The spike (S) protein of SARS-CoV-2 has been observed in three distinct pre-fusion conformations: locked, closed and open. Of these, the function of the locked conformation remains poorly understood. Here we engineered a SARS-CoV-2 S protein construct “S-R/x3” to arrest SARS-CoV-2 spikes in the locked conformation by a disulfide bond. Using this construct we determined high-resolution structures confirming that the x3 disulfide bond has the ability to stabilize the otherwise transient locked conformations. Structural analyses reveal that wild-type SARS-CoV-2 spike can adopt two distinct locked-1 and locked-2 conformations. For the D614G spike, based on which all variants of concern were evolved, only the locked-2 conformation was observed. Analysis of the structures suggests that rigidified domain D in the locked conformations interacts with the hinge to domain C and thereby restrains RBD movement. Structural change in domain D correlates with spike conformational change. We propose that the locked-1 and locked-2 conformations of S are present in the acidic high-lipid cellular compartments during virus assembly and egress. In this model, release of the virion into the neutral pH extracellular space would favour transition to the closed or open conformations. The dynamics of this transition can be altered by mutations that modulate domain D structure, as is the case for the D614G mutation, leading to changes in viral fitness. The S-R/x3 construct provides a tool for the further structural and functional characterization of the locked conformations of S, as well as how sequence changes might alter S assembly and regulation of receptor binding domain dynamics.

The spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is an important target for vaccine and drug development. However, the rapid emergence of variant strains with mutated S proteins has rendered many treatments ineffective. Cleavage of the S protein by host proteases is essential for viral infection. Here, we discovered that the S protein contains two previously unidentified Cathepsin L (CTSL) cleavage sites (CS-1 and CS-2). Both sites are highly conserved among all known SARS-CoV-2 variants. Our structural studies revealed that CTSL cleavage promoted S to adopt receptor-binding domain (RBD) “up” activated conformations, facilitating receptor-binding and membrane fusion. We confirmed that CTSL cleavage is essential during infection of all emerged SARS-CoV-2 variants (including the recently emerged Omicron variant) by pseudovirus (PsV) infection experiment. Furthermore, we found CTSL-specific inhibitors not only blocked infection of PsV/live virus in cells but also reduced live virus infection of ex vivo lung tissues of both human donors and human ACE2-transgenic mice. Finally, we showed that two CTSL-specific inhibitors exhibited excellent In vivo effects to prevent live virus infection in human ACE2-transgenic mice. Our work demonstrated that inhibition of CTSL cleavage of SARS-CoV-2 S protein is a promising approach for the development of future mutation-resistant therapy.

Population antibody response is thought to be important in selection of virus variants. We report that SARS-CoV-2 infection elicits a population immune response that is mediated by a lineage of VH1-69 germline antibodies. A representative antibody R1-32 from this lineage was isolated. By cryo-EM, we show that it targets a semi-cryptic epitope in the spike receptor-binding domain. Binding to this non-ACE2 competing epitope results in spike destruction, thereby inhibiting virus entry. On the basis of epitope location, neutralization mechanism and analysis of antibody binding to spike variants, we propose that recurrent substitutions at 452 and 490 are associated with immune evasion of the identified population antibody response. These substitutions, including L452R (present in the Delta variant), disrupt interactions mediated by the VH1-69-specific hydrophobic HCDR2 to impair antibody-antigen association, enabling variants to escape. The first Omicron variants were sensitive to antibody R1-32 but subvariants that harbour L452R quickly emerged and spread. Our results provide insights into how SARS-CoV-2 variants emerge and evade host immune responses.

No abstract

SARS-CoV-2 Omicron variants feature highly mutated spike proteins with extraordinary abilities in evading antibodies isolated earlier in the pandemic. Investigation of memory B cells from patients primarily with breakthrough infections with the Delta variant enables isolation of a number of neutralizing antibodies cross-reactive to heterologous variants of concern (VOCs) including Omicron variants (BA.1-BA.4). Structural studies identify altered complementarity determining region (CDR) amino acids and highly unusual heavy chain CDR2 insertions respectively in two representative cross-neutralizing antibodies—YB9-258 and YB13-292. These features are putatively introduced by somatic hypermutation and they are heavily involved in epitope recognition to broaden neutralization breadth. Previously, insertions/deletions were rarely reported for antiviral antibodies except for those induced by HIV-1 chronic infections. These data provide molecular mechanisms for cross-neutralization of heterologous SARS-CoV-2 variants by antibodies isolated from Delta variant infected patients with implications for future vaccination strategy.

SARS-CoV-2 spike protein (S) is structurally dynamic and has been observed by cryo-EM to adopt a variety of prefusion conformations that can be categorized as locked, closed and open. The locked conformations feature tightly packed trimers with structural elements incompatible with RBD in "up" position. For SARS-CoV-2 S, it has been shown that the locked conformations are transient under neutral pH. Probably due to their transience, locked conformations remain largely uncharacterized for SARS-CoV-1 S. Intriguingly, locked conformations were the only conformations captured for S proteins of bat and pangolin origin SARS-related coronaviruses. In this study, we introduced x1, x2, and x3 disulfides into SARS-CoV-1 S. Some of these disulfides have been shown to preserve rare locked conformations when introduced to SARS-CoV-2 S. Introduction of these disulfides allowed us to image a variety of locked and other rare conformations for SARS-CoV-1 S by cryo-EM. We identified bound cofactors and structural features that are associated with SARS-CoV-1 S locked conformations. We compare newly determined structures to other available spike structures of Sarbecoviruses to identify conserved features and discuss their possible functions.

Currently circulating SARS-CoV-2 Omicron variants feature highly mutated spike proteins with extraordinary abilities in evading acute-infection-induced germline antibodies isolated earlier in the pandemic. We identified that memory B cells from Delta variant breakthrough-infection patients expressed antibodies with more extensive somatic hypermutations (SHMs) allowing isolation of a number of broadly neutralizing antibodies with activities against heterologous variants of concerns (VOCs) including Omicron variant. Structural studies identified that SHM introduced altered amino acids and highly unusual HCDR2 insertions respectively in two representative broadly neutralizing antibodies - YB9-258 and YB13-292. Previously, insertion/deletion were rarely reported for antiviral antibodies except for those induced by HIV-1 chronic infections. Identified SHMs involved heavily in epitope recognition, they broadened neutralization breadth by rendering antibodies resistant to VOC mutations highly detrimental to previously isolated antibodies targeting similar epitopes. These data provide molecular mechanisms for enhanced immunity to heterologous SARS-CoV-2 variants after repeated antigen exposures with implications for future vaccination strategy.

Population antibody response is believed to be important in selection of new variant viruses. We identified that SARS-CoV-2 infections elicit a population immune response mediated by a lineage of VH1-69 germline antibodies. The representative antibody R1-32 targets a novel semi-cryptic epitope defining a new class of RBD targeting antibodies. Binding to this non-ACE2 competing epitope leading to spike destruction impairing virus entry. Based on epitope location, neutralization mechanism and analysis of antibody binding to spike variants we propose that recurrent substitutions at 452 and 490 are associated with immune evasion of this population antibody response. These substitutions, including L452R found in the Delta variant, disrupt interaction mediated by the VH1-69 specific hydrophobic HCDR2 to impair antibody-antigen association allowing variants to escape. Lacking 452/490 substitutions, the Omicron variant is sensitive to this class of antibodies. Our results provide new insights into SARS-CoV-2 variant genesis and immune evasion.