An official website of the United States government
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein binds the receptor angiotensin converting enzyme 2 (ACE2) and drives virus-host membrane fusion through refolding of its S2 domain. Whereas the S1 domain contains high sequence variability, the S2 domain is conserved and is a promising pan-betacoronavirus vaccine target. We applied cryo–electron tomography to capture intermediates of S2 refolding and understand inhibition by antibodies to the S2 stem-helix. Subtomogram averaging revealed ACE2 dimers cross-linking spikes before transitioning into S2 intermediates, which were captured at various stages of refolding. Pan-betacoronavirus neutralizing antibodies targeting the S2 stem-helix bound to and inhibited refolding of spike prehairpin intermediates. Combined with molecular dynamics simulations, these structures elucidate the process of SARS-CoV-2 entry and reveal how pan-betacoronavirus S2-targeting antibodies neutralize infectivity by arresting prehairpin intermediates.
more »
« less
Structural insights into the modulation of coronavirus spike tilting and infectivity by hinge glycans
Abstract Coronavirus spike glycoproteins presented on the virion surface mediate receptor binding, and membrane fusion during virus entry and constitute the primary target for vaccine and drug development. How the structure dynamics of the full-length spikes incorporated in viral lipid envelope correlates with the virus infectivity remains poorly understood. Here we present structures and distributions of native spike conformations on vitrified human coronavirus NL63 (HCoV-NL63) virions without chemical fixation by cryogenic electron tomography (cryoET) and subtomogram averaging, along with site-specific glycan composition and occupancy determined by mass spectrometry. The higher oligomannose glycan shield on HCoV-NL63 spikes than on SARS-CoV-2 spikes correlates with stronger immune evasion of HCoV-NL63. Incorporation of cryoET-derived native spike conformations into all-atom molecular dynamic simulations elucidate the conformational landscape of the glycosylated, full-length spike that reveals a role of hinge glycans in modulating spike bending. We show that glycosylation at N1242 at the upper portion of the stalk is responsible for the extensive orientational freedom of the spike crown. Subsequent infectivity assays implicated involvement of N1242-glyan in virus entry. Our results suggest a potential therapeutic target site for HCoV-NL63.
more »
« less
- Award ID(s):
- 1942763
- PAR ID:
- 10473146
- Publisher / Repository:
- Nature Publishing Group
- Date Published:
- Journal Name:
- Nature Communications
- Volume:
- 14
- Issue:
- 1
- ISSN:
- 2041-1723
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
We develop a generalizable AI-driven workflow that leverages heterogeneous HPC resources to explore the time-dependent dynamics of molecular systems. We use this workflow to investigate the mechanisms of infectivity of the SARS-CoV-2 spike protein, the main viral infection machinery. Our workflow enables more efficient investigation of spike dynamics in a variety of complex environments, including within a complete SARS-CoV-2 viral envelope simulation, which contains 305 million atoms and shows strong scaling on ORNL Summit using NAMD. We present several novel scientific discoveries, including the elucidation of the spike’s full glycan shield, the role of spike glycans in modulating the infectivity of the virus, and the characterization of the flexible interactions between the spike and the human ACE2 receptor. We also demonstrate how AI can accelerate conformational sampling across different systems and pave the way for the future application of such methods to additional studies in SARS-CoV-2 and other molecular systems.more » « less
-
Site specific N- and O-glycosylation mapping of the spike proteins of SARS-CoV-2 variants of concernAbstract The glycosylation on the spike (S) protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, modulates the viral infection by altering conformational dynamics, receptor interaction and host immune responses. Several variants of concern (VOCs) of SARS-CoV-2 have evolved during the pandemic, and crucial mutations on the S protein of the virus have led to increased transmissibility and immune escape. In this study, we compare the site-specific glycosylation and overall glycomic profiles of the wild type Wuhan-Hu-1 strain (WT) S protein and five VOCs of SARS-CoV-2: Alpha, Beta, Gamma, Delta and Omicron. Interestingly, both N- and O-glycosylation sites on the S protein are highly conserved among the spike mutant variants, particularly at the sites on the receptor-binding domain (RBD). The conservation of glycosylation sites is noteworthy, as over 2 million SARS-CoV-2 S protein sequences have been reported with various amino acid mutations. Our detailed profiling of the glycosylation at each of the individual sites of the S protein across the variants revealed intriguing possible association of glycosylation pattern on the variants and their previously reported infectivity. While the sites are conserved, we observed changes in the N- and O-glycosylation profile across the variants. The newly emerged variants, which showed higher resistance to neutralizing antibodies and vaccines, displayed a decrease in the overall abundance of complex-type glycans with both fucosylation and sialylation and an increase in the oligomannose-type glycans across the sites. Among the variants, the glycosylation sites with significant changes in glycan profile were observed at both theN-terminal domain and RBD of S protein, with Omicron showing the highest deviation. The increase in oligomannose-type happens sequentially from Alpha through Delta. Interestingly, Omicron does not contain more oligomannose-type glycans compared to Delta but does contain more compared to the WT and other VOCs. O-glycosylation at the RBD showed lower occupancy in the VOCs in comparison to the WT. Our study on the sites and pattern of glycosylation on the SARS-CoV-2 S proteins across the VOCs may help to understand how the virus evolved to trick the host immune system. Our study also highlights how the SARS-CoV-2 virus has conserved bothN- andO- glycosylation sites on the S protein of the most successful variants even after undergoing extensive mutations, suggesting a correlation between infectivity/ transmissibility and glycosylation.more » « less
-
Virus-like particles (VLPs) have been proposed as an attractive tool in SARS-CoV-2 vaccine development, both as (1) a vaccine candidate with high immunogenicity and low reactogenicity and (2) a substitute for live virus in functional and neutralization assays. Though multiple SARS-CoV-2 VLP designs have already been explored in Sf9 insect cells, a key parameter ensuring VLPs are a viable platform is the VLP spike yield (i.e., spike protein content in VLP), which has largely been unreported. In this study, we show that the common strategy of producing SARS-CoV-2 VLPs by expressing spike protein in combination with the native coronavirus membrane and/or envelope protein forms VLPs, but at a critically low spike yield (~0.04–0.08 mg/L). In contrast, fusing the spike ectodomain to the influenza HA transmembrane domain and cytoplasmic tail and co-expressing M1 increased VLP spike yield to ~0.4 mg/L. More importantly, this increased yield translated to a greater VLP spike antigen density (~96 spike monomers/VLP) that more closely resembles that of native SARS-CoV-2 virus (~72–144 Spike monomers/virion). Pseudotyping further allowed for production of functional alpha (B.1.1.7), beta (B.1.351), delta (B.1.617.2), and omicron (B.1.1.529) SARS-CoV-2 VLPs that bound to the target ACE2 receptor. Finally, we demonstrated the utility of pseudotyped VLPs to test neutralizing antibody activity using a simple, acellular ELISA-based assay performed at biosafety level 1 (BSL-1). Taken together, this study highlights the advantage of pseudotyping over native SARS-CoV-2 VLP designs in achieving higher VLP spike yield and demonstrates the usefulness of pseudotyped VLPs as a surrogate for live virus in vaccine and therapeutic development against SARS-CoV-2 variants.more » « less
-
The SARS-CoV-2 virion has shown remarkable resilience, capable of mutating to escape immune detection and re-establishing infectious capabilities despite new vaccine rollouts. Therefore, there is a critical need to identify relatively immutable epitopes on the SARS-CoV-2 virion that are resistant to future mutations the virus may accumulate. While hACE2 has been identified as the receptor that mediates SARS-CoV-2 susceptibility, it is only modestly expressed in lung tissue. C-type lectin receptors like DC-SIGN can act as attachment sites to enhance SARS-CoV-2 infection of cells with moderate or low hACE2 expression. We developed an easy-to-implement assay system that allows for the testing of SARS-CoV-2 trans-infection. Using our assay, we assessed how SARS-CoV-2 Spike S1-domain glycans and spike proteins from different strains affected the ability of pseudotyped lentivirions to undergo DC-SIGN-mediated trans-infection. Through our experiments with seven glycan point mutants, two glycan cluster mutants and four strains of SARS-CoV-2 spike, we found that glycans N17 and N122 appear to have significant roles in maintaining COVID-19′s infectious capabilities. We further found that the virus cannot retain infectivity upon the loss of multiple glycosylation sites, and that Omicron BA.2 pseudovirions may have an increased ability to bind to other non-lectin receptor proteins on the surface of cells. Taken together, our work opens the door to the development of new therapeutics that can target overlooked epitopes of the SARS-CoV-2 virion to prevent C-type lectin-receptor-mediated trans-infection in lung tissue.more » « less
