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Authors: Walls et al.

Link to paper: https://www.cell.com/cell/fulltext/S0092-8674(20)30262-2

Journal/ Pre-Print: Cell

Key Words: viral entry, spike glycoprotein (S), neutralizing antibody, cryo-EM

Summary 

This study determines the Cryo-EM structure of SARS-CoV-2 spike protein ectodomain trimer and reveals two conformations of its receptor-binding domain during viral entry. Using multiple sequence alignment and biolayer interferometry, the study shows that SARS-CoV and SARS-CoV-2 spike proteins bind to hACE2 with comparable affinity, suggesting an explanation for the efficient spread of SARS-CoV-2. Sequence analysis also reveals a unique furin cleavage site in SARS-CoV-2 spike protein, which upon abrogation mildly affects viral entry. Lastly, polyclonal antibodies generated against SARS-CoV spike protein are shown to have potent inhibitory effect in SARS-CoV-2 spike protein-mediated viral entry.

Research highlights:

1. Cryo-EM data reveal structure of the SARS-CoV-2 spike protein ectodomain trimer and show the closed and open conformations of SARS-CoV-2 spike glycoprotein.

2. SARS-CoV-2 spike protein is shown to have a similar binding affinity to hACE2 as SARS-CoV spike protein.

3. SARS-CoV-2 spike-mediated entry is shown to be inhibited by the polyclonal antibodies generated against SARS-CoV.

Research Impact:

· Understand the virology and/or cell biology of SARS-CoV2/COVID19: improves knowledge on SARS-CoV-2 entry process

· Develop a vaccine for SARS-CoV2/COVID19: paves the way for designing broadly protective vaccines against the SARS and SARS-related coronaviruses

· Inhibition of SARS-CoV2/COVID19 transmission and treatment of SARS-CoV2/COVID19 positive individuals: provides a structural framework for drugs against viral entry

· Understand the immune response to SARS-CoV2/COVID19: the existence of cross-inhibiting antibodies suggests that exposure to one of the SARS-CoV or SARS-CoV-2 may potentially elicit cross-reactive antibodies against the other virus

Methodology:

· In vitro biochemistry study

· In vitro structural study

· In vitro cell culture and virology study

Strengths and limitation of the paper:

Novelty:

- First to show structures of the two conformations (open and closed) of SARS-CoV-2 spike SB domain during viral entry.

- Along with Wrapp et al. and Lan et al., this study is the first to publish a high-resolution structure of the SARS-CoV-2 spike protein.

- Along with Coutard et al., this study is the first to report the unique furin-like cleavage site in SARS-CoV-2 spike protein.

- Similar comparisons between the receptor binding affinity of SARS-CoV spike protein and SARS-CoV-2 spike protein have been shown in recent literature.

- Similar SARS-CoV polyclonal antibody cross-neutralizing analyses have been performed in recent literature.

- The paper provides further evidence for hACE2 being a functional entry receptor of SARS-CoV-2.

Standing in the field: Some controversies.

- The determined structure of the SARS-CoV-2 spike protein agrees with that proposed in other studies.

- The furin-cleavage site identified in this study agrees with that identified in Coutard et al.

- Wrapp et al. suggests an at-least-10 times higher hACE binding affinity of the SARS-CoV-2 spike protein compared to SARS-CoV spike protein.

- Ou et al. showed that polyclonal anti-SARS-CoV spike protein antibody or serum from SARS-CoV recovered patient have no inhibition in SARS-CoV-2 viral entry, and vice versa.

Appropriate statistics:

- Western-blots and biolayer interferometry assays were performed multiple times with at least two independent preparations of each sample.

- Most other quantification experiment have included at least three technical replicates.

- No methods were used to determine whether the data met assumptions of the statistical approach.

Viral model used: SARS-CoV-2 and SARS-CoV S pseudoviruses using murine leukemia virus (MLV) backbone

Translatability:

- The structural data provided a blueprint for the design of vaccines and drugs of viral entry

- The cross-neutralizing antibody inhibition experiment offers insight into design of a broadly protective vaccine.

Main limitations:

- Did not directly look at the antigenicity of SARS-CoV-2 spike protein, as stated in the title. Instead, studied the cross-reactivity of SARS-CoV-2 spike protein and the previously generated antibodies against SARS-CoV spike protein.

- Cell types used are not respiratory epithelial lining cells, which are naturally infected cell types during an infection.

- Technical replicates instead of biological replicates are used, lowering statistical power.

- Direct binding of the polyclonal antibodies against SARS-CoV-2 spike protein is not shown and binding site on spike protein is not identified.

- The conclusion that ‘the binding affinity of SARS-CoV-2 spike protein and SARS-CoV spike protein to hACE2 receptor is comparable’ is drawn, when the binding responses and KD values for both proteins against the same concentration of hACE2 appear to be numerically dissimilar.

- MLV pseudoviruses are used to mimic viral infection, which may have simplified the situation in cell culture experiments.

- It is unclear and not well explained why in Figure 1A. furin mutation increases viral entry.