MARY KATHERINE BRADFORD PLIMACK
$864,475
BOSTON CHILDREN'S HOSPITAL
Massachusetts
National Institute of Allergy and Infectious Diseases (NIAID)
The COVID-19 pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), raising alarms about all pathogenic coronaviruses with pandemic potential. The first critical step for coronaviruses to enter host cells is viral membrane fusion mediated by their spike (S) proteins. Among them, the best-studied spike protein is the SARS-CoV-2 spike, which is a heavily glycosylated type I membrane protein anchored in the viral membrane. It is first produced as a precursor that trimerizes and then cleaved by a furin-like protease into two fragments: the receptor-binding fragment S1 and the fusion fragment S2. Binding of the receptor-binding domain (RBD) in S1 to the host cell receptor ACE2 and further proteolytic cleavage at a second site in S2 (S2’ site) are believed to trigger dissociation of S1 and irreversible refolding of S2 into a postfusion conformation. These large structural rearrangements bring together the viral and cellular membranes, ultimately leading to fusion of the two bilayers. Previous studies have revealed both pre- and post-fusion conformations of the soluble fragments of many spike proteins, but less is known for structures of their transmembrane and membrane- proximal regions in the context of lipid bilayers. There is strong evidence for functional roles of these membrane- interacting regions in viral fusion, and yet mechanistic studies on how they exert their functions remain scarce. We hypothesize that high-resolution structural studies of distinct conformational states of the membrane-bound coronavirus spike proteins in membrane will reveal novel structure features that are critical for our understanding of viral entry. We have determined a structure of the SARS-CoV-2 postfusion spike containing all the membrane-interacting regions reconstituted in a lipid bilayer. Our structure has revealed some unexpected findings that the internal fusion peptide forms a hairpin-like wedge spanning almost the entire lipid bilayer and the transmembrane segment wraps around the fusion peptide at the last stage of membrane fusion, demonstrating the importance of membrane in studying these viral fusion proteins. In this project, we plan to determine structures of the distinct conformational states of the coronavirus S proteins reconstituted in lipid nanodiscs. We will also define roles of critical structural elements of the S proteins in membrane fusion and determine impact of other viral structural proteins by mutagenesis and functional studies. Our goal is to visualize novel structural features of the spike proteins in the context of membrane and elucidate their functions to inform development of intervention strategies. We will pursue the following specific aims: 1) we will determine structure of the S2’ fragment in the postfusion conformation in membrane; 2) we will determine structure of the full-length prefusion S proteins in membrane; 3) we will elucidate roles of novel structural features of the full-length S proteins in their stability, function and antigenicity, and impact of other viral components.