Date of Award

2012

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Microbiology and Immunology

Abstract

Acute respiratory tract infections in humans are responsible for significant morbidity and mortality especially in children, elderly, and the immunocompromised. Virus infection is the primary cause of acute respiratory tract infections. Infection with coronaviruses can cause disease ranging from common colds to severe acute respiratory syndrome. Currently no coronavirus-specific antivirals are available. Patients are given symptomatic treatment, or are prescribed inappropriately antibiotics which do not target the underlying virus infection. The identification of specific inhibitors of coronaviruses or knowledge about how coronaviruses interact with the innate immune system could provide new avenues for developing therapeutics. I investigated the replication of human coronavirus NL63 (HCoV-NL63) in primary human airway epithelial cultures and characterized the activity of the viral papain-like protease 2 (PLP2) domain. Using quantitative real time PCR and imaging techniques I showed that HCoV-NL63 undergoes productive replication in the ciliated bronchial epithelial cells. To identify potential broad spectrum inhibitors of HCoVs, small molecule inhibitors of SARS-CoV were screened for cross-reactivity against HCoV-NL63 PLP2 and I identified a compound, GRL-0737, which blocks HCoV-NL63 with an EC50 ~5µM. I also investigated HCoV-NL63 mediated antagonism of interferon-β (IFN-β) induction. I showed that HCoV-NL63 infection of bronchial epithelial cells results in a small, but measurable release of IFN-β by enzyme linked immunosorbent assay (ELISA). I confirmed that HCoV-NL63 PLP2 is an IFN-β antagonist, and abrogates the ability of the newly identified innate immune signaling protein STING (stimulator of interferon genes) to induce expression of an IRF-3 dependent luciferase reporter. Furthermore, using immunofluorescence I demonstrated that HCoV-NL63 non-structural protein 3 (viral protein in which PLP2 resides) co-localizes with STING suggesting that HCoV-NL63 PLP2 may be targeting STING activation of the IRF-3 arm of the type I IFN response to virus infection. This work demonstrates that HCoV-NL63 PLP2 plays an important role in evasion of the host immune response and that HCoV-NL63 replication can be disrupted by inhibitors of the PLP2 protease, making this protease an attractive target for antiviral treatments of coronavirus infection.

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Creative Commons License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License.

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