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Inter-domain communication in SARS-CoV-2 spike proteins controls protease-triggered cell entry.

ABSTRACT: SARS-CoV-2 continues to evolve into variants of concern (VOC), with greatest variability in the multidomain, entry-facilitating spike proteins. To recognize the significance of adaptive spike protein changes, we compare variant SARS-CoV-2 virus particles in several assays reflecting authentic virus-cell entry. Virus particles with adaptive changes in spike amino-terminal domains (NTDs) are hypersensitive to proteolytic activation of membrane fusion, an essential step in virus-cell entry. Proteolysis is within fusion domains (FDs), at sites over 10 nm from the VOC-specific NTD changes, indicating allosteric inter-domain control of fusion activation. In addition, NTD-specific antibodies block FD cleavage, membrane fusion, and virus-cell entry, suggesting restriction of inter-domain communication as a neutralization mechanism. Finally, using structure-guided mutagenesis, we identify an inter-monomer β sheet structure that facilitates NTD-to-FD transmissions and subsequent fusion activation. This NTD-to-FD axis that sensitizes viruses to infection and to NTD-specific antibody neutralization provides new context for understanding selective forces driving SARS-CoV-2 evolution.

SUBMITTER: Qing E

PROVIDER: S-EPMC9015963 | biostudies-literature | 2022 May

REPOSITORIES: biostudies-literature

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Inter-domain communication in SARS-CoV-2 spike proteins controls protease-triggered cell entry.

Qing Enya E Li Pengfei P Cooper Laura L Schulz Sebastian S Jäck Hans-Martin HM Rong Lijun L Perlman Stanley S Gallagher Tom T

Cell reports 20220419 5

SARS-CoV-2 continues to evolve into variants of concern (VOC), with greatest variability in the multidomain, entry-facilitating spike proteins. To recognize the significance of adaptive spike protein changes, we compare variant SARS-CoV-2 virus particles in several assays reflecting authentic virus-cell entry. Virus particles with adaptive changes in spike amino-terminal domains (NTDs) are hypersensitive to proteolytic activation of membrane fusion, an essential step in virus-cell entry. Proteol ...[more]

PMID: 35477024

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Project description:Evolved SARS-CoV-2 variants of concern (VOCs) spread through human populations in succession. Major virus variations are in the entry-facilitating viral spike (S) proteins; Omicron VOCs have 29-40 S mutations relative to ancestral D614G viruses. The impacts of this Omicron divergence on S protein structure, antigenicity, cell entry pathways, and pathogenicity have been extensively evaluated, yet gaps remain in correlating specific alterations with S protein functions. In this study, we compared the functions of ancestral D614G and Omicron VOCs using cell-free assays that can reveal differences in several distinct steps of the S-directed virus entry process. Relative to ancestral D614G, Omicron BA.1 S proteins were hypersensitized to receptor activation, to conversion into intermediate conformational states, and to membrane fusion-activating proteases. We identified mutations conferring these changes in S protein character by evaluating domain-exchanged D614G/Omicron recombinants in the cell-free assays. Each of the three functional alterations was mapped to specific S protein domains, with the recombinants providing insights on inter-domain interactions that fine-tune S-directed virus entry. Our results provide a structure-function atlas of the S protein variations that may promote the transmissibility and infectivity of current and future SARS-CoV-2 VOCs. IMPORTANCE Continuous SARS-CoV-2 adaptations generate increasingly transmissible variants. These succeeding variants show ever-increasing evasion of suppressive antibodies and host factors, as well as increasing invasion of susceptible host cells. Here, we evaluated the adaptations enhancing invasion. We used reductionist cell-free assays to compare the entry steps of ancestral (D614G) and Omicron (BA.1) variants. Relative to D614G, Omicron entry was distinguished by heightened responsiveness to entry-facilitating receptors and proteases and by enhanced formation of intermediate states that execute virus-cell membrane fusion. We found that these Omicron-specific characteristics arose from mutations in specific S protein domains and subdomains. The results reveal the inter-domain networks controlling S protein dynamics and efficiencies of entry steps, and they offer insights on the evolution of SARS-CoV-2 variants that arise and ultimately dominate infections worldwide.

Project description:Selective pressures drive adaptive changes in the coronavirus spike proteins directing virus-cell entry. These changes are concentrated in the amino-terminal domains (NTDs) and the receptor-binding domains (RBDs) of complex modular spike protein trimers. The impact of this hypervariability on virus entry is often unclear, particularly with respect to sarbecovirus NTD variations. Therefore, we constructed indels and substitutions within hypervariable NTD regions and used severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus-like particles and quantitative virus-cell entry assays to elucidate spike structures controlling this initial infection stage. We identified NTD variations that increased SARS-CoV-2 spike protein-mediated membrane fusion and cell entry. Increased cell entry correlated with greater presentation of RBDs to ACE2 receptors. This revealed a significant allosteric effect, in that changes within the NTDs can orient RBDs for effective virus-cell binding. Yet, those NTD changes elevating receptor binding and membrane fusion also reduced interdomain associations, leaving spikes on virus-like particles susceptible to irreversible inactivation. These findings parallel those obtained decades ago, in which comparisons of murine coronavirus spike protein variants established inverse relationships between membrane fusion potential and virus stability. Considerable hypervariability in the SARS-CoV-2 spike protein NTDs also appear to be driven by counterbalancing pressures for effective virus-cell entry and durable extracellular virus infectivity. These forces may selectively amplify SARS-CoV-2 variants of concern. IMPORTANCE Adaptive changes that increase SARS-CoV-2 transmissibility may expand and prolong the coronavirus disease 2019 (COVID-19) pandemic. Transmission requires metastable and dynamic spike proteins that bind viruses to cells and catalyze virus-cell membrane fusion. Using newly developed assays reflecting these two essential steps in virus-cell entry, we focused on adaptive changes in SARS-CoV-2 spike proteins and found that deletions in amino-terminal domains reset spike protein metastability, rendering viruses less stable yet more poised to respond to cellular factors that prompt entry and subsequent infection. The results identify adjustable control features that balance extracellular virus stability with facile virus dynamics during cell entry. These equilibrating elements warrant attention when monitoring the evolution of pandemic coronaviruses.

Project description:SARS-CoV-2 infection or vaccination produces neutralizing antibody responses that contribute to better clinical outcomes. The receptor binding domain (RBD) and the N-terminal domain (NTD) of the spike trimer (S) constitute the two major neutralizing targets for the antibody system. Neutralizing antibodies targeting the RBD bind to several different sites on this domain. In contrast, most neutralizing antibodies to NTD characterized to date bind to a single supersite, however these antibodies were obtained by methods that were not NTD specific. Here we use NTD specific probes to focus on anti-NTD memory B cells in a cohort of pre-omicron infected individuals some of which were also vaccinated. Of 275 NTD binding antibodies tested 103 neutralized at least one of three tested strains: Wuhan-Hu-1, Gamma, or PMS20, a synthetic variant which is extensively mutated in the NTD supersite. Among the 43 neutralizing antibodies that were further characterized, we found 6 complementation groups based on competition binding experiments. 58% targeted epitopes outside the NTD supersite, and 58% neutralized either Gamma or Omicron, but only 14% were broad neutralizers. Three of the broad neutralizers were characterized structurally. C1520 and C1791 recognize epitopes on opposite faces of the NTD with a distinct binding pose relative to previously described antibodies allowing for greater potency and cross-reactivity with 7 different variants including Beta, Delta, Gamma and Omicron. Antibody C1717 represents a previously uncharacterized class of NTD-directed antibodies that recognizes the viral membrane proximal side of the NTD and SD2 domain, leading to cross-neutralization of Beta, Gamma and Omicron. We conclude SARS-CoV-2 infection and/or Wuhan-Hu-1 mRNA vaccination produces a diverse collection of memory B cells that produce anti-NTD antibodies some of which can neutralize variants of concern. Rapid recruitment of these cells into the antibody secreting plasma cell compartment upon re-infection likely contributes to the relatively benign course of subsequent infections with SARS-CoV-2 variants including omicron.

Dataset Information

Inter-domain communication in SARS-CoV-2 spike proteins controls protease-triggered cell entry.

Publications

Inter-domain communication in SARS-CoV-2 spike proteins controls protease-triggered cell entry.

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