An Interdisciplinary Approach to Pandemic Preparedness: Understanding Coronavirus Spike Protein Structure-Function Relationships and Viral Pathogenesis for the Development of Broad-Based Therapeutic and Control Strategies
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The Coronaviridae family is known for its extensive diversity and remarkable ability to cross species barriers, exemplified by three major spillover events into human populations: SARS-CoV, MERS-CoV, and SARS-CoV-2. The increasing identification of animal coronaviruses capable of infecting humans underscores the need for a One Health approach to understanding viral entry mechanisms and developing broad-based vaccines and antivirals. This thesis encompasses studies ranging from the epidemiology of SARS-CoV-2 prevalence in feline populations during the early pandemic to investigations into how coronavirus spike proteins mediate host cell entry and strategies to counteract this process. Key findings include the identification of host cell glutaminase as a potential pan-coronavirus antiviral target and the development of monoclonal antibodies targeting the highly conserved stalk domain of the spike protein, which demonstrated broad-spectrum reactivity and anti-fusogenic activity. This research also uncovered novel mechanisms of spike protein-mediated entry, including the critical role of the 0 domain in regulating fusogenicity and infection efficiency. A groundbreaking discovery in this work is the identification of a previously unreported protease cleavage site within the feline coronavirus spike protein, located in Domain D of the spike protein. As a Class I fusion protein, the spike protein requires cleavage by host proteases to become fusion-competent. Traditionally, Alphacoronaviruses, including feline coronaviruses, were thought to rely solely on the universal S2’ cleavage site. This discovery challenges that paradigm by revealing an additional cleavage site. Notably, mutations at this site dramatically reduce infectivity, emphasizing its essential role in facilitating viral entry. This discovery led to the mapping of currently available coronavirus cleavage sites onto the structure, revealing that many of these sites are located within Domain D, a finding that had never been recognized before. Based on our findings, we propose a functional role for Domain D as a hotspot for protease cleavage and gateway mutations. Prior to this study, the role of Domain D was underappreciated, highlighting the significance of this work in advancing our understanding of coronavirus biology.