Biology and Biomedical Sciences: Immunology
Date of Award
Doctor of Philosophy (PhD)
Chair and Committee
Deborah J Lenschow
Abstract of the Dissertation
A Role for Interferon Stimulated Gene-15: ISG15) During Chikungunya Virus Infection
Scott William Werneke
Doctor of Philosophy in Biology and Biomedical Science
Washington University in St. Louis, 2013
Professor Deborah J. Lenschow, Chairperson
Chikungunya fever is caused by Chikungunya virus: CHIKV), an infectious disease that is characterized by severe joint and muscle pain in humans. The latest outbreak of CHIKV, which began in 2005, has affected millions of people across India, Singapore, and the Indian Ocean Island region. Type I interferon: IFN), which mediates protection against many different viruses through the upregulation of interferon stimulated genes: ISGs), has been shown to be essential for the control of CHIKV infection. As the role of ISGs during CHIKV infection is largely unknown, we investigated the activity of the interferon stimulated gene ISG15.
ISG15 is a ubiquitin like molecule that has the ability to conjugate to intracellular proteins and can also be found as a free form both intra- and extracellularly. ISG15 conjugation has previously been shown to be essential for protection against influenza B and Sindbis virus infections. We demonstrate that neonatal ISG15-/- mice are profoundly more susceptible to CHIKV infection compared to WT mice. Unlike other viral models, mice lacking the ability to form ISG15 conjugates through the deletion of the E1 enzyme UbE1L, do not display an increase in CHIKV induced lethality. In addition, we observed no differences in viral loads between wild type and ISG15-/- mice during the course of the infection. Instead, ISG15-/- mice displayed a dramatic increase in proinflammatory cytokines and chemokines. Our data suggests that the role of ISG15 during CHIKV infection is conjugation independent and that ISG15 protection is mediated not through its action as an antiviral molecule, but through regulation of the immune response.
To identify potential mechanisms of action for unconjugated free ISG15, we characterized both intra- and extracellular forms of ISG15 using mass spectrometry. We identify phosphorylation as a potential post-translational modification for extracellular ISG15 and identify the secreted 30kDa ISG15 conjugate as ISG15 bound to hemoglobin beta. We also identify over 140 potential non-covalent binding partners for intracellular ISG15 that are involved in many different cellular pathways, including innate immunity, vesicular sorting, and cell signaling. Based on our findings, we have generated preliminary data examining a role for ISG15 in autophagy as well as a role for ISG15 in the regulation of cytokine production. Our characterization of both intra- and extracellular ISG15 will hopefully guide future experiments needed to determine the mechanism by which ISG15 regulates the host response to viral infection.
Werneke, Scott, "A Role for Interferon Stimulated Gene-15 (ISG15) During Chikungunya Virus Infection" (2013). All Theses and Dissertations (ETDs). 1059.