Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation
- Viroscience Department, Erasmus Medical Center, Netherlands
- Department of Biochemistry, University of Illinois at Urbana-Champaign, United States
- Center for Biophysics and Quantitative Biology, University of Illinois at Urbana-Champaign, United States
- Carl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, United States
Figures
Figure 1 with 1 supplement
SARS-CoV-2 rapidly acquires multibasic cleavage site mutations when propagated on VeroE6 cells.
(A–C) Deep-sequencing analysis of VeroE6 passage 2 (A), passage 3 (B), and passage 4 (C) virus stocks. In each graph, the amino acid sequence logo of the multibasic cleavage site is shown. (D–F) Sanger sequencing chromatograms of VeroE6 passage 2 (D), passage 3 (E), and passage 4 (F) viruses. Multibasic cleavage site mutations identified by deep-sequencing are indicated with arrows. Translated sequences are indicated below Sanger reads. (G) Plaque size analysis of VeroE6 passage 2–4 virus stocks on VeroE6 cells. Red arrow heads indicate small plaques. Scale bar indicates 1 cm.
Figure 1—figure supplement 1
Deep-sequencing analysis of VeroE6 passage 1 virus multibasic cleavage site and full genome deep-sequencing analysis of passage 1–4 viruses.
(A) Deep-sequencing analysis of the VeroE6 passage 1 virus stock. In each graph, the amino acid sequence logo of the multibasic cleavage site is shown. (B) Full genome deep-sequencing analysis of VeroE6 passage 1, 2, 3, and passage 4 viruses.
Figure 2
Mutations in the multibasic cleavage site and the adjacent serine residue (S686) abrogate S1/S2 cleavage.
(A) Analysis of S1/S2 cleavage by S1 immunoblot of SARS-CoV-2 S (WT), multibasic cleavage site (MBCS) mutant and S686G mutant pseudoviruses. (B) Quantification of S1 cleavage from four independent pseudovirus productions. (C) Analysis of S1/S2 cleavage by multiplex S1 (red) and S2 (green) immunoblot of SARS-CoV-2 S (WT) and S686G mutant pseudoviruses. S0 indicates uncleaved spike; S1 indicates the S1 domain of cleaved spike; VSV-N indicates VSV nucleoprotein (production control). Numbers indicate the molecular weight (kDa) of bands of the protein standard. (D) Quantification of S2 cleavage from four independent pseudovirus productions. Error bars indicate SD. EV = empty vector. WT = wild type. kDa = kilo dalton.
Figure 3
The SARS-CoV-2 multibasic cleavage site and the adjacent serine residue (S686) enhance infectivity and serine protease mediated entry on Calu-3 and VeroE6-TMPRSS2 cells.
(A–B) SARS-CoV-2 (WT), multibasic cleavage site (MBCS) mutant and S686G pseudovirus infectious titers on (A) VeroE6 and (B) Calu-3 cells. (C) Fold change in SARS-CoV-2, MBCS mutant and S686G pseudovirus infectious titers on Calu-3 cells over infectious titers on VeroE6 cells. (D) SARS-CoV-2, MBCS mutant and S686G pseudovirus infectious titers on VeroE6-TMPRSS2 cells. (E) Fold change in SARS-CoV-2, MBCS mutant and S686G pseudovirus infectious titers on VeroE6-TMPRSS2 cells over infectious titers on VeroE6 cells. One-way ANOVA was performed for statistical analysis comparing all groups with WT. (F–I) SARS-CoV-2, MBCS mutant and S686G pseudovirus entry into (F and G) VeroE6 cells or (H and I) VeroE6-TMPRSS2 cells pre-treated with a concentration range of either (F and H) camostat mesylate or (G and I) E64D. Two-way ANOVA, followed by a bonferroni post hoc test was performed for statistical analysis comparing all groups to WT. WT pseudovirus entry into VeroE6 cells treated with 10 µM E64D was significantly different from del-RRAR, R682A, R685A and S686G pseudovirus entry. * indicates statistical significance (p<0.05) compared to WT (A–E). * indicates statistical significance (p<0.05) compared to WT at the highest inhibitor concentration (F–I). Experiments were performed in triplicate. Representative experiments from at least two independent experiments are shown. Error bars indicate SD. WT = wild type.
Figure 4
Multibasic cleavage site mutations and the adjacent serine residue (S686) impair spike protein fusogenicity.
(A–C) Fusogenicity of wild type SARS-CoV-2 spike and spike mutants was assessed after 18 hr by measuring the sum of all GFP+ pixels per well in a GFP-complementation fusion assay on VeroE6-GFP1-10 (A), VeroE6-TMPRSS2-GFP1-10 (B), and Calu-3-GFP1-10 (C) cells. The experiment was performed in triplicate. A representative experiment from two independent experiments is shown. Statistical analysis was performed by one-way ANOVA. * indicates a significant difference compared to WT (p<0.05). Error bars indicate SD. EV = empty vector. WT = wild type.
Figure 5 with 1 supplement
SARS-CoV-2 propagation in Calu-3 cells efficiently prevents SARS-CoV-2 cell culture adaptation.
(A) Deep-sequencing analysis of Calu-3 passage 2 virus from a VeroE6 passage 1. (B) Deep-sequencing analysis of Calu-3 passage 3 virus from the Calu-3 passage 2 in A. (C) Deep-sequencing analysis of Calu-3 passage 3 virus grown from a VeroE6 passage 2 stock (Figure 1A). Deep-sequencing analysis of Calu-3 passage 5 virus from a Calu-3 passage 3 stock in C. In each graph, the amino acid sequence logo of the multibasic cleavage site is shown.
Figure 5—figure supplement 1
Multibasic cleavage site deep-sequencing analysis of passage 4 Calu-3 viruses from an adapted VeroE6 P3 stock and full genome deep-sequencing analysis of Calu-3-propagated viruses.
(A) Deep-sequencing analysis of Calu-3 passage 4 virus from a VeroE6 passage 3 stock (from Figure 1B). (B) Deep-sequencing analysis of Calu-3 passage 4 virus from a VeroE6 passage 3 stock produced in the presence of 10 μM E64D. In each graph, the amino acid sequence logo of the multibasic cleavage site is shown. (C) Full genome deep-sequencing analysis of Calu-3-propagated viruses.
Figure 6 with 1 supplement
Serine protease expression prevents MBCS mutations.
(A–B) Deep-sequencing analysis of VeroE6 passage 4 virus from a VeroE6 passage 3 (A is a redisplay of Figure 1C) mock-treated or treated with 10 μM camostat. (C–D) Deep-sequencing analysis of VeroE6-TMPRSS2 passage 4 virus from a VeroE6 passage 3 mock-treated or treated with 10 μM camostat. In each graph the amino acid sequence logo of the multibasic cleavage site is shown.
Figure 6—figure supplement 1
Multibasic cleavage site and full genome deep-sequencing analysis of passage 4 VeroE6 and VeroE6-TMPRSS2 viruses.
(A–B) Deep-sequencing analysis of VeroE6 passage 4 virus from a VeroE6 passage 3, trypsin-treated (A) or treated with 10% FBS (B). (C–D) Deep-sequencing analysis of VeroE6-TMPRSS2 passage 4 virus from a VeroE6 passage 3, trypsin-treated (C) or treated with 10% FBS (D). In each graph, the amino acid sequence logo of the multibasic cleavage site is shown. (E) Full genome deep-sequencing analysis of VeroE6 and VeroE6-TMPRSS2-propagated viruses. In E VeroE6 P4 (mock) is a redisplay of VeroE6 P4 in Figure 1—figure supplement 1B.
Figure 7
A 2D air-liquid interface human airway organoid model for SARS-CoV-2 propagation.
(A) Human airway organoids were dissociated and plated onto 12 mm transwell inserts. After an 8–12 week differentiation period at air-liquid interface cultures contained ciliated, non-ciliated and basal cells as shown on a hematoxylin-eosin stain. (B) Air-exposed cells, but not basal cells, expressed the priming protease TMPRSS2 as shown by immunohistochemistry. (C) Immunofluorescent staining indicated that in these cultures, ciliated cells (acetylated tubulin+ or AcTUB+ cells) were infected by SARS-CoV-2. (D and E) At 5 days post-infection, whole-well confocal imaging indicated the infection was widespread (D) and cytopathic effects, including cilia damage (D and E) and syncytial cells (E) were visible. Scale bars indicate 20 µm in A, B, C; 2 mm in D; and 100 µm in E.
Figure 8 with 1 supplement
2D air-liquid interface human airway organoids produce high titer stocks without multibasic cleavage site mutations.
(A–B) Deep-sequencing analysis (A) and Sanger chromatogram (B) of Organoid passage 3 virus from a VeroE6 passage 2 stock (Figure 1A). The amino acid sequence logo of the multibasic cleavage site is shown. The translated sequence is indicated below the Sanger read. Arrows indicate where cell culture adaptations to VeroE6 cells occur. (C) Plaque size analysis of VeroE6 passage 2 and Organoid passage 3 virus (the VeroE6 data is a redisplay of Figure 1G). Red arrow heads indicate large plaques. Scale bar indicates 1 cm. (D) Full genome deep-sequencing analysis of VeroE6 passage 2 and organoid passage three stocks. In D VeroE6 P2 is a redisplay of VeroE6 P2 in Figure 1—figure supplement 1B. (E) Immunoblot analysis of VeroE6 passage 2 and 3, Calu-3 passage 3 and Organoid passage 3 stocks. S0 indicates uncleaved spike; S1 indicates the S1 domain of cleaved spike; NP indicates nucleoprotein. Numbers indicate the molecular weight (kDa) of bands of the protein standard. (F) Quantification of cleavage from three immunoblots. Error bars indicate SD.
Figure 8—figure supplement 1
Schematic workflow for the production of SARS-CoV-2 stocks on 2D air-liquid interface differentiated airway organoids.
Step 1. 3D self-renewing airway organoids are grown from human lung tissue. Next, these are dissociated to single cells and differentiated at air-liquid interface for 4–12 weeks. Step 2. Differentiated cultures are infected at a multiplicity of infection of 0.05 and washed daily for 5 days. The washes from day 2 to day 5 are collected and stored at 4°C. Step 3. Virus collections are cleared by centrifugation and filtered to remove debris larger than 0.45 μm. Next, the medium is exchanged three times using Amicon columns to remove cytokines and debris smaller than 100 kDa. Purified virus preparations are then stored at −80°C in aliquots. Step 4. Stocks can be characterized using plaque assays, Sanger sequencing and deep-sequencing. Created with BioRender.com.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Rabbit-anti-SARS-CoV NP (polyclonal) | Sino Biological | Cat# 40143-T62 | IF (1:1000) |
| Antibody | Mouse anti-TMPRSS2 (monoclonal) | Santa Cruz | Cat# sc-515727 | IHC (1:200) |
| Antibody | Goat-anti-mouse | Dako | Cat# P0260 | IF (1:400) |
| Antibody | Goat anti-rabbit IgG (H+L) Alexa Fluor Plus 594 | Invitrogen | Cat# A32740 | IF (1:400) |
| Antibody | Goat anti-mouse IgG (H+L) Alexa Fluor 488 | Invitrogen | Cat# A11029 | IF (1:2000) |
| Antibody | Mouse-anti-AcTub IgG2A Alexa Fluor 488 (monoclonal) | Santa Cruz Biotechnology | Cat# sc-23950 AF488 | IF (1:100) |
| Antibody | Mouse anti-nucleocapsid | Sinobiological | Cat# 40143-MM05 | IF (1:1000) |
| Antibody | Rabbit anti-SARS-CoV S1 (polyclonal) | Sinobiological | Cat# 40150-T62 | WB (1:1000) |
| Antibody | Mouse-anti-SARS-CoV-2 S2 (monoclonal) | Genetex | Cat# GTX632604 | WB (1:1000) |
| Antibody | Mouse-anti-VSV-N (monoclonal) | Absolute Antibody | Cat# Ab01403-2.0 | WB (1:1000) |
| Biological sample (Homo sapiens) | Airway organoids | Mykytyn et al., 2021 | ||
| Cell line (Cercopithecus aethiops) | VeroE6 | ATCC | CRL 1586TM | |
| Cell line (Cercopithecus aethiops) | VeroE6 TMPRSS2 | Mykytyn et al., 2021 | ||
| Cell line (Cercopithecus aethiops) | VeroE6 GFP1-10 | Mykytyn et al., 2021 | ||
| Cell line (Cercopithecus aethiops) | VeroE6 GFP1-10 TMPRSS2 | Mykytyn et al., 2021 | ||
| Cell line (Homo sapiens) | Calu-3 | ATCC | HTB 55 | |
| Cell line (Homo sapiens) | Calu-3 GFP1-10 | Mykytyn et al., 2021 | ||
| Chemical compound, drug | E64D | MedChemExpress | Cat# HY-100229 | |
| Chemical compound, drug | Camostat mesylate | Sigma | Cat# SML0057 | |
| Chemical compound, drug | Polyethylenimine linear | Polysciences | Cat# 23966 | |
| Chemical compound, drug | Hygromycin B | Invitrogen | Cat# 10843555001 | |
| Chemical compound, drug | Geneticin | Invitrogen | Cat# 10131035 | |
| Chemical compound, drug | Avicel | FMC biopolymers | - | |
| Chemical compound, drug | Laemmli | BioRad | Cat# 1610747 | |
| Commercial assay or kit | SuperScript IV Reverse Transcriptase | Invitrogen | Cat# 18090200 | |
| Commercial assay or kit | Pfu Ultra II Fusion HS DNA Polymerase | Agilent Technologies | Cat# 600674 | |
| Commercial assay or kit | Qiaquick PCR Purification Kit | QIAGEN | Cat# 28104 | |
| Commercial assay or kit | BigDye Terminator v3.1 Cycle Sequencing Kit | Applied Biosystems | Cat# 4337456 | |
| Commercial assay or kit | ProtoScript II Reverse Transcriptase | New England BioLabs | Cat# NEB M0368X | |
| Commercial assay or kit | KAPA HyperPlus | Roche | Cat# 7962428001 | |
| Other | Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-100 membrane | Millipore | Cat# UFC910024 | |
| Other | Opti-MEM I (1X) + GlutaMAX | Gibco | Cat# 51985–042 | |
| Other | Advanced DMEM/F12 | Thermo Fisher scientific | Cat# 12634–010 | |
| Other | AO medium | Sachs et al., 2019 | N/A | |
| Other | Pneumacult ALI medium | Stemcell | Cat # 05001 | |
| Other | TryplE | Thermo Fisher scientific | Cat# 12605010 | |
| Other | Basement membrane extract | R and D Systems | Cat# 3533-005-02 | |
| Other | Transwell inserts | Corning | Cat# 3460 | |
| Other | Collagen Type I, High concentration Rat tail | Corning | Cat# 354249 | |
| Other | 0.45 μm low protein binding filter | Millipore | Cat# SLHV033RS | |
| Other | Hoechst | Thermo Fisher | Cat# H1399 | |
| Other | Ampure XP Beads | Beckman Coulter | Cat# A63882 | |
| Other | Illumina sequencer V3 MiSeq flowcell | Illumina | ||
| Other | ABI PRISM 3100 Genetic Analyzer | Applied Biosystems | ||
| Other | Odyssey CLx | Licor | ||
| Other | Amersham Typhoon Biomolecular Image | GE Healthcare | ||
| Other | Amersham Imager 600 | GE Healthcare | ||
| Other | LSM700 confocal microscope | Zeiss | ||
| Other | Carl ZEISS Vert.A1 | Zeiss | ||
| Software, algorithm | ZEN | Zeiss | ||
| Software, algorithm | ImageQuant TL 8.2 | GE Healthcare | ||
| Software, algorithm | Studio Lite Ver 5.2 | Licor | ||
| Software, algorithm | GraphPad PRISM 8, 9 | GraphPad | ||
| Software, algorithm | Illustrator | Adobe inc | ||
| Strain, strain background (SARS-CoV-2) | SARS-CoV-2 BavPat-1 | Dr. Christian Drosten | European Virus Archive Global #026 V-03883 |
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Human airway cells prevent SARS-CoV-2 multibasic cleavage site cell culture adaptation
eLife 10:e66815.
https://doi.org/10.7554/eLife.66815
