broad antiviral activity, the exact mechanism of IFITM3 viral fusion interference, and whether it directly interacts with the fusion environment remains unknown. To better understand the physiological conditions of IFITM3 antiviral activity, we required an improved understanding of the endogenous levels of S-fatty acylation. While earlier work in our lab has shown this post-translational modification to be critical for IFITM3 activity, it was previously impossible to distinguish between different modified populations. I therefore developed the acyl PEG exchange (APE) assay. Utilizing cysteine selective mass tags, APE detects different levels of fatty acylated cysteines within a protein population, which allows us to probe the lipidated states of endogenous proteins for the first time. Using this assay, I have shown that the majority of endogenous human IFITM3 is dually S-fatty acylated. To investigate the mechanism of IFITM3 antiviral activity, I generated recombinant, native, synthetically lipidated protein for structural and fusion-based studies. We applied an in vitro viral fusion model that detects the lipid mixing of viral envelopes with liposomes. We demonstrate that viral fusion is mitigated with the inclusion of recombinant IFITM3 liposomes. Furthermore, when IFITM3 is modified with maleimide palmitate to mimic fatty-acylation at cysteine 105, lipid mixing is inhibited more than the unmodified IFITM3. Overall, our recombinant model of viral fusion provides an in vitro approach to investigate IFITM3 function. The assay provides the first evidence that IFITM3 directly alters the membrane fusion environment, and that cysteine palmitoylation enhances protein function as well. In the future, these studies will be complemented with more accurate in vitro assays to further elucidate its critical mechanism.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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