Proteolytic fusion activation at the S2 site, which occurs for all those coronaviruses, can take place in several cellular compartments (20)
Proteolytic fusion activation at the S2 site, which occurs for all those coronaviruses, can take place in several cellular compartments (20). this medically important computer virus family. (SARS-CoV) and (MERS-CoV) are the causative brokers of these fatal pneumonias that exhibited that coronaviruses could cross 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 occur. Coronavirus entry is usually mediated by the trimeric transmembrane spike (S) glycoprotein, which is responsible for receptor binding and fusion of the viral and host membranes. S is usually a class I viral fusion protein that is synthesized as a single-chain precursor of 1 1,300 amino acids and trimerizes upon folding. It forms an extensive crown decorating the computer virus surface and is the main H3B-6545 Hydrochloride target of neutralizing antibodies upon contamination. Coronavirus S proteins are comprised of two functional subunits, termed S1 and S2 (5). S1 mediates binding to the host receptor and exhibits the most diversity among coronaviruses, partially accounting for the wide host range of this computer virus family. S2 induces fusion of the viral envelope with cellular membranes and is conserved among coronaviruses. The S glycoprotein exists as a metastable prefusion trimer at the viral surface, and its 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 host membrane (12, 13). The subsequent irreversible refolding of the fusion machinery provides the energy required to juxtapose the viral and host membranes, promoting 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 fragments spanning the heptad-repeat motifs (14C16). H3B-6545 Hydrochloride The data are limited to a small portion of the fusion machinery and do not reveal how most of the S2 subunit refolds. A detailed knowledge of the conformational changes driving fusion is usually 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 targeting 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 Rabbit polyclonal to IL20 conformation. Our data reveal that 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 weak sequence conservation, the structure demonstrates structural similarity to paramyxovirus F proteins, thereby reinforcing the relatedness of their fusion mechanisms and their evolutionary connection. Finally, the results H3B-6545 Hydrochloride provide a structural framework to rationalize the mode of neutralization of antibodies targeting the conserved fusion H3B-6545 Hydrochloride machinery. Results Protease-Mediated Fusion Activation of Coronavirus S Proteins. Coronavirus S proteins harbor up to two protease cleavage sites located at the boundary between the S1 and S2 subunits (S1/S2 site) and upstream from the fusion peptide (S2 site) (Fig. 1(MHV) or MERS-CoV (20). This cleavage event, along with subsequent binding to the host receptor, is essential to promote cleavage at the S2 site and fusion activation in the case of MERS-CoV (12). The critical importance of cleavage at the S1/S2 site is also exemplified by the (Bat-CoV) HKU4. Bat-CoV HKU4 shares a high degree of sequence similarity with MERS-CoV and can H3B-6545 Hydrochloride bind to the same human receptor (DPP4), although it is unable to infect human cells (3). Engineering 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 entry into human cells (4). These results demonstrate that both receptor and protease specificity are important determinants of host range. Proteolytic fusion activation at the S2 site, which occurs for all coronaviruses, can take place in several cellular compartments (20). For instance, transmembrane protease/serine protease (TMPRSS) processing of SARS-CoV and MERS-CoV S at the cell membrane, furin-mediated processing of human coronavirus (HCoV)-NL63 and MERS-CoV S in the.