This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research Article
Taha Azad
Ottawa Hospital Research Institute
Corresponding Author
ORCiD: https://orcid.org/0000-0002-9498-0657
Ragunath Singaravelu
Ottawa Hospital Research Institute
Corresponding Author
John C. Bell
Ottawa Hospital Research Institute
Corresponding Author
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Declarations:
Declarations
- This study involved human subjects.
- The author confirmed that all appropriate ethical guidelines for the use of human subjects have been followed, any necessary IRB and/or ethics committee review has been obtained, and information about the IRB/ethics committee is included in the manuscript.
- The author has confirmed that all necessary patient/participant consent or assent has been obtained and the appropriate institutional forms have been archived. If the IRB/ethics committee waived the requirement for patient/participant consent or assent, an explanation for the waiver is included in the text.
- The author has confirmed that a statement listing potential conflicts of interest or lack thereof is included in the text.
The ongoing COVID-19 pandemic has highlighted the` immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 lifecycle. We developed a bioluminescence-based biosensor to interrogate the interaction between the SARS-CoV-2 viral spike protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The biosensor assay is based on a Nanoluciferase complementation reporter, composed of two subunits, Large BiT and Small BiT, fused to the spike receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this biosensor, we uncovered a critical role for glycosylation of asparagine residues within the RBD in mediating successful binding to the cellular ACE2 receptor and subsequent virus infection. Our findings support RBD glycosylation as a therapeutic and vaccine target to blunt SARSCoV- 2 infections.
Keywords
coronavirus, bioluminescence, biosensor, viral entry, high-throughput screening, 2019-nCoV, SARS-CoV-2, COVID-19
Figure 1
Figure 2
Figure 3
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryTable1.pdf
Supplementary Table 1. Plasmid and primer sequences
- SupplementaryFigure1.jpg
Supplemental Figure 1. Structural comparison of SARS CoV and CoV-2 spike protein RBDs. (A) Sequence alignment of SARS CoV and CoV-2 spike RBDs performed with ClustalW. Yellow indicates complete identity, while blue depicts residues with similar side chain properties. The receptor binding motif (RBM) is highlighted beneath. Residues participating in the interaction with ACE2 are indicated with an asterisk if at a common site in both SARS CoV and CoV-2 or a box if unique to one RBD or another. (B) Alignment of tertiary structures of SARS CoV (green; PDB Id: 2AJF) and CoV-2 (blue; PDB Id: 6M0J) RBDs in complex with ACE2 (pink and orange, respectively) (Lan et al. 2020; Li et al. 2005a). Interaction sites of SARS CoV (C) and CoV-2 (D) RBDs (blue) with ACE2 (orange). Black dashed lines depict polar contacts.
- SupplementaryFigure2.jpg
Supplemental Figure 2. SARS-CoV-2 NanoBiT is compatible with alternate imaging systems. (A) 293T cells were co-transfected with constructs expressing the indicated constructs (left panel). 48 hours post-transfection, cells were lysed and FMZ was added to each well. Luminescence was visualized using the IVIS CCD camera.(B) Optimized ACE2-RBD biosensor produces robust luminescent signal observable to naked eye. 293T cells were co-transfected with SmBiT-ACE2 and RBD-LgBiT. 50 g of cell lysate was incubated with FMZ (1:50 ratio) at room temperature and imaged using a standard 12MP digital phone camera.
- SupplementaryFigure3.jpg
Supplemental Figure 3. Comparison of substrates for Nanoluc-based reporter assay. (A) 293T cells were transfected as indicated (SmBiT-ACE2, RBD-LgBiT or co- transfected). 20 ug of lysates were read by the addition of equal volume of substrate: coelenterazine (CTZ, blue) or furimazine (FMZ, brown). (n=3, mean±SD; one-way ANOVA, *** p < 0.005 relative to RBD-LgBiT alone, Dunnett’s correction for multiple comparisons). Assays performed with CTZ and FMZ were analyzed independently. (B) 40 ul of supernatant from transfected 293T cells in panel A were harvested and read similarly (n=3, mean±SD; one-way ANOVA, *** p < 0.005, ** p < 0.01 relative to RBD- LgBiT alone, Dunnett’s correction for multiple comparisons). Assays performed with CTZ and FMZ were analyzed independently.
- SupplementaryFigure4.jpg
Supplemental Figure 4. Development of SARS-CoV biosensor. Biosensor (BS) assay was performed on supernatants of 293T cells co-transfected with SmBiT-ACE2 and either SARS-CoV or SARS-CoV-2 RBD-LgBiT constructs.
- SupplementaryFigure5.jpg
Supplemental Figure 5. N-linked glycosylation of SARS-CoV-2 Spike RBD is critical to its interaction with ACE2. (A) Recombinant RBD purified from E. coli was incubated for 15 minutes with cell lysate containing SmBiT-ACE2 at room temperature. Equal amounts of lysates containing RBD-LgBiT were then added and incubated for 5 minutes. Luciferase assay was performed using CTZ as substrate. (n=3, mean±SD; one-way ANOVA, *** p < 0.005, Tukey’s correction for multiple comparisons.) (B) 293T cells were transfected with RBD-LgBiT and subsequently treated with tunicamycin. Lysates were combined with lysates from 293T cells transfected with SmBiT-ACE2 and BS assay was performed. (C) BS assay was performed on lysates from 293T cells co- transfected with the indicated RBD-glycosylation site mutant-LgBiT constructs and SmBiT-ACE2 constructs. (D) BS assay was performed on supernatants from 293T cells co-transfected with RBD-glycosylation site mutant-LgBiT constructs and SmBiT-ACE2 constructs. (E) ) SARS-CoV-2 S pseudotyped lentivirus encoding ZsGreen and luciferase reporters was incubated with PBS, PNGase F, or Endo H for 1 hour, and then used to infect HEK293-ACE2 cells. 48 hours post-transduction, cells were evaluated for GFP. (F) Lentivirus levels of Spike mutant pseudotypes were titered via p24 ELISA.
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research Article
Taha Azad
Ottawa Hospital Research Institute
Corresponding Author
ORCiD: https://orcid.org/0000-0002-9498-0657
Ragunath Singaravelu
Ottawa Hospital Research Institute
Corresponding Author
John C. Bell
Ottawa Hospital Research Institute
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 14 Aug, 2020
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Declarations:
Declarations
- This study involved human subjects.
- The author confirmed that all appropriate ethical guidelines for the use of human subjects have been followed, any necessary IRB and/or ethics committee review has been obtained, and information about the IRB/ethics committee is included in the manuscript.
- The author has confirmed that all necessary patient/participant consent or assent has been obtained and the appropriate institutional forms have been archived. If the IRB/ethics committee waived the requirement for patient/participant consent or assent, an explanation for the waiver is included in the text.
- The author has confirmed that a statement listing potential conflicts of interest or lack thereof is included in the text.
The ongoing COVID-19 pandemic has highlighted the` immediate need for the development of antiviral therapeutics targeting different stages of the SARS-CoV-2 lifecycle. We developed a bioluminescence-based biosensor to interrogate the interaction between the SARS-CoV-2 viral spike protein and its host entry receptor, angiotensin-converting enzyme 2 (ACE2). The biosensor assay is based on a Nanoluciferase complementation reporter, composed of two subunits, Large BiT and Small BiT, fused to the spike receptor-binding domain (RBD) of the SARS-CoV-2 S protein and ACE2 ectodomain, respectively. Using this biosensor, we uncovered a critical role for glycosylation of asparagine residues within the RBD in mediating successful binding to the cellular ACE2 receptor and subsequent virus infection. Our findings support RBD glycosylation as a therapeutic and vaccine target to blunt SARSCoV- 2 infections.
Figure 1
Figure 2
Figure 3
This is a list of supplementary files associated with this preprint. Click to download.
- SupplementaryTable1.pdf
Supplementary Table 1. Plasmid and primer sequences
- SupplementaryFigure1.jpg
Supplemental Figure 1. Structural comparison of SARS CoV and CoV-2 spike protein RBDs. (A) Sequence alignment of SARS CoV and CoV-2 spike RBDs performed with ClustalW. Yellow indicates complete identity, while blue depicts residues with similar side chain properties. The receptor binding motif (RBM) is highlighted beneath. Residues participating in the interaction with ACE2 are indicated with an asterisk if at a common site in both SARS CoV and CoV-2 or a box if unique to one RBD or another. (B) Alignment of tertiary structures of SARS CoV (green; PDB Id: 2AJF) and CoV-2 (blue; PDB Id: 6M0J) RBDs in complex with ACE2 (pink and orange, respectively) (Lan et al. 2020; Li et al. 2005a). Interaction sites of SARS CoV (C) and CoV-2 (D) RBDs (blue) with ACE2 (orange). Black dashed lines depict polar contacts.
- SupplementaryFigure2.jpg
Supplemental Figure 2. SARS-CoV-2 NanoBiT is compatible with alternate imaging systems. (A) 293T cells were co-transfected with constructs expressing the indicated constructs (left panel). 48 hours post-transfection, cells were lysed and FMZ was added to each well. Luminescence was visualized using the IVIS CCD camera.(B) Optimized ACE2-RBD biosensor produces robust luminescent signal observable to naked eye. 293T cells were co-transfected with SmBiT-ACE2 and RBD-LgBiT. 50 g of cell lysate was incubated with FMZ (1:50 ratio) at room temperature and imaged using a standard 12MP digital phone camera.
- SupplementaryFigure3.jpg
Supplemental Figure 3. Comparison of substrates for Nanoluc-based reporter assay. (A) 293T cells were transfected as indicated (SmBiT-ACE2, RBD-LgBiT or co- transfected). 20 ug of lysates were read by the addition of equal volume of substrate: coelenterazine (CTZ, blue) or furimazine (FMZ, brown). (n=3, mean±SD; one-way ANOVA, *** p < 0.005 relative to RBD-LgBiT alone, Dunnett’s correction for multiple comparisons). Assays performed with CTZ and FMZ were analyzed independently. (B) 40 ul of supernatant from transfected 293T cells in panel A were harvested and read similarly (n=3, mean±SD; one-way ANOVA, *** p < 0.005, ** p < 0.01 relative to RBD- LgBiT alone, Dunnett’s correction for multiple comparisons). Assays performed with CTZ and FMZ were analyzed independently.
- SupplementaryFigure4.jpg
Supplemental Figure 4. Development of SARS-CoV biosensor. Biosensor (BS) assay was performed on supernatants of 293T cells co-transfected with SmBiT-ACE2 and either SARS-CoV or SARS-CoV-2 RBD-LgBiT constructs.
- SupplementaryFigure5.jpg
Supplemental Figure 5. N-linked glycosylation of SARS-CoV-2 Spike RBD is critical to its interaction with ACE2. (A) Recombinant RBD purified from E. coli was incubated for 15 minutes with cell lysate containing SmBiT-ACE2 at room temperature. Equal amounts of lysates containing RBD-LgBiT were then added and incubated for 5 minutes. Luciferase assay was performed using CTZ as substrate. (n=3, mean±SD; one-way ANOVA, *** p < 0.005, Tukey’s correction for multiple comparisons.) (B) 293T cells were transfected with RBD-LgBiT and subsequently treated with tunicamycin. Lysates were combined with lysates from 293T cells transfected with SmBiT-ACE2 and BS assay was performed. (C) BS assay was performed on lysates from 293T cells co- transfected with the indicated RBD-glycosylation site mutant-LgBiT constructs and SmBiT-ACE2 constructs. (D) BS assay was performed on supernatants from 293T cells co-transfected with RBD-glycosylation site mutant-LgBiT constructs and SmBiT-ACE2 constructs. (E) ) SARS-CoV-2 S pseudotyped lentivirus encoding ZsGreen and luciferase reporters was incubated with PBS, PNGase F, or Endo H for 1 hour, and then used to infect HEK293-ACE2 cells. 48 hours post-transduction, cells were evaluated for GFP. (F) Lentivirus levels of Spike mutant pseudotypes were titered via p24 ELISA.
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