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Defining the Biochemical Factors Regulating IFITM3-Mediated Antiviral Activity

Chesarino, Nicholas M

Abstract Details

2016, Doctor of Philosophy, Ohio State University, Biomedical Sciences.
Influenza virus infects 5 to 20 percent of the global population each year, and is one of the top ten causes of human deaths. Furthermore, the ability of influenza viruses to evolve into novel strains, such as the 2009 pandemic H1N1 virus, leaves the human population exposed to more devastating pandemics, underscoring the importance of uncovering broadly-acting immune inhibitors of viral infection. For this reason, our lab focuses on a cellular protein known as interferon-induced transmembrane protein 3 (IFITM3), which inhibits the fundamental cellular entry process of all strains of influenza virus tested to date. Importantly, several studies have linked a polymorphism in IFITM3, which leads to a truncated variant of the protein, to increased influenza severity. Despite the essential role of IFITM3 in inhibiting influenza virus infections, a better understanding of its mechanism of action, cellular trafficking, and post-translational regulation are needed in order to develop IFITM3-based antiviral therapies. Through investigation of Fyn kinase-mediated phosphorylation of IFITM3, we discovered that IFITM3 traffics to the plasma membrane and enters into the endocytic pathway, where it blocks influenza virus from entering the cell. We learned that this trafficking is disrupted upon IFITM3 phosphorylation, which we found to block an endocytic motif, sequestering IFITM3 at the plasma membrane and leaving cells more vulnerable to viral infection. In the course of these studies, we found that phosphorylation of IFITM3 also blocks a NEDD4 E3 ubiquitin ligase recognition motif, preventing its ubiquitination in addition to its endocytosis. Research into the NEDD4-IFITM3 interaction uncovered several key mechanisms of IFITM3 regulation and activity. We determined that NEDD4 and IFITM3 directly interact, leading to poly-ubiquitination of IFITM3 and ultimately degradation in the lysosome. We demonstrated the ability to manipulate basal levels of IFITM3 by knocking down or knocking out NEDD4, and showed that downregulating NEDD4 greatly increases resistance to infection by several viruses tested. Finally, prediction of IFITM3 secondary structure using a bioinformatics approach allowed us to identify a highly-conserved, short amphipathic helix within a hydrophobic region previously thought to be a transmembrane domain. Consistent with the known ability of amphipathic helices to alter membrane properties, we show that this helix and its amphipathicity are required for inhibition of influenza virus, Zika virus, vesicular stomatitis virus, Ebola virus, and human immunodeficiency virus infections by IFITM3. Further, we specifically show that the amphipathic helix is required for blockade of influenza virus hemagglutinin-mediated membrane fusion by IFITM3. Overall, this work has revealed several mechanistic insights into the IFITM proteins that should prove useful in the development of broadly-acting, novel IFITM-based antiviral therapies.
Jacob Yount (Advisor)
Ian Davis (Committee Chair)
Amal Amer (Committee Member)
Joanne Turner (Committee Member)
Mark Wewers (Committee Member)
174 p.

Recommended Citations

Citations

  • Chesarino, N. M. (2016). Defining the Biochemical Factors Regulating IFITM3-Mediated Antiviral Activity [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1480426112676394

    APA Style (7th edition)

  • Chesarino, Nicholas. Defining the Biochemical Factors Regulating IFITM3-Mediated Antiviral Activity. 2016. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1480426112676394.

    MLA Style (8th edition)

  • Chesarino, Nicholas. "Defining the Biochemical Factors Regulating IFITM3-Mediated Antiviral Activity." Doctoral dissertation, Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1480426112676394

    Chicago Manual of Style (17th edition)