Finally, the results provide a structural framework to rationalize the mode of neutralization of antibodies targeting the conserved fusion machinery. Results Protease-Mediated Fusion Activation of Coronavirus S Proteins. coronaviruses could mix the species barrier from bats, camels, raccoons, or palm civets to humans (1C4). These observations, along with surveillance studies, suggest that additional emergence events could happen. Coronavirus entry is definitely mediated from the trimeric transmembrane spike (S) glycoprotein, which is responsible for receptor binding and fusion of the viral and sponsor membranes. S is a class I viral fusion protein that is synthesized like a single-chain precursor of 1 1,300 amino acids and trimerizes upon folding. It G6PD activator AG1 forms an extensive crown decorating the disease surface and is the main target of neutralizing antibodies upon illness. Coronavirus S proteins are comprised of two practical subunits, termed S1 and S2 (5). S1 mediates binding to the sponsor receptor and exhibits the most diversity among coronaviruses, partially accounting for the wide sponsor range of this disease family. S2 induces fusion of the viral envelope with cellular membranes and is conserved among coronaviruses. The S glycoprotein is present like a metastable prefusion trimer in the viral surface, and its G6PD activator AG1 structure has recently been characterized (6C11). Receptor binding and proteolytic processing promote large-scale conformational changes allowing initiation of the fusion reaction by insertion of the hydrophobic fusion peptide into the sponsor membrane (12, 13). The subsequent irreversible refolding of the fusion machinery G6PD activator AG1 provides the energy required to juxtapose the viral and sponsor membranes, advertising fusion and delivery of the viral genome into the cytoplasm. The only available structural information about the conformational changes undergone by coronavirus fusion machinery comes from X-ray crystallography studies of short polypeptide Cdc42 fragments spanning the heptad-repeat motifs (14C16). The data are limited to a small portion of the fusion machinery and don’t reveal how most of the S2 subunit refolds. A detailed knowledge of the conformational changes driving fusion is important to define the convenience of epitopes targeted by neutralizing antibodies and to engineer improved subunit vaccine candidates, as was reported for the (RSV) fusion (F) protein (17C19). Alternatively, heptad-repeatCmimicking peptides have been successfully used to inhibit type I fusion machineries, including coronavirus S glycoproteins (5). Furthering our understanding of the structural rearrangements underlying fusion bears the promise of developing next-generation inhibitors focusing on this viral family. We report here the characterization of the molecular determinants associated with the triggering of several -coronavirus S glycoproteins using a combination of limited proteolysis, mass spectrometry, and single-particle EM. We describe a near-atomic-resolution cryoEM reconstruction of a coronavirus fusion machinery ectodomain in the postfusion conformation. Our data reveal the postfusion S trimer adopts a 180-?-long cone-shaped architecture arranged around a prominent central triple-helical bundle and is the longest structure observed for any class I fusion protein. Despite fragile sequence conservation, the structure demonstrates structural similarity to paramyxovirus F proteins, therefore reinforcing the relatedness of their fusion mechanisms and their evolutionary connection. Finally, the results provide a structural platform to rationalize the mode of neutralization of G6PD activator AG1 antibodies focusing on the conserved fusion machinery. Results Protease-Mediated Fusion Activation of Coronavirus S Proteins. Coronavirus S proteins harbor up to two protease cleavage sites located in the boundary between the S1 and S2 subunits (S1/S2 site) and upstream from your fusion peptide (S2 site) (Fig. 1(MHV) or MERS-CoV (20). This cleavage event, along with subsequent binding to the sponsor receptor, is essential to promote cleavage in the S2 site and fusion activation in the case of MERS-CoV (12). The essential importance of cleavage in the S1/S2 site is also exemplified from the (Bat-CoV) HKU4. Bat-CoV HKU4 shares a high degree of sequence similarity with MERS-CoV and may bind to the same human being receptor (DPP4), although it is unable to infect human being cells (3). Executive two point mutations in the Bat-CoV HKU4 S1/S2 region, which introduces two protease cleavage sites similar to the ones found in the MERS-CoV S sequence, is sufficient to allow efficient access into human being cells (4). These results demonstrate that both receptor and protease specificity are important determinants of sponsor range. Proteolytic fusion activation in the S2 site, which happens for those coronaviruses, can take place in several cellular compartments (20). For instance, transmembrane protease/serine protease (TMPRSS) control of SARS-CoV and MERS-CoV S in the cell membrane, G6PD activator AG1 furin-mediated control of human being coronavirus (HCoV)-NL63 and MERS-CoV S in the early endosomes, or lysosomal protease-mediated triggering of SARS-CoV S (cathepsin L) or MHV S are key events that enable fusion activation and coronavirus access into sponsor cells.