Structures, conformations and distributions of SARS-CoV-2 spike protein trimers on intact virions

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virions are surrounded by a lipid bilayer from which spike (S) protein trimers protrude. Heavily glycosylated S trimers bind the ACE2 receptor and mediate entry of virions into target cells. S exhibits extensive conformational flexibility: it modulates the exposure of its receptor binding site and later undergoes complete structural rearrangement to drive fusion of viral and cellular membranes. The structures and conformations of soluble, overexpressed, purified S proteins have been studied in detail using cryo-electron microscopy. The structure and distribution of S on the virion surface, however, has not been characterised. Here we applied cryo-electron microscopy and tomography to image intact SARS-CoV-2 virions, determining the high-resolution structure, conformational flexibility and distributions of S trimers in situ on the virion surface. These results provide a basis for understanding the conformations of S present on the virion, and for studying their interactions with neutralizing antibodies.

Stabilizing the Closed SARS-CoV-2 Spike Trimer

“…The viral spike is mostly closed (Ke, Oton et al 2020) and although many neutralizing antibodies are directed against RBD, the antibodies with the highest neutralizing activity bind RBD in the down state (Brouwer, Caniels et al 2020, Robbiani, Gaebler et al 2020). Similar to other class I fusion proteins, a closed conformation may be more reminiscent of transmitted virus and antibodies that recognize the closed state may be more important for protection as has been shown for HIV (Sanders and Moore 2017).…”

“…Stabilization of the hinge loop of the S proteins of SARS-CoV and MERS-CoV has been achieved by mutation of two consecutive residues to proline in the S2 subunit between the central helix (CH) and heptad repeat 1 (HR1) (Pallesen, Wang et al 2017, Kirchdoerfer, Wang et al 2018 and this approach (2P) has successfully been applied into the SARS-CoV-2 S protein (Wrapp, Wang et al 2020). However, the S protein carrying these substitutions with additional furin site mutations (S-2P) remains unstable and strategies have recently been described to improve stability (Henderson, Edwards et al 2020, McCallum, Walls et al 2020, Xiong, Qu et al 2020. Comparison of the structure of SARS-CoV-2 S-2P (Walls, Park et al 2020, Wrapp, Wang et al 2020 with that of native SARS-CoV-2 S (Cai, Zhang et al 2020, Ke, Oton et al 2020 shows that the former adopts a more open conformation with one or more of the RBDs in the 'up' conformation.…”

“…However, the S protein carrying these substitutions with additional furin site mutations (S-2P) remains unstable and strategies have recently been described to improve stability (Henderson, Edwards et al 2020, McCallum, Walls et al 2020, Xiong, Qu et al 2020. Comparison of the structure of SARS-CoV-2 S-2P (Walls, Park et al 2020, Wrapp, Wang et al 2020 with that of native SARS-CoV-2 S (Cai, Zhang et al 2020, Ke, Oton et al 2020 shows that the former adopts a more open conformation with one or more of the RBDs in the 'up' conformation. Although neutralizing antibodies have been mapped to the RBD in the up as well as in the down state, the antibodies that bind the conserved epitopes on RBD in the down state were described to have the highest neutralizing potency (Brouwer, Caniels et al 2020, Robbiani, Gaebler et al 2020.…”

An ultra-potent synthetic nanobody neutralizes SARS-CoV-2 by locking Spike into an inactive conformation

“…Coronavirus virions are bounded by a membrane envelope that contains ~25 copies of the homotrimeric transmembrane spike glycoprotein (Spike) responsible for virus entry into the host cell (9). The surface-exposed portion of Spike is composed of two domains, S1 and S2 (10).…”

“…Binding to ACE2 requires the RBD in the up-state and enables cleavage by host proteases TMPRSS2 or cathepsin, triggering a dramatic conformational change in S2 that enables viral entry (16). In SARS-CoV-2 virions, Spike oscillates between an active, open conformation with at least one RBD in the up-state and an inactive, closed conformation with all RBDs in the down-state (9,11,14,15).…”