April 2020 Nsp3 abcam ab181620 Nucleocapsid Sino Biological 40143-MM05 Validation Commercial antibodies validated as per manufacturers website: Beta actin Sigma A5441 Immunoblot on chicken fibroblast cell extracts Spike Abcam Ab252690 Validated by ELISA on free peptide from SARS-CoV-1 Nsp3 abcam ab181620 Validated by western blot on SARS-CoV-1 infected cells Nucleocapsid Sino Biological 40143-MM05 Validated by western blot with corresponding viruses Eukaryotic cell lines Policy information about cell lines Cell line source(s) VeroE6 cells were obtained from ATCC, Calu-3 cells were obtained from Manfred Frey, originally from ATCC. Authentication Cells were not further authenticated Mycoplasma contamination Cells have been tested and are free of mycoplasma.

Graphical Abstract Highlights d MERS-CoV/SARS-CoV S composite glycan shields analyzed by cryo-EM and mass spectrometry d Structures of MERS-CoV/SARS-CoV S with neutralizing antibodies from survivors d LCA60 inhibits receptor binding by interacting with MERS-CoV S protein/glycans d S230 blocks receptor binding and triggers fusogenic rearrangements via functional mimicry In Brief Structural analysis of the SARS-CoV S and MERS-CoV S glycoproteins in complex with neutralizing antibodies from human survivors sheds light into the mechanisms of membrane fusion and neutralization Walls et al., SUMMARYRecent outbreaks of severe acute respiratory syndrome and Middle East respiratory syndrome, along with the threat of a future coronavirus-mediated pandemic, underscore the importance of finding ways to combat these viruses. The trimeric spike transmembrane glycoprotein S mediates entry into host cells and is the major target of neutralizing antibodies. To understand the humoral immune response elicited upon natural infections with coronaviruses, we structurally characterized the SARS-CoV and MERS-CoV S glycoproteins in complex with neutralizing antibodies isolated from human survivors. Although the two antibodies studied blocked attachment to the host cell receptor, only the anti-SARS-CoV S antibody triggered fusogenic conformational changes via receptor functional mimicry. These results provide a structural framework for understanding coronavirus neutralization by human antibodies and shed light on activation of coronavirus membrane fusion, which takes place through a receptor-driven ratcheting mechanism.

The tremendous pandemic potential of coronaviruses was demonstrated twice in the past few decades by two global outbreaks of deadly pneumonia. The coronavirus spike (S) glycoprotein initiates infection by promoting fusion of the viral and cellular membranes through conformational changes that remain largely uncharacterized. Here we report the cryoEM structure of a coronavirus S glycoprotein in the postfusion state, showing large-scale secondary, tertiary, and quaternary rearrangements compared with the prefusion trimer and rationalizing the free-energy landscape of this conformational machine. We also biochemically characterized the molecular events associated with refolding of the metastable prefusion S glycoprotein to the postfusion conformation using limited proteolysis, mass spectrometry, and single-particle EM. The observed similarity between postfusion coronavirus S and paramyxovirus F structures demonstrates that a conserved refolding trajectory mediates entry of these viruses and supports the evolutionary relatedness of their fusion subunits. Finally, our data provide a structural framework for understanding the mode of neutralization of antibodies targeting the fusion machinery and for engineering next-generation subunit vaccines or inhibitors against this medically important virus family.

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