CoChat
Sign Up
Cited by 3,142SARS-CoV-2 and COVID-19 ResearchCOVID-19 Clinical Research StudiesAnimal Virus Infections Studies

The DOI matches its Crossref record; 48 of the 48 references it cites were found in a registry or an index.

Cell entry mechanisms of SARS-CoV-2

Open access
Shang, Jian, Wan, Yushun, Luo, Chuming, Ye, Gang, Geng, Qibin, Auerbach, Ashley, Li, Fang·2020·Proceedings of the National Academy of Sciences
DOI 10.1073/pnas.2003138117PMC PMC7260975
Open this paper in CoChat — free account

Abstract

Significance: A key to curbing SARS-CoV-2 is to understand how it enters cells. SARS-CoV-2 and SARS-CoV both use human ACE2 as entry receptor and human proteases as entry activators. Using biochemical and pseudovirus entry assays and SARS-CoV as a comparison, we have identified key cell entry mechanisms of SARS-CoV-2 that potentially contribute to the immune evasion, cell infectivity, and wide spread of the virus. This study also clarifies conflicting reports from recent studies on cell entry of SARS-CoV-2. Finally, by highlighting the potency and the evasiveness of SARS-CoV-2, the study provides insight into intervention strategies that target its cell entry mechanisms.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.1073/pnas.2003138117

Cites

48 of 48 references verified0 citation errors · 0 not checkable

Statistics

Figures

Fig. 1. PPC motif in SARS-CoV-2 spike protein. (A) Different stages of coronavirus entry where host cellular proteases may activate coronavirus spikes. (B) Schematic drawing of the three-dimensional (3D) structure of coronavirus spike. S1, receptor-binding subunit; S2, membrane fusion subunit; TM, transmembrane anchor; IC, intracellular tail. (C) Schematic drawing of the 1D structure of coronavirus spike. NTD, N-terminal domain. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are structural units in coronavirus S2 that function in membrane fusion. (D) Sequence comparison of the spike proteins from SARS-CoV-2, SARS-CoV, and two bat SARS-like coronaviruses in a region at the S1/S2 boundary. Only SARS-CoV-2 spike contains a putative PPC motif—RRAR (residues in the box). The assumed PPC cleavage site is in front of the arginine residue labeled in red. The spike region mutated from SARS-CoV-2 sequence (TNSPRRA) to SARS-CoV sequence (SLL) is labeled in blue. GenBank accession numbers are QHD43416.1 for SARS-CoV-2 spike, AFR58740.1 for SARS-CoV spike, MG916901.1 for bat Rs3367 spike, and QHR63300.2 for bat RaTG13 spike.
Fig. 1. PPC motif in SARS-CoV-2 spike protein. (A) Different stages of coronavirus entry where host cellular proteases may activate coronavirus spikes. (B) Schematic drawing of the three-dimensional (3D) structure of coronavirus spike. S1, receptor-binding subunit; S2, membrane fusion subunit; TM, transmembrane anchor; IC, intracellular tail. (C) Schematic drawing of the 1D structure of coronavirus spike. NTD, N-terminal domain. FP (fusion peptide), HR1 (heptad repeat 1), and HR2 (heptad repeat 2) are structural units in coronavirus S2 that function in membrane fusion. (D) Sequence comparison of the spike proteins from SARS-CoV-2, SARS-CoV, and two bat SARS-like coronaviruses in a region at the S1/S2 boundary. Only SARS-CoV-2 spike contains a putative PPC motif—RRAR (residues in the box). The assumed PPC cleavage site is in front of the arginine residue labeled in red. The spike region mutated from SARS-CoV-2 sequence (TNSPRRA) to SARS-CoV sequence (SLL) is labeled in blue. GenBank accession numbers are QHD43416.1 for SARS-CoV-2 spike, AFR58740.1 for SARS-CoV spike, MG916901.1 for bat Rs3367 spike, and QHR63300.2 for bat RaTG13 spike.CC BY · PubMed Central
Fig. 2. Role of PPC motif in SARS-CoV-2 spike-mediated cell entry. (A) Cleavage state of SARS-CoV-2 spike on the surface of pseudoviruses. Packaged SARS-CoV-2 pseudoviruses were subjected to Western blot analysis for detection of the cleavage state of SARS-CoV-2 spike. SARS-CoV-2 spike fragments were detected using anti-C9 antibody targeting the C-terminal C9 tag of the spike protein. (Left) Wild-type (WT) SARS-CoV-2 pseudoviruses. (Right) SARS-CoV-2 pseudoviruses where the PPC motif in the spike protein had been mutated to the corresponding sequence in SARS-CoV spike (see Fig. 1D for details). (B) SARS-CoV-2 pseudovirus entry into three types of target cells. The two types of pseudoviruses correspond to the pseudoviruses in A. Pseudovirus entry efficiency was characterized as luciferase signal accompanying entry. The entry efficiency of wild-type SARS-CoV-2 pseudoviruses was taken as 100%. Error bars indicate SD (n = 4). ***P < 0.001; *P < 0.05.
Fig. 2. Role of PPC motif in SARS-CoV-2 spike-mediated cell entry. (A) Cleavage state of SARS-CoV-2 spike on the surface of pseudoviruses. Packaged SARS-CoV-2 pseudoviruses were subjected to Western blot analysis for detection of the cleavage state of SARS-CoV-2 spike. SARS-CoV-2 spike fragments were detected using anti-C9 antibody targeting the C-terminal C9 tag of the spike protein. (Left) Wild-type (WT) SARS-CoV-2 pseudoviruses. (Right) SARS-CoV-2 pseudoviruses where the PPC motif in the spike protein had been mutated to the corresponding sequence in SARS-CoV spike (see Fig. 1D for details). (B) SARS-CoV-2 pseudovirus entry into three types of target cells. The two types of pseudoviruses correspond to the pseudoviruses in A. Pseudovirus entry efficiency was characterized as luciferase signal accompanying entry. The entry efficiency of wild-type SARS-CoV-2 pseudoviruses was taken as 100%. Error bars indicate SD (n = 4). ***P < 0.001; *P < 0.05.CC BY · PubMed Central
Fig. 3. Effect of PPCs on SARS-CoV-2 spike-mediated cell entry. (A) SARS-CoV-2 pseudovirus entry into three types of target cells in the presence of PPCi. The pseudoviruses were packaged in the presence of different concentrations of PPCi before they were subjected to cell entry; (-) control: no pseudovirus was added. Also shown is the Western blot result of the corresponding pseudoviruses (packaged in the presence of different concentrations of PPCi). The entry efficiency of SARS-CoV-2 pseudoviruses without any treatment was taken as 100%. Error bars indicate SD (n = 4). ***P < 0.001; **P 0.01; *P < 0.05. (B) SARS-CoV pseudovirus entry into three types of target cells in the presence of PPCi. The experiments were performed in the same way as in A, except that SARS-CoV spike replaced SARS-CoV-2 spike in pseudoviruses. The entry efficiency of SARS-CoV pseudoviruses without any treatment was taken as 100%. (C) Western blot result of SARS-CoV-2 pseudoviruses packaged in cells treated with siRNA. (Left) Pseudoviruses packaged in cells treated with siRNA-negative control. (Right) Pseudoviruses packaged in cells treated with furin-targeting siRNA. (D) Western blot result of SARS-CoV-2 pseudoviruses packaged in cells treated with MMP inhibitor. (Left) Pseudoviruses packaged in cells not treated with MMP inhibitor. (Right) Pseudoviruses packaged in cells treated with MMP inhibitor.
Fig. 3. Effect of PPCs on SARS-CoV-2 spike-mediated cell entry. (A) SARS-CoV-2 pseudovirus entry into three types of target cells in the presence of PPCi. The pseudoviruses were packaged in the presence of different concentrations of PPCi before they were subjected to cell entry; (-) control: no pseudovirus was added. Also shown is the Western blot result of the corresponding pseudoviruses (packaged in the presence of different concentrations of PPCi). The entry efficiency of SARS-CoV-2 pseudoviruses without any treatment was taken as 100%. Error bars indicate SD (n = 4). ***P < 0.001; **P 0.01; *P < 0.05. (B) SARS-CoV pseudovirus entry into three types of target cells in the presence of PPCi. The experiments were performed in the same way as in A, except that SARS-CoV spike replaced SARS-CoV-2 spike in pseudoviruses. The entry efficiency of SARS-CoV pseudoviruses without any treatment was taken as 100%. (C) Western blot result of SARS-CoV-2 pseudoviruses packaged in cells treated with siRNA. (Left) Pseudoviruses packaged in cells treated with siRNA-negative control. (Right) Pseudoviruses packaged in cells treated with furin-targeting siRNA. (D) Western blot result of SARS-CoV-2 pseudoviruses packaged in cells treated with MMP inhibitor. (Left) Pseudoviruses packaged in cells not treated with MMP inhibitor. (Right) Pseudoviruses packaged in cells treated with MMP inhibitor.CC BY · PubMed Central
Fig. 4. Effect of other protease inhibitors on SARS-CoV-2 entry. (A) SARS-CoV-2 pseudovirus entry into three types of target cells in the presence of protease inhibitors. For pseudoviruses treated with PPCi, the pseudoviruses were packaged in the presence of PPCi (5 µM) before they were subjected to cell entry. For pseudoviruses treated with TMPRSS2 inhibitor camostat or lysosomal protease inhibitor E64d, pseudovirus entry was performed in the presence of camostat (50 µM) or E64d (50 µM). The cleavage state of SARS-CoV-2 spike was the same as in Fig. 3A (5 µM PPCi condition). The entry efficiency of SARS-CoV-2 pseudoviruses without any treatment was taken as 100%. Error bars indicate SD (n = 4). ***P < 0.001; *P < 0.05. (B) SARS-CoV pseudovirus entry into three types of target cells. The treatments were done in the same way as in A.
Fig. 4. Effect of other protease inhibitors on SARS-CoV-2 entry. (A) SARS-CoV-2 pseudovirus entry into three types of target cells in the presence of protease inhibitors. For pseudoviruses treated with PPCi, the pseudoviruses were packaged in the presence of PPCi (5 µM) before they were subjected to cell entry. For pseudoviruses treated with TMPRSS2 inhibitor camostat or lysosomal protease inhibitor E64d, pseudovirus entry was performed in the presence of camostat (50 µM) or E64d (50 µM). The cleavage state of SARS-CoV-2 spike was the same as in Fig. 3A (5 µM PPCi condition). The entry efficiency of SARS-CoV-2 pseudoviruses without any treatment was taken as 100%. Error bars indicate SD (n = 4). ***P < 0.001; *P < 0.05. (B) SARS-CoV pseudovirus entry into three types of target cells. The treatments were done in the same way as in A.CC BY · PubMed Central
Fig. 5. Comparison of receptor binding affinity and cell entry efficiency of SARS-CoV-2 and SARS-CoV. (A) Spike pull-down assay using hACE2 as the bait and cell-associated coronavirus spike molecules as the targets. (Top) Cell-expressed coronavirus spike molecules including SARS-CoV-2 spike, SARS-CoV-2 spike containing a mutant furin site as in Fig. 2A, SARS-CoV spike, and MERS-CoV spike. These spike molecules all contain a C-terminal C9 tag. (Middle) Pull-down result using His6-tagged hACE2. (Bottom) Pull-down result using Fc-tagged hACE2. (B) RBD pull-down assay using Fc-tagged hACE2 as the bait and soluble coronavirus RBDs as the targets. These RBD molecules all contain a C-terminal His6 tag. (C) (Left) Entry of SARS-CoV-2 and SARS-CoV pseudoviruses into three types of target cells. (Right) Western blot of SARS-CoV-2 and SARS-CoV pseudoviruses used in the cell entry assay.
Fig. 5. Comparison of receptor binding affinity and cell entry efficiency of SARS-CoV-2 and SARS-CoV. (A) Spike pull-down assay using hACE2 as the bait and cell-associated coronavirus spike molecules as the targets. (Top) Cell-expressed coronavirus spike molecules including SARS-CoV-2 spike, SARS-CoV-2 spike containing a mutant furin site as in Fig. 2A, SARS-CoV spike, and MERS-CoV spike. These spike molecules all contain a C-terminal C9 tag. (Middle) Pull-down result using His6-tagged hACE2. (Bottom) Pull-down result using Fc-tagged hACE2. (B) RBD pull-down assay using Fc-tagged hACE2 as the bait and soluble coronavirus RBDs as the targets. These RBD molecules all contain a C-terminal His6 tag. (C) (Left) Entry of SARS-CoV-2 and SARS-CoV pseudoviruses into three types of target cells. (Right) Western blot of SARS-CoV-2 and SARS-CoV pseudoviruses used in the cell entry assay.CC BY · PubMed Central
Fig. 6. Summary of cell entry mechanisms of SARS-CoV-2. (A) A schematic view of three unique features of SARS-CoV-2 entry: hidden RBD in the spike for immune evasion, RBD’s high hACE2 binding affinity for efficient entry, and furin preactivation of the spike for enhanced entry into some cells. (B) Implications of the cell entry mechanisms of SARS-CoV-2.
Fig. 6. Summary of cell entry mechanisms of SARS-CoV-2. (A) A schematic view of three unique features of SARS-CoV-2 entry: hidden RBD in the spike for immune evasion, RBD’s high hACE2 binding affinity for efficient entry, and furin preactivation of the spike for enhanced entry into some cells. (B) Implications of the cell entry mechanisms of SARS-CoV-2.CC BY · PubMed Central

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Show all 48 references

Research works printed but not linked: 2

  1. R. Woelfel . Clinical presentation and virological assessment of hospitalized cases of coronavirus disease 2019 in a travel-associated transmission cluster. medRxiv:2020.2003.2005.20030502 (8 March 2020).
  2. 10.1016/j.cell.2020.02.058 · DOI · Paper page

Cited by

Sorted by citations

See all 3,142

Related

Metadata

Authors
Shang, J., Wan, Y., Luo, C., Ye, G., Geng, Q., Auerbach, A., & Li, F.
Affiliations
University of Minnesota (US)
Corresponding author
Li, Fang
ORCID
Venue
Proceedings of the National Academy of Sciences
Publisher
National Academy of Sciences
Published
2020-05-06
Volume
117
Issue
21
Pages
11727-11734
ISSN
  • 0027-8424 (ISSN-L)
  • 1091-6490
DOI
10.1073/pnas.2003138117
PMC
PMC7260975
OpenAlex
W3022435472
Cited by
3,142
References
48
Citations by year
2026: 78 · 2025: 171 · 2024: 257 · 2023: 444 · 2022: 746 · 2021: 993 · 2020: 450
FWCI
87.09
Type
journal article
Language
en
Open access
Licence
Locations
  • Proceedings of the National Academy of Sciences (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
Division of Intramural Research, National Institute of Allergy and Infectious Diseases (R01AI089728, R01AI110700)
Topics
  • Health Sciences › Medicine › Infectious Diseases › SARS-CoV-2 and COVID-19 Research
  • Health Sciences › Medicine › Infectious Diseases › COVID-19 Clinical Research Studies
  • Life Sciences › Agricultural and Biological Sciences › Animal Science and Zoology › Animal Virus Infections Studies
Keywords
SARS-CoV-2, COVID-19 pandemic, viral entry, spike protein, receptor binding domain, ACE2 receptor, furin protease, proteolytic activation, SARS-CoV, receptor binding affinity, immune evasion, pseudovirus entry
MeSH terms
Protein Domains, Receptors, Coronavirus, Angiotensin-Converting Enzyme 2, Cell Line, Humans, Peptidyl-Dipeptidase A, Receptors, Virus, Virus Activation, Severe acute respiratory syndrome-related coronavirus, Virus Internalization, Immune Evasion, Spike Glycoprotein, Coronavirus

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review