The pandemic coronavirus SARS-CoV-2 threatens public health worldwide. The viral spike protein mediates SARS-CoV-2 entry into host cells and harbors a S1/S2 cleavage site containing multiple arginine ...residues (multibasic) not found in closely related animal coronaviruses. However, the role of this multibasic cleavage site in SARS-CoV-2 infection is unknown. Here, we report that the cellular protease furin cleaves the spike protein at the S1/S2 site and that cleavage is essential for S-protein-mediated cell-cell fusion and entry into human lung cells. Moreover, optimizing the S1/S2 site increased cell-cell, but not virus-cell, fusion, suggesting that the corresponding viral variants might exhibit increased cell-cell spread and potentially altered virulence. Our results suggest that acquisition of a S1/S2 multibasic cleavage site was essential for SARS-CoV-2 infection of humans and identify furin as a potential target for therapeutic intervention.
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•The spike protein of SARS-CoV-2 harbors a multibasic S1/S2 site•The host cell protease furin cleaves the SARS-CoV-2 spike protein at the S1/S2 site•Cleavage at the S1/S2 site is essential for spike-driven viral entry into lung cells
Coronavirus spike proteins are activated by host cell proteases. Hoffmann and colleagues show that the pandemic SARS-CoV-2 harbors a highly cleavable S1/S2 cleavage site not found in closely related coronaviruses. Cleavage at this site is mediated by furin and is required for viral entry into human lung cells.
The recent emergence of the novel, pathogenic SARS-coronavirus 2 (SARS-CoV-2) in China and its rapid national and international spread pose a global health emergency. Cell entry of coronaviruses ...depends on binding of the viral spike (S) proteins to cellular receptors and on S protein priming by host cell proteases. Unravelling which cellular factors are used by SARS-CoV-2 for entry might provide insights into viral transmission and reveal therapeutic targets. Here, we demonstrate that SARS-CoV-2 uses the SARS-CoV receptor ACE2 for entry and the serine protease TMPRSS2 for S protein priming. A TMPRSS2 inhibitor approved for clinical use blocked entry and might constitute a treatment option. Finally, we show that the sera from convalescent SARS patients cross-neutralized SARS-2-S-driven entry. Our results reveal important commonalities between SARS-CoV-2 and SARS-CoV infection and identify a potential target for antiviral intervention.
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•SARS-CoV-2 uses the SARS-CoV receptor ACE2 for host cell entry•The spike protein of SARS-CoV-2 is primed by TMPRSS2•Antibodies against SARS-CoV spike may offer some protection against SARS-CoV-2
The emerging SARS-coronavirus 2 (SARS-CoV-2) threatens public health. Hoffmann and coworkers show that SARS-CoV-2 infection depends on the host cell factors ACE2 and TMPRSS2 and can be blocked by a clinically proven protease inhibitor. These findings might help to establish options for prevention and treatment.
The rapid spread of the SARS-CoV-2 Omicron variant suggests that the virus might become globally dominant. Further, the high number of mutations in the viral spike protein raised concerns that the ...virus might evade antibodies induced by infection or vaccination. Here, we report that the Omicron spike was resistant against most therapeutic antibodies but remained susceptible to inhibition by sotrovimab. Similarly, the Omicron spike evaded neutralization by antibodies from convalescent patients or individuals vaccinated with the BioNTech-Pfizer vaccine (BNT162b2) with 12- to 44-fold higher efficiency than the spike of the Delta variant. Neutralization of the Omicron spike by antibodies induced upon heterologous ChAdOx1 (Astra Zeneca-Oxford)/BNT162b2 vaccination or vaccination with three doses of BNT162b2 was more efficient, but the Omicron spike still evaded neutralization more efficiently than the Delta spike. These findings indicate that most therapeutic antibodies will be ineffective against the Omicron variant and that double immunization with BNT162b2 might not adequately protect against severe disease induced by this variant.
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•Omicron uses human and animal ACE2 for host cell entry•Omicron is resistant against neutralization by several therapeutic antibodies•Omicron efficiently evades antibodies from infected or 2 × BNT-vaccinated patients•Omicron moderately evades antibodies induced by 3 × BNT or heterologous vaccination
The SARS-CoV-2 Omicron variant is rapidly spreading worldwide and a public health concern. Experiments show that this variant is resistant against several therapeutic antibodies for COVID-19 and efficiently evades antibodies induced upon infection or double BNT162b2 vaccination, but not triple BNT162b2 or ChAdOx1/BNT162b2 vaccination.
•Host cell proteases activate the spike protein of the SARS-coronavirus.•Activation is essential for viral infectivity and a potential target for intervention.•Cathepsin L activates the spike protein ...in host cell endosomes.•TMPRSS2 activates the spike protein at the plasma membrane.•SARS- and MERS-coronavirus exploit the same proteases for activation.
The severe acute respiratory syndrome (SARS) pandemic revealed that zoonotic transmission of animal coronaviruses (CoV) to humans poses a significant threat to public health and warrants surveillance and the development of countermeasures. The activity of host cell proteases, which cleave and activate the SARS-CoV spike (S) protein, is essential for viral infectivity and constitutes a target for intervention. However, the identities of the proteases involved have been unclear. Pioneer studies identified cathepsins and type II transmembrane serine proteases as cellular activators of SARS-CoV and demonstrated that several emerging viruses might exploit these enzymes to promote their spread. Here, we will review the proteolytic systems hijacked by SARS-CoV for S protein activation, we will discuss their contribution to viral spread in the host and we will outline antiviral strategies targeting these enzymes. This paper forms part of a series of invited articles in Antiviral Research on “From SARS to MERS: 10years of research on highly pathogenic human coronaviruses.’’
Multivalent lectin-glycan interactions are widespread in biology and are often exploited by pathogens to bind and infect host cells. Glycoconjugates can block such interactions and thereby prevent ...infection. The inhibition potency strongly depends on matching the spatial arrangement between the multivalent binding partners. However, the structural details of some key lectins remain unknown and different lectins may exhibit overlapping glycan specificity. This makes it difficult to design a glycoconjugate that can potently and specifically target a particular multimeric lectin for therapeutic interventions, especially under the challenging in vivo conditions. Conventional techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) can provide quantitative binding thermodynamics and kinetics. However, they cannot reveal key structural information, e.g., lectin’s binding site orientation, binding mode, and interbinding site spacing, which are critical to design specific multivalent inhibitors. Herein we report that gold nanoparticles (GNPs) displaying a dense layer of simple glycans are powerful mechanistic probes for multivalent lectin-glycan interactions. They can not only quantify the GNP-glycan-lectin binding affinities via a new fluorescence quenching method, but also reveal drastically different affinity enhancing mechanisms between two closely related tetrameric lectins, DC-SIGN (simultaneous binding to one GNP) and DC-SIGNR (intercross-linking with multiple GNPs), via a combined hydrodynamic size and electron microscopy analysis. Moreover, a new term, potential of assembly formation (PAF), has been proposed to successfully predict the assembly outcomes based on the binding mode between GNP-glycans and lectins. Finally, the GNP-glycans can potently and completely inhibit DC-SIGN-mediated augmentation of Ebola virus glycoprotein-driven cell entry (with IC50 values down to 95 pM), but only partially block DC-SIGNR-mediated virus infection. Our results suggest that the ability of a glycoconjugate to simultaneously block all binding sites of a target lectin is key to robust inhibition of viral infection.
Coronaviruses make use of a large envelope protein called spike (S) to engage host cell receptors and catalyze membrane fusion. Because of the vital role that these S proteins play, they represent a ...vulnerable target for the development of therapeutics. Here, we describe the isolation of single-domain antibodies (VHHs) from a llama immunized with prefusion-stabilized coronavirus spikes. These VHHs neutralize MERS-CoV or SARS-CoV-1 S pseudotyped viruses, respectively. Crystal structures of these VHHs bound to their respective viral targets reveal two distinct epitopes, but both VHHs interfere with receptor binding. We also show cross-reactivity between the SARS-CoV-1 S-directed VHH and SARS-CoV-2 S and demonstrate that this cross-reactive VHH neutralizes SARS-CoV-2 S pseudotyped viruses as a bivalent human IgG Fc-fusion. These data provide a molecular basis for the neutralization of pathogenic betacoronaviruses by VHHs and suggest that these molecules may serve as useful therapeutics during coronavirus outbreaks.
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•VHHs isolated from a llama immunized with prefusion-stabilized coronavirus spikes•Structural characterization of VHHs reveals conserved mechanism of neutralization•SARS-CoV-1 S-directed VHH cross-reacts with SARS-CoV-2 S•Bivalent VHH neutralizes SARS-CoV-2 pseudoviruses
Using llamas immunized with prefusion-stabilized betacoronavirus spike proteins, Wrapp et al. identify neutralizing cross-reactive single-domain camelid antibodies, which may serve not only as useful reagents for researchers studying the viruses causing MERS, SARS, and COVID-19, but also potential therapeutic candidates. Crystal structures further reveal how these antibodies bind spike proteins to prevent virus entry into cells.
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