Membrane fusion levels correlated precisely with the extents of S2 cleavage (Figures?4C and 4D). hypersensitive to proteolytic activation of membrane fusion, an essential step in virus-cell entry. Proteolysis is within fusion domains (FDs), at sites over 10?nm from the VOC-specific NTD changes, indicating allosteric inter-domain control of fusion activation. In addition, NTD-specific antibodies block FD cleavage, membrane fusion, and virus-cell entry, suggesting restriction of inter-domain communication as a neutralization mechanism. Finally, using structure-guided mutagenesis, we identify an inter-monomer sheet structure that facilitates NTD-to-FD transmissions and subsequent fusion activation. This NTD-to-FD axis that sensitizes viruses to infection and to NTD-specific antibody neutralization provides new context for understanding selective forces driving SARS-CoV-2 evolution. Keywords: coronavirus, SARS-CoV-2, spike protein, virus entry, membrane CDDO-EA fusion, virus neutralization, virus evolution, virus variation Graphical abstract Open in a separate window Qing et?al. identify connections between N-terminal and C-terminal domains of SARS-CoV-2 spike proteins that control the proteolytic activation of membrane fusion and show mechanisms of N-terminal domain-specific antibody neutralization. Introduction Even with available vaccines, antiviral treatments, and mitigation measures, SARS-CoV-2 continues to spread through human populations, with adaptive viruses becoming increasing transmissible and potentially able to resist vaccine-induced immunity. Highly contagious variants of concern (VOC) emerge, first D614G, then , , , , and variants. Conceivably a genetically stable variant with maximum transmissibility into both naive and immunized humans will eventually predominate (Burioni and Topol, 2021), yet this is not certain, making for current missions to predict ongoing SARS-CoV-2 evolutionary trajectories. Aims are in place to identify transmissibility determinants in past and current VOC and further elucidate VOC resistance to vaccine antibodies and antiviral agents. This study addresses a part of these aims by assessing VOC responses to host transmissibility determinants and by explicating antibody neutralization mechanisms. VOC have acquired adaptive mutations throughout the 30 kb RNA genome, yet most are present in the spike (S)?gene. Variations in S proteins adapt viruses to diverse host factors conferring virus-cell entry. The principal host factors are receptors and proteases. Receptor binding domains (RBDs) adhere virus particles to target cell receptors, hence RBD mutations adapt viruses to human and animal orthologs of ACE2, the SARS-CoV-2 receptor (Niu et?al., 2021; Ren et?al., 2021; Wang et?al., 2021b). Receptor-bound S proteins acquire conformations that are poised for membrane fusion (Benton et?al., 2020; Jackson et?al., 2022; Peng et?al., 2021), and are then cleaved by host cell proteases to generate fragments that undergo large-scale multidomain conformational transitions. These transitory intermediate structures tether virus and cell membranes together and pull the two into coalescence (Jackson et?al., 2022; Peng et?al., 2021; Shang et?al., 2020b). CDDO-EA Mutations at or near protease cleavage sites increase or decrease spike fragmentation, in turn affecting proteolytic activation of membrane fusion (Hoffmann et?al., 2020; Shang et?al., 2020b; Walls et?al., 2020). Other adaptive S protein mutations affect virus stability and fusion activation distinctly, for example, a powerfully selected D614G substitution in all VOC operates to stabilize S proteins in so-called pre-fusion conformations, increasing the durability of extracellular virus CDDO-EA infectivity (Fernandez, 2020; Zhang et?al., 2020, 2021a). Several more recently acquired VOC mutations alter epitopes, allowing viruses to escape neutralization by antibodies binding to RBDs and other domains (Gobeil et?al., 2021; Graham et?al., 2021; Planas et?al., 2021; Wang et?al., 2021a). Amino-terminal domains (NTDs) of SARS-CoV-2 proteins are among the most hypervariable, with both indel and missense mutations in past and present VOC. This level of variation is puzzling in light of currently obscure NTD functions. While several studies suggest that the NTDs bind viruses to cellular ligands (Baker CDDO-EA et?al., 2020; Qing et?al., 2021; Wei et?al., 2020), the significance of these interactions is often unclear, as they cannot substitute for ACE2-directed virus-cell entry (Baker et?al., 2020; Qing et?al., 2021; Wei et?al., 2020). In addition, the NTDs contain an antigenic supersite that is recognized by a prominent class of neutralizing antibodies (Cerutti et?al., 2021; Graham et?al., 2021; McCallum et?al., 2021). This neutralization demonstrates the functional relevance of NTDs in virus entry, but the mechanism by which antibody binding to a domain apparently unnecessary for virus-cell binding or membrane fusion is hard to discern. Finally, there is the question of whether NTD variation is driven by a requirement for antibody escape. While it is definitely conceivable that variants overcoming antibody restriction are Cxcr7 positively selected, the majority of acute SARS-CoV-2 infections take place within the unvaccinated (Cdcgov, 2021; Linsenmeyer et?al., 2021; Muhsen et?al., 2021; Ng et?al., 2021; Singanayagam et?al., 2022), raising the likelihood that VOC NTD variations offer fitness advantages that are CDDO-EA independent of antibody evasion. Here,.
