A multibasic cleavage site in the spike protein of SARS-CoV-2 is essential for infection of human lung cells

Authors: Hoffman, M. et al.

Link to paper: https://www.cell.com/molecular-cell/fulltext/S1097-2765(20)30264-1

Journal/ Pre-Print: Molecular Cell

Tags: Cell Biology, Molecular biology, Structural biology, Virology

Research Highlights 

1. The spike protein of SARS-CoV-2 harbours a multibasic (arginine-rich) S1/S2 cleavage site

2. The host cell protease furin cleaves the SARS-CoV-2 spike (S) protein at the S1/S2 site

3. Cleavage at the S1/S2 site is essential for spike-driven viral entry into lung cells

Summary 

The authors found that SARS-CoV-2 spike (S) protein contains a unique multibasic motif at the S1/S2 cleavage site. Replacement of this motif with monobasic sequences such as the SARS-CoV S1/S2 site, or deletion of arginine residues in the SARS-CoV-2-S motif, reduced furin-mediated cleavage of SARS-CoV-2-S (although further repeats are required to determine the robustness of furin dependence). Addition of arginine residues increased cleavage. Similarly, mutation of SARS-CoV-2-S reduced cell-cell fusion and viral entry in human lung cell lines, but not in Vero (monkey kidney) cells. SARS-CoV-S with the SARS-CoV-2 S1/S2 motif rescued cell-cell fusion relative to WT SARS-CoV-S, but this did not enhance viral entry in human lung cells.

Impact for SARS-CoV2/COVID19 research efforts

Understand the virology and/or cell biology of SARS-CoV2/COVID19

Inhibit of SARS-CoV2/COVID19 transmission

Study Type

· In silico study / bioinformatics study (amino acid sequence analysis of S proteins)

· In vitro study

Strengths and limitations of the paper

Novelty: The authors were the first to experimentally show the role of the multibasic motif in SARS-CoV-2-S S1/S2 cleavage site in cell-cell fusion and viral entry

Standing in the field: Consistent with previous findings that furin and TMPRSS2 are required for SARS-CoV-2-S processing and viral entry.

Appropriate statistics: Correct statistical test used. Did the minimum number of repeats (n=3) in all experiments except one (n=4)

Viral model used: SARS-CoV-2 S protein expressed in HEK293 cells and incorporated into VSV particles

Translatability: No. However, use of furin inhibitor in vitro showed reduction of SARS-COV-2-S cleavage. Therefore, furin inhibition could be potential therapeutic approach if used for short periods of time, which has shown to be safe in mice (Sarac et al., 2004). Additionally, testing this furin inhibitor with authentic SARS-CoV-2 virus could help to increase translatability

Main limitations:

· Could increase n to improve robustness of results

· Could quantify imaging to detect cell-cell fusion, for ease of comparison between S protein mutants

· Western blot loading controls show unequal loading in representative blots – either increase n or quantify and normalise to loading control signal.

· Experimental design to detect S protein cleavage by Western blot is quite complex – unclear where proteases are coming from (endogenous from HEK293 or exogenous trypsin?)

· Following this, methods section and figure legends could have more detail

· Do not use authentic SARS-CoV-2 virus, although as authors mention, currently no technology available for replacing/deleting specific residues in authentic virus

· Lacking positive and negative biological controls for viral entry in Calu-3 cells experiment

· Have some contradictory findings (e.g. Figure 2, panel E results do not support panel D, specifically effect of optimising S1/S2 cleavage site) but do not comment on potential reasons for this

· Also in Figure 2E, the authors do not fully discuss the SARS-S and SARS-2 results, which are slightly conflicting (e.g. SARS-2-S with SARS S1/S2 motif has reduced entry versus SARS WT (which also contains SARS S1/S2 motif), indicating that this motif is potentially not as important for cleavage as the authors suggest)