Structural and Functional Analysis of the Coronavirus Spike Protein Fusion Peptide

Enveloped viruses access their host cells by binding to receptors on the plasma membrane and then undergoingfusion with the host membrane. Both binding and fusion are mediated by a specific viral "spike" protein that istypically primed for fusion activation by proteolytic cleavage to expose the fusion peptide. Coronavirus fusionspike protein (CoV S) is a complex biomolecular machine that has a novel fusion peptide with has a great dealof inherent flexibility in its fusion reaction. This is exploited by these viruses in their diverse entry pathways andis a primary determinant of viral tropism. We have pioneered the concept that that the proteolytic cleavage eventsin S that lead to membrane fusion occur both at the interface of the receptor binding (S1) and fusion (S2) domains(called S1/S2), as well as adjacent to a structurally and functionally novel fusion peptide within S2 (called S2').Thus, there are notable differences between CoV S and most other class I fusion proteins including: 1) that theproteolytic events liberating the fusion peptide are diverse, and 2) that the fusion peptide itself is atypical insequence compared to other fusion peptides, containing a mixture of important hydrophobic and negatively-charged residues, and may represent a larger than normal fusion "platform" instead of a defined "peptide". Thusfusion peptide activity is likely controlled by reorganization of the fusion platform, based on both hydrophobic(i.e. lipid-binding) and ionic (i.e. Ca2+ and pH) interactions. Despite the recent availability of two S structures,there remains a very limited mechanistic understanding of membrane fusion for the CoV family, or any structuralinformation to correlate structural biology aspects of S to its function in membrane fusion. This information iscritical to understanding viral pathogenesis and CoV emergence into the Human population. We propose todevelop a panel of monoclonal antibodies to the SARS-CoV-2/COVID-19 fusion peptide that will be used as toolsto understand the fusion mechanism of coronaviruses, and which will be integrated into our biophysical,biochemical, and in vivo approach to study the unique cleavage-activated regulation of CoV S protein. Theseantibodies will also provide a platform for development of novel broadly-acting therapeutic antibodies for SARS-CoV-2 and other coronaviruses. Moving the field forward with these innovative studies will provide criticalknowledge about CoV entry and tropism needed to safeguard human health from an emerging pathogen likelyto cause severe outbreaks, and for which few or no medical countermeasures exist.