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HKU1 is a human betacoronavirus that causes mild yet prevalent respiratory disease, and is related to the zoonotic SARS and MERS betacoronaviruses, which have high fatality rates and pandemic potential. Cell tropism and host range is determined in part by the coronavirus spike (S) protein, which binds cellular receptors and mediates membrane fusion. As the largest known class I fusion protein, its size and extensive glycosylation have hindered structural studies of the full ectodomain, thus preventing a molecular understanding of its function and limiting development of effective interventions. Here we present the 4.0 Å resolution structure of the trimeric HKU1 S protein determined using single-particle cryo-electron microscopy. In the pre-fusion conformation, the receptor-binding subunits, S1, rest above the fusion-mediating subunits, S2, preventing their conformational rearrangement. Surprisingly, the S1 C-terminal domains are interdigitated and form extensive quaternary interactions that occlude surfaces known in other coronaviruses to bind protein receptors. These features, along with the location of the two protease sites known to be important for coronavirus entry, provide a structural basis to support a model of membrane fusion mediated by progressive S protein destabilization through receptor binding and proteolytic cleavage. These studies should also serve as a foundation for the structure-based design of betacoronavirus vaccine immunogens.

Middle East respiratory syndrome coronavirus (MERS-CoV) emerged into the human population in 2012 and has caused substantial morbidity and mortality. Potently neutralizing antibodies targeting the receptor-binding domain (RBD) on MERS-CoV spike (S) protein have been characterized, but much less is known about antibodies targeting non-RBD epitopes. Here, we report the structural and functional characterization of G2, a neutralizing antibody targeting the MERS-CoV S1 N-terminal domain (S1-NTD). Structures of G2 alone and in complex with the MERS-CoV S1-NTD define a site of vulnerability comprising two loops, each of which contain a residue mutated in G2-escape variants. Cell-surface binding studies and in vitro competition experiments demonstrate that G2 strongly disrupts the attachment of MERS-CoV S to its receptor, dipeptidyl peptidase-4 (DPP4), with the inhibition requiring the native trimeric S conformation. These results advance our understanding of antibody-mediated neutralization of coronaviruses and should facilitate the development of immunotherapeutics and vaccines against MERS-CoV. • The epitope for the neutralizing antibody G2 is confined to the apex of the MERS-CoV S1-NTD • G2 epitope is relatively well conserved • G2 IgG and Fab both neutralize pseudotyped and authentic MERS-CoV • G2 neutralizes by preventing the binding of DPP4 to trimeric S protein Wang et al. report the structural and functional characterization of the Middle East respiratory syndrome coronavirus (MERS-CoV)-neutralizing antibody G2. G2 recognizes a conserved epitope on the MERS-CoV S1 N-terminal domain (S1-NTD) and neutralizes MERS-CoV by interfering with binding to host receptor dipeptidyl peptidase-4 (DPP4). The findings are relevant for understanding the viral attachment mechanism and for the development of S1-NTD-based vaccines. [ABSTRACT FROM AUTHOR]