CD147-spike protein is a novel route for SARS-CoV-2 infection to host cells methods
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SARS-CoV-2 employs the CD147 receptor for host cell entry
To explore the role of CD147 in SARS-CoV-2 infection, CD147 stable knockdown cells, Vero E6-shCD147, and BEAS-2B-shCD147, were infected with virus for 48 h. Quantitative PCR analysis showed that virus copy number was markedly decreased in CD147 knockdown group (Fig. ). In contrast, CD147 overexpression in BEAS-2B cells promoted virus infection (Fig. ). More importantly, the expression of human CD147 allows SARS-CoV-2 to enter into non-susceptible BHK-21 cells (Fig. ). The consistent findings were obtained in BEAS-2B-shCD147, BEAS-2B-CD147, and BHK-21-CD147 cells using SARS-CoV-2 pseudovirus (Fig. ). Moreover, human CD147 extracellular fragment inhibited pseudovirus entry into BHK-21-CD147 cells, suggesting the fragment could compete with membrane receptor CD147 when binding to spike protein (Fig. ). In the meanwhile, the virus-induced cytopathic effect was inhibited by an anti-C...
CD147 is an alternative receptor for SARS-CoV-2 infection in ACE2-deficient cell types
As both CD147 and ACE2 contribute to virus infection for host cells by binding to spike protein, we wonder how CD147 works alongside ACE2. We found no interaction between CD147 and ACE2 by Co-IP assay (Fig. ), which was verified by fluorescence resonance energy transfer (FRET) assay (Fig. ). Furthermore, the co-localizations between CD147 and spike protein, and ACE2 and spike protein were detected in lung tissues from patient with COVID-19, but no co-localization between CD147 and ACE2 was observed (Fig. ). Previous study provides the evidences that lymphocytes are infected and injured by SARS-CoV, leading to decreased circulating lymphocytes. Interestingly, in our study, SARS-CoV-2 virions were observed in lymphocytes in lung tissue from COVID-19 patient (Fig. ). T cells derived from human peripheral blood mononuclear cells (PBMC) were collected to analyze the expressions of CD147 an...
In vitro antiviral tests
Vero E6 cells (1 × 10 4 ) were cultured in a 96-well plate at 37 °C overnight, the supernatant was discarded and 100 µl of medium (containing 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1000 µg/mL Meplazumab) was added into the plates to incubate for 1 h. Then the cells were infected with SARS-CoV-2 (100 Median Tissue Culture Infectious Dose, TCID 50 ). After 1 h infection, the supernatants were removed and 200 µl of medium (containing 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1000 µg/mL Meplazumab) was added, the cells were cultured to observe the cytopathic changes for 3 days by crystal violet staining to calculate EC 50. The same method was employed with different antibody concentration (containing 3.125, 6.25, 12.5, 25, 50, 100, 150, 200 µg/mL Meplazumab). The supernatants were harvested to d...
Plaque assay
Vero E6 cells (1 × 10 5 ) were cultured in a 24-well plate at 37 °C overnight, the supernatant was discarded and 300 µl of medium (containing 100, 200, 400 µg/mL Meplazumab) was added into the plates to incubate for 1 h. Then the cells were infected with SARS-CoV-2 (25 TCID 50 ). After 1 h infection, the supernatants were removed and 500 µl of medium (containing 100, 200, 400 µg/mL Meplazumab) was added. The cells were cultured for 3 days and then the virus titer was detected by plaque assay.
Co-IP assay
Co-IP assay was performed using Pierce ® Co-Immunoprecipitation Kit (26149, Thermo Fisher Scientific, USA) according to the manufacturer's protocol. Mouse anti-human CD147 antibody (Jiangsu Pacific Meinuoke Biopharmceutical Co. Ltd, China, 50 µg), anti-SARS-CoV-2 spike antibody (40150-R007, Sino Biological, China, 50 µg) and anti-ACE2 antibody (80031-R003, Sino Biological, China, 50 µg) were used for antibody immobilization for Co-IP. The eluted proteins were detected by western blot. After boiling for 5-10 min, the eluted proteins were loaded to 12% SDS-PAGE gel and then transferred to PVDF membranes (Millipore, Boston, USA). After blocking with 5% non-fat milk for 1 h, the membrane was incubated with the corresponding primary antibodies at 4 °C overnight. The images were developed after incubation with the seco...
OpNS-EM specimen preparation
Specimens were prepared for electron microscopy by the OpNS-EM protocol. In brief, CD147 (2.26 mg/mL) and spike(RBD) (1.04 mg/mL) were diluted to 0.02 and 0.01 mg/mL with PBS. By incubating CD147 and spike(RBD) with 1:1 molar ratio, i.e., 1.5 mg/mL of spike(RBD) and 1.5 mg/mL of CD147, at 4 °C for 4 h, the CD147-spike(RBD) complexes were formed and then diluted to a concentration of 0.015 mg/mL with PBS buffer. Then an aliquot (~3 µl) was placed on an ultra-thin carbon film-coated 200-mesh copper grid (Cu-200UL, Electron Microscopy Sciences, USA) which had been glow discharged. After ~1 min, excess CD147-spike(RBD) complexes solution was blotted with a piece of filter paper. Then the grid was quickly washed by touching the grid surface with a drop (~35 µl) of distilled water on parafilm and blotting dry w...
Electron microscopy data collection and image processing
OpNS-EM micrographs were acquired at room temperature on a FEI CETA 16 M CMOS camera by a FEI F200C TEM operating at × 120,000 magnification, 200 kV high-tension, and low-electron-dose conditions under a defocus in a range of 0.15-0.65 µm. Under such conditions, each pixel of the raw micrographs corresponded to 1.00 Å in the specimens. The collected TEM Images were then processed with EMAN, SPIDER, and FREALIGN software packages. The parameters of the contrast transfer function (CTF) for each raw micrograph were fitted and determined with ctffind3 in FREALIGN and then corrected with SPIDER software. For particle selecting, the e2boxer.py program in EMAN2 were used, and then the automatically selected particles were examined and extracted with the EMAN package (boxer program). A 2D reference-free class-averaging program was then applied. Afte...
Immuno-electron microscope
The lung and kidney tissues were collected from a patient with COVID-19 in Tangdu Hospital of Fourth Military Medical University. Vero E6 cells infected with SARS-CoV-2, and lung and kidney tissues were harvested and fixed at 4 °C. The sections were prepared through dehydration and embedding. After washing with ultrapure water, the sections were treated with 1% H 2 O 2 for 10 min. Having been blocked with goat serum for 30 min, the sections were incubated with corresponding primary antibodies (rabbit anti-human CD147 antibody, produced by our laboratory; mouse anti-2019-nCoV Spike antibody, Sino Biological, China; mouse anti-ACE2 antibody, Proteintech, USA, rabbit anti-SARS-CoV-2 Spike antibody, 40150-R007, Sino Biological, China) for 16 h at 4 °C overnight. Subsequently, the sections were washed with PBS for 5 min, and treated with PBS (c...
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Quantify the primary readouts for this experiment: Protein expression by western blot; Microscopy-based tissue or cell-state imaging.
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Evidence quotes (8)
To explore the role of CD147 in SARS-CoV-2 infection, CD147 stable knockdown cells, Vero E6-shCD147, and BEAS-2B-shCD147, were infected with virus for 48 h. Quantitative PCR analysis showed that virus copy number was markedly decreased in CD147 knockdown group (Fig. ). In contrast, CD147 overexpression in BEAS-2B cells promoted virus infection (Fig. ). More importantly, the expression of human CD147 allows SARS-CoV-2 to enter into non-susceptible BHK-21 cells (Fig. ). The consistent findings were obtained in BEAS-2B-shCD147, BEAS-2B-CD147, and BHK-21-CD147 cells using SARS-CoV-2 pseudovirus (Fig. ). Moreover, human CD147 extracellular fragment inhibited pseudovirus entry into BHK-21-CD147 cells, suggesting the fragment could compete with membrane receptor CD147 when binding to spike protein (Fig. ). In the meanwhile, the virus-induced cytopathic effect was inhibited by an anti-CD147 antibody, Meplazumab, in a dose-dependent manner with EC 50 of 24.86 µg/mL (Fig. ). The virus copy number was also suppressed by Meplazumab with half-maximal inhibitory concentration (IC 50 ) of 15.16 µg/mL (Fig. ). Plaque assay demonstrated that Meplazumab significantly i...
As both CD147 and ACE2 contribute to virus infection for host cells by binding to spike protein, we wonder how CD147 works alongside ACE2. We found no interaction between CD147 and ACE2 by Co-IP assay (Fig. ), which was verified by fluorescence resonance energy transfer (FRET) assay (Fig. ). Furthermore, the co-localizations between CD147 and spike protein, and ACE2 and spike protein were detected in lung tissues from patient with COVID-19, but no co-localization between CD147 and ACE2 was observed (Fig. ). Previous study provides the evidences that lymphocytes are infected and injured by SARS-CoV, leading to decreased circulating lymphocytes. Interestingly, in our study, SARS-CoV-2 virions were observed in lymphocytes in lung tissue from COVID-19 patient (Fig. ). T cells derived from human peripheral blood mononuclear cells (PBMC) were collected to analyze the expressions of CD147 and ACE2 by real-time PCR assay, which demonstrated that CD147 expression was detected in CD4+ and CD8+ T cells, but no expression of ACE2 was detected (Fig. ). It has been reported that the destruction of lung cells by SARS-CoV-2 infection triggers inflammatory storm, including T cell immune response...
Vero E6 cells (1 × 10 4 ) were cultured in a 96-well plate at 37 °C overnight, the supernatant was discarded and 100 µl of medium (containing 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1000 µg/mL Meplazumab) was added into the plates to incubate for 1 h. Then the cells were infected with SARS-CoV-2 (100 Median Tissue Culture Infectious Dose, TCID 50 ). After 1 h infection, the supernatants were removed and 200 µl of medium (containing 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1000 µg/mL Meplazumab) was added, the cells were cultured to observe the cytopathic changes for 3 days by crystal violet staining to calculate EC 50. The same method was employed with different antibody concentration (containing 3.125, 6.25, 12.5, 25, 50, 100, 150, 200 µg/mL Meplazumab). The supernatants were harvested to detect the gene copy number of the virus with quantitative PCR to calculate IC 50 (QIAampViral RNA Mini Kit, QIAGEN, Germany; One-Step RT-PCR kit, RR064A, Takara, Japan). A virus control group and a blank control group were set at the same time. The primers and probe sequences were listed as follows:
Vero E6 cells (1 × 10 5 ) were cultured in a 24-well plate at 37 °C overnight, the supernatant was discarded and 300 µl of medium (containing 100, 200, 400 µg/mL Meplazumab) was added into the plates to incubate for 1 h. Then the cells were infected with SARS-CoV-2 (25 TCID 50 ). After 1 h infection, the supernatants were removed and 500 µl of medium (containing 100, 200, 400 µg/mL Meplazumab) was added. The cells were cultured for 3 days and then the virus titer was detected by plaque assay.
Co-IP assay was performed using Pierce ® Co-Immunoprecipitation Kit (26149, Thermo Fisher Scientific, USA) according to the manufacturer's protocol. Mouse anti-human CD147 antibody (Jiangsu Pacific Meinuoke Biopharmceutical Co. Ltd, China, 50 µg), anti-SARS-CoV-2 spike antibody (40150-R007, Sino Biological, China, 50 µg) and anti-ACE2 antibody (80031-R003, Sino Biological, China, 50 µg) were used for antibody immobilization for Co-IP. The eluted proteins were detected by western blot. After boiling for 5-10 min, the eluted proteins were loaded to 12% SDS-PAGE gel and then transferred to PVDF membranes (Millipore, Boston, USA). After blocking with 5% non-fat milk for 1 h, the membrane was incubated with the corresponding primary antibodies at 4 °C overnight. The images were developed after incubation with the secondary antibodies (goat anti-mouse IgG(H + L) antibody, 31430, Thermo Fisher Scientific, MA, USA, dilution ratio 1:5000; goat anti-rabbit IgG(H + L) antibody; 31460, Thermo Fisher Scientific, MA, USA, dilution ratio 1:5000) at room temperature for 1 h.
Specimens were prepared for electron microscopy by the OpNS-EM protocol. In brief, CD147 (2.26 mg/mL) and spike(RBD) (1.04 mg/mL) were diluted to 0.02 and 0.01 mg/mL with PBS. By incubating CD147 and spike(RBD) with 1:1 molar ratio, i.e., 1.5 mg/mL of spike(RBD) and 1.5 mg/mL of CD147, at 4 °C for 4 h, the CD147-spike(RBD) complexes were formed and then diluted to a concentration of 0.015 mg/mL with PBS buffer. Then an aliquot (~3 µl) was placed on an ultra-thin carbon film-coated 200-mesh copper grid (Cu-200UL, Electron Microscopy Sciences, USA) which had been glow discharged. After ~1 min, excess CD147-spike(RBD) complexes solution was blotted with a piece of filter paper. Then the grid was quickly washed by touching the grid surface with a drop (~35 µl) of distilled water on parafilm and blotting dry with another piece of filter paper. Such touching and blotting step was repeated three times. For each time, a clean drop of clean distilled water was used. Then three drops of freshly prepared ~1% (w/v) uranyl formate (UF) negative stain (Electron Microscopy Sciences, USA) solution on parafilm were...
OpNS-EM micrographs were acquired at room temperature on a FEI CETA 16 M CMOS camera by a FEI F200C TEM operating at × 120,000 magnification, 200 kV high-tension, and low-electron-dose conditions under a defocus in a range of 0.15-0.65 µm. Under such conditions, each pixel of the raw micrographs corresponded to 1.00 Å in the specimens. The collected TEM Images were then processed with EMAN, SPIDER, and FREALIGN software packages. The parameters of the contrast transfer function (CTF) for each raw micrograph were fitted and determined with ctffind3 in FREALIGN and then corrected with SPIDER software. For particle selecting, the e2boxer.py program in EMAN2 were used, and then the automatically selected particles were examined and extracted with the EMAN package (boxer program). A 2D reference-free class-averaging program was then applied. After quality evaluation, particle images from each sample (6681 CD147; 5073 spike(RBD); 12,426 CD147-spike(RBD) complexes) were selected from the corrected micrographs and extracted as 128 × 128-pixel images by using boxer software. Then the contrast of the particle images was normaliz...
The lung and kidney tissues were collected from a patient with COVID-19 in Tangdu Hospital of Fourth Military Medical University. Vero E6 cells infected with SARS-CoV-2, and lung and kidney tissues were harvested and fixed at 4 °C. The sections were prepared through dehydration and embedding. After washing with ultrapure water, the sections were treated with 1% H 2 O 2 for 10 min. Having been blocked with goat serum for 30 min, the sections were incubated with corresponding primary antibodies (rabbit anti-human CD147 antibody, produced by our laboratory; mouse anti-2019-nCoV Spike antibody, Sino Biological, China; mouse anti-ACE2 antibody, Proteintech, USA, rabbit anti-SARS-CoV-2 Spike antibody, 40150-R007, Sino Biological, China) for 16 h at 4 °C overnight. Subsequently, the sections were washed with PBS for 5 min, and treated with PBS (containing 1% bovine serum albumin, BSA, pH8.2) for 7 min. Then the gold colloid-labeling method (20 nm InnovaCoat® GOLD (10 OD) Goat Anti Rabbit IgG Conjugate, 219-1000, Expedeon; InnovaCoat® GOLD-40 nm Goat Anti Mouse nanoparticles, 216-1000, Expedeon; InnovaCoat® G...
Machine-readable layer
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"text": "To explore the role of CD147 in SARS-CoV-2 infection, CD147 stable knockdown cells, Vero E6-shCD147, and BEAS-2B-shCD147, were infected with virus for 48 h. Quantitative PCR analysis showed that virus copy number was markedly decreased in CD147 knockdown group (Fig. ). In contrast, CD147 overexpression in BEAS-2B cells promoted virus infection (Fig. ). More importantly, the expression of human CD147 allows SARS-CoV-2 to enter into non-susceptible BHK-21 cells (Fig. ). The consistent findings were obtained in BEAS-2B-shCD147, BEAS-2B-CD147, and BHK-21-CD147 cells using SARS-CoV-2 pseudovirus (Fig. ). Moreover, human CD147 extracellular fragment inhibited pseudovirus entry into BHK-21-CD147 cells, suggesting the fragment could compete with membrane receptor CD147 when binding to spike protein (Fig. ). In the meanwhile, the virus-induced cytopathic effect was inhibited by an anti-C..."
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"name": "CD147 is an alternative receptor for SARS-CoV-2 infection in ACE2-deficient cell types",
"text": "As both CD147 and ACE2 contribute to virus infection for host cells by binding to spike protein, we wonder how CD147 works alongside ACE2. We found no interaction between CD147 and ACE2 by Co-IP assay (Fig. ), which was verified by fluorescence resonance energy transfer (FRET) assay (Fig. ). Furthermore, the co-localizations between CD147 and spike protein, and ACE2 and spike protein were detected in lung tissues from patient with COVID-19, but no co-localization between CD147 and ACE2 was observed (Fig. ). Previous study provides the evidences that lymphocytes are infected and injured by SARS-CoV, leading to decreased circulating lymphocytes. Interestingly, in our study, SARS-CoV-2 virions were observed in lymphocytes in lung tissue from COVID-19 patient (Fig. ). T cells derived from human peripheral blood mononuclear cells (PBMC) were collected to analyze the expressions of CD147 an..."
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"text": "Vero E6 cells (1 × 10 4 ) were cultured in a 96-well plate at 37 °C overnight, the supernatant was discarded and 100 µl of medium (containing 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1000 µg/mL Meplazumab) was added into the plates to incubate for 1 h. Then the cells were infected with SARS-CoV-2 (100 Median Tissue Culture Infectious Dose, TCID 50 ). After 1 h infection, the supernatants were removed and 200 µl of medium (containing 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1000 µg/mL Meplazumab) was added, the cells were cultured to observe the cytopathic changes for 3 days by crystal violet staining to calculate EC 50. The same method was employed with different antibody concentration (containing 3.125, 6.25, 12.5, 25, 50, 100, 150, 200 µg/mL Meplazumab). The supernatants were harvested to d..."
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"text": "Vero E6 cells (1 × 10 5 ) were cultured in a 24-well plate at 37 °C overnight, the supernatant was discarded and 300 µl of medium (containing 100, 200, 400 µg/mL Meplazumab) was added into the plates to incubate for 1 h. Then the cells were infected with SARS-CoV-2 (25 TCID 50 ). After 1 h infection, the supernatants were removed and 500 µl of medium (containing 100, 200, 400 µg/mL Meplazumab) was added. The cells were cultured for 3 days and then the virus titer was detected by plaque assay."
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"text": "Co-IP assay was performed using Pierce ® Co-Immunoprecipitation Kit (26149, Thermo Fisher Scientific, USA) according to the manufacturer's protocol. Mouse anti-human CD147 antibody (Jiangsu Pacific Meinuoke Biopharmceutical Co. Ltd, China, 50 µg), anti-SARS-CoV-2 spike antibody (40150-R007, Sino Biological, China, 50 µg) and anti-ACE2 antibody (80031-R003, Sino Biological, China, 50 µg) were used for antibody immobilization for Co-IP. The eluted proteins were detected by western blot. After boiling for 5-10 min, the eluted proteins were loaded to 12% SDS-PAGE gel and then transferred to PVDF membranes (Millipore, Boston, USA). After blocking with 5% non-fat milk for 1 h, the membrane was incubated with the corresponding primary antibodies at 4 °C overnight. The images were developed after incubation with the seco..."
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"text": "Specimens were prepared for electron microscopy by the OpNS-EM protocol. In brief, CD147 (2.26 mg/mL) and spike(RBD) (1.04 mg/mL) were diluted to 0.02 and 0.01 mg/mL with PBS. By incubating CD147 and spike(RBD) with 1:1 molar ratio, i.e., 1.5 mg/mL of spike(RBD) and 1.5 mg/mL of CD147, at 4 °C for 4 h, the CD147-spike(RBD) complexes were formed and then diluted to a concentration of 0.015 mg/mL with PBS buffer. Then an aliquot (~3 µl) was placed on an ultra-thin carbon film-coated 200-mesh copper grid (Cu-200UL, Electron Microscopy Sciences, USA) which had been glow discharged. After ~1 min, excess CD147-spike(RBD) complexes solution was blotted with a piece of filter paper. Then the grid was quickly washed by touching the grid surface with a drop (~35 µl) of distilled water on parafilm and blotting dry w..."
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"text": "OpNS-EM micrographs were acquired at room temperature on a FEI CETA 16 M CMOS camera by a FEI F200C TEM operating at × 120,000 magnification, 200 kV high-tension, and low-electron-dose conditions under a defocus in a range of 0.15-0.65 µm. Under such conditions, each pixel of the raw micrographs corresponded to 1.00 Å in the specimens. The collected TEM Images were then processed with EMAN, SPIDER, and FREALIGN software packages. The parameters of the contrast transfer function (CTF) for each raw micrograph were fitted and determined with ctffind3 in FREALIGN and then corrected with SPIDER software. For particle selecting, the e2boxer.py program in EMAN2 were used, and then the automatically selected particles were examined and extracted with the EMAN package (boxer program). A 2D reference-free class-averaging program was then applied. Afte..."
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"text": "The lung and kidney tissues were collected from a patient with COVID-19 in Tangdu Hospital of Fourth Military Medical University. Vero E6 cells infected with SARS-CoV-2, and lung and kidney tissues were harvested and fixed at 4 °C. The sections were prepared through dehydration and embedding. After washing with ultrapure water, the sections were treated with 1% H 2 O 2 for 10 min. Having been blocked with goat serum for 30 min, the sections were incubated with corresponding primary antibodies (rabbit anti-human CD147 antibody, produced by our laboratory; mouse anti-2019-nCoV Spike antibody, Sino Biological, China; mouse anti-ACE2 antibody, Proteintech, USA, rabbit anti-SARS-CoV-2 Spike antibody, 40150-R007, Sino Biological, China) for 16 h at 4 °C overnight. Subsequently, the sections were washed with PBS for 5 min, and treated with PBS (c..."
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