he ongoing global pandemic of the novel SARS-CoV-2 coronavirus presents an urgent need for the development of effective preventative and treatment therapies. The viral spike (S)-protein is a prime target for such therapies because of its critical role in the virus life cycle. The transmembrane CoV S-protein spike trimer is composed of interwoven protomers that include an N-terminal receptor-binding S1 subunit and a C-terminal S2 subunit that contains the fusion elements (Fig. 1a,b) 1. The S1 subunit is subdivided into the N-terminal domain (NTD) followed by the receptor-binding domain (RBD) and two structurally conserved subdomains (SD1 and SD2). Together, these domains cap the S2 subunit, protecting the conserved fusion machinery. Binding to the host receptor via the RBD in S1 is followed by proteolytic cleavage of the spike by host proteases 2. Large conformational changes in the S-protein result in S1 shedding and exposure of the fusion machinery in S2. Class I fusion proteins, such as the CoV-2 S-protein, undergo large conformational changes during the fusion process and must, by necessity, be highly flexible and dynamic. Indeed, cryo-EM structures of the SARS-CoV-2 spike reveal considerable flexibility and dynamics in the S1 subunit 1,2 , especially around the RBD, which exhibits two discrete conformational states-a 'down' state that is shielded from receptor binding and an 'up' state that is receptor-accessible. The wealth of structural information for β-CoV S-proteins, including the recently determined cryo-EM structures of the SARS-CoV-2 spike 1-11 , has provided a rich source of detailed geometric information from which to begin precise examination of the macromolecular transitions underlying triggering of this fusion machine. Several structures of soluble ectodomain constructs that retain the complete S1 subunit and the surface-exposed S2 subunit have been determined. These include SARS-CoV-2 1,3 , SARS 4-8 , MERS 4,9 and other human 1,10 and murine 11 β-CoV S-proteins.

The SARS-CoV-2 spike (S) protein is the target of vaccine design efforts to end the COVID-19 pandemic. Despite a low mutation rate, isolates with the D614G substitution in the S protein appeared early during the pandemic, and are now the dominant form worldwide. Here, we explore spike conformational changes and the effects of the D614G mutation on a soluble S ectodomain construct. Cryo-EM structures reveal altered RBD disposition; antigenicity and proteolysis experiments reveal structural changes and enhanced furin cleavage efficiency of the G614 variant. Furthermore, furin cleavage alters the up/down ratio of the Receptor Binding Domains (RBD) in the G614 S ectodomain, demonstrating an allosteric effect on RBD positioning triggered by changes in the SD2 region, that harbors residue 614 and the furin cleavage site. Our results elucidate SARS-CoV-2 spike conformational landscape and allostery, and have implications for vaccine design.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with multiple spike mutations enable increased transmission and antibody resistance. We combined cryo–electron microscopy (cryo-EM), binding, and computational analyses to study variant spikes, including one that was involved in transmission between minks and humans, and others that originated and spread in human populations. All variants showed increased angiotensin-converting enzyme 2 (ACE2) receptor binding and increased propensity for receptor binding domain (RBD)–up states. While adaptation to mink resulted in spike destabilization, the B.1.1.7 (UK) spike balanced stabilizing and destabilizing mutations. A local destabilizing effect of the RBD E484K mutation was implicated in resistance of the B.1.1.28/P.1 (Brazil) and B.1.351 (South Africa) variants to neutralizing antibodies. Our studies revealed allosteric effects of mutations and mechanistic differences that drive either interspecies transmission or escape from antibody neutralization.

SARS-CoV-2 neutralizing antibodies (NAbs) protect against COVID-19. A concern regarding SARS-CoV-2 antibodies is whether they mediate disease enhancement. Here, we isolated NAbs against the receptor-binding domain (RBD) and the N-terminal domain (NTD) of SARS-CoV-2 spike from individuals with acute or convalescent SARS-CoV-2 or a history of SARS-CoV infection. Cryo-electron microscopy of RBD and NTD antibodies demonstrated function-specific modes of binding. Select RBD NAbs also demonstrated Fc receptor-γ (FcγR)-mediated enhancement of virus infection in vitro , while five non-neutralizing NTD antibodies mediated FcγR-independent in vitro infection enhancement. However, both types of infection-enhancing antibodies protected from SARS-CoV-2 replication in monkeys and mice. Three of 46 monkeys infused with enhancing antibodies had higher lung inflammation scores compared to controls. One monkey had alveolar edema and elevated bronchoalveolar lavage inflammatory cytokines. Thus, while in vitro antibody-enhanced infection does not necessarily herald enhanced infection in vivo , increased lung inflammation can rarely occur in SARS-CoV-2 antibody-infused macaques.

The SARS-CoV-2 Spike protein acquired a D614G mutation early in the pandemic that confers greater infectivity and is now the globally dominant form. To determine whether D614G might also mediate neutralization-escape that could compromise vaccine efficacy, sera from Spike-immunized mice, nonhuman primates and humans were evaluated for neutralization of pseudoviruses bearing either D614 or G614 spike. In all cases, the G614 pseudovirus was moderately more susceptible to neutralization. The G614 pseudovirus also was more susceptible to neutralization by receptor binding domain (RBD) monoclonal antibodies and convalescent sera from people infected with either form of the virus. Negative stain electron microscopy revealed a higher percentage of the 1-RBD “up” conformation in the G614 spike, suggesting increased epitope exposure as a mechanism of enhanced vulnerability to neutralization. Based on these findings, the D614G mutation is not expected to be an obstacle for current vaccine development.