D614G mutation of SARS-CoV-2 spike protein enhances viral infectivity
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Abstract
Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The spike (S) protein that mediates SARS-CoV-2 entry into host cells is a major target for vaccines and therapeutics. Thus, insights into its sequence variations are key to understanding the infection and antigenicity of SARS-CoV-2. A dominant mutational variant at position 614 of the S protein (aspartate to glycine, D614G mutation) was observed in the SARS-CoV-2 genome sequence obtained from the Nextstrain database. Using a pseudovirus-based assay, we identified that S-D614 and S-G614 protein pseudotyped viruses share a common receptor, human angiotensin-converting enzyme 2 (ACE2), which could be blocked by recombinant ACE2 with the fused Fc region of human IgG1. However, S-D614 and S-G614 protein demonstrated functional differences. First, S-G614 protein could be cleaved by serine protease elastase-2 more efficiently. Second, S-G614 pseudovirus infected 293T-ACE2 cells significantly more efficiently than did the S-D614 pseudovirus, especially in the presence of elastase-2. Third, an elastase inhibitor approved for clinical use blocked elastase-enhanced S-G614 pseudovirus infection. Moreover, 93% (65/70) convalescent sera from patients with COVID-19 could neutralize both S-D614 and S-G614 pseudoviruses with comparable efficiencies, but about 7% (5/70) convalescent sera showed reduced neutralizing activity against the S-G614 pseudovirus. These findings have important implications for SARS-CoV-2 transmission and immune interventions.
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SciScore for 10.1101/2020.06.20.161323: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethical approval: The study was approved by the Ethics Commission of Chongqing Medical University (ref. no. 2020003).
Consent: Written informed consent was waived by the Ethics Commission of the designated hospital for emerging infectious diseases.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Antibodies and inhibitors: The anti-RBD monoclonal antibody against the SARS-CoV-2 S protein was kindly provided by Prof. Aishun Jin from Chongqing Medical University anti-RBDsuggested: NoneExperimental Models: Cell Lines S… SciScore for 10.1101/2020.06.20.161323: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement IRB: Ethical approval: The study was approved by the Ethics Commission of Chongqing Medical University (ref. no. 2020003).
Consent: Written informed consent was waived by the Ethics Commission of the designated hospital for emerging infectious diseases.Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Antibodies and inhibitors: The anti-RBD monoclonal antibody against the SARS-CoV-2 S protein was kindly provided by Prof. Aishun Jin from Chongqing Medical University anti-RBDsuggested: NoneExperimental Models: Cell Lines Sentences Resources Western blot analysis of SARS-CoV-2 S protein expression: To analyze S protein expression in cells, S-D614- and S-G614-expressing plasmids were transfected into HEK 293T cells. HEK 293Tsuggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)Neutralization and inhibition assays: The 293T-ACE2 cells (2 × 104 cells/well) were seeded on 96-well plates. 293T-ACE2suggested: RRID:CVCL_YZ65)Software and Algorithms Sentences Resources The nucleotide and amino acid sequences of S were aligned with multiple sequence alignment software MUltiple Sequence Comparison by Log-Expectation (MUSCLE) separately. MUSCLEsuggested: (MUSCLE, RRID:SCR_011812)Protein bands were visualized using SuperSignal West Pico Chemiluminescent Substrate kits (Bio-Rad, Hercules, CA, USA) and quantified by densitometry using ImageJ software (NCBI, Bethesda, MD, USA). ImageJsuggested: (ImageJ, RRID:SCR_003070)Luciferase activity was measured 72 h after infection and the percentage of neutralization was calculated using GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA, USA). GraphPadsuggested: (GraphPad Prism, RRID:SCR_002798)Statistical analyses: Statistical analyses of the data were performed using GraphPad Prism version 6.0 software. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:Our study had some limitations. First, 19 amino acids of S protein on the C-terminal were not included in order to improve the packaging efficiency of the SARS-CoV-2 S protein pseudotyped virus, and this pseudovirus only recapitulates viral entry events. Therefore, additional assays with authentic SARS-CoV-2 viruses are required. Second, we only tested neutralizing antibodies against the S protein. Previous studies on SARS-CoV indicated that only a small fraction of memory B cells specific for SARS-CoV antigens are directed against neutralizing epitopes present on the S protein27. Third, in addition to D614G, further studies on other mutations in the S protein are needed to evaluate their impact on SARS-CoV-2 infectivity, pathogenicity, and immunogenicity. Further studies are needed to determine the impact of these mutations on the severity of COVID-19. In summary, we established a SARS-CoV-2 S protein-mediated pseudoviral entry assay and explored the cellular entry of S-D614 and S-G614 pseudotyped viruses. Our study provided evidence that the D614G mutation introduces an additional elastase-2 cut site in the S protein, thereby promoting its cleavage and viral cell entry, resulting in SARS-CoV-2 becoming more infectious. Importantly, the D614G mutation reduced the sensitivity of the virus to serum neutralizing antibodies in 7% convalescent patients with COVID-19. Our study will be helpful for understanding SARS-CoV-2 transmission and for the design of vaccines and therapeutic...
Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
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SciScore for 10.1101/2020.06.20.161323: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement The study was approved by the Ethics Commission of Chongqing Medical University (ref. no. 2020003). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The anti-RBD (receptor-binding domain) monoclonal antibody against the SARS-CoV-2 S protein was kindly provided by Prof. Aishun Jin from Chongqing Medical University. anti-RBD (receptor-binding domain)suggested: NoneExperimental Models: Cell Lines Sentences Resources Second, S-G614 pseudovirus infected … SciScore for 10.1101/2020.06.20.161323: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Institutional Review Board Statement The study was approved by the Ethics Commission of Chongqing Medical University (ref. no. 2020003). Randomization not detected. Blinding not detected. Power Analysis not detected. Sex as a biological variable not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The anti-RBD (receptor-binding domain) monoclonal antibody against the SARS-CoV-2 S protein was kindly provided by Prof. Aishun Jin from Chongqing Medical University. anti-RBD (receptor-binding domain)suggested: NoneExperimental Models: Cell Lines Sentences Resources Second, S-G614 pseudovirus infected 293T-ACE2 cells significantly more efficiently than the S-D614 pseudovirus, Moreover, 93% (38/41) sera from convalescent COVID-19 patients could neutralize both S-D614 and S-G614 pseudotyped viruses with comparable efficiencies, but about 7% (3/41) convalescent sera showed decreased neutralizing activity against S-G614 pseudovirus. 293T-ACE2suggested: NoneLuc.R-E- was co-transfected with pS-D614, pS-G614, respectively to package the SARS-CoV-2 S pseudotyped single-round Luc virus in HEK293T cells. HEK293Tsuggested: KCB Cat# KCB 200744YJ, CVCL_0063Since we observed that S-G614 could be more efficiently cleaved by host protease when exogenously expressed in 293T cells, we assumed that host proteases may be involved in the enhancement of S-G614 virus entry. 293Tsuggested: NoneSoftware and Algorithms Sentences Resources All complete SARS-CoV-2 S gene sequences were downloaded from NCBI website (https://www.ncbi.nlm.nih.gov/sars-cov-2/) on Jun 1, 2020. NCBIsuggested: (NCBI, SCR_006472)The nucleotide and amino acid sequences of S were aligned with multiple sequence alignment software MUSCLE separately. MUSCLEsuggested: (MUSCLE, SCR_011812)Protein bands were visualized using SuperSignal™ West Pico Chemiluminescent Substrate kits (Bio-Rad, Hercules, CA, USA) and quantified by densitometry using ImageJ software (National Center for Biotechnology Information [NCBI], Bethesda, MD, USA). ImageJsuggested: (ImageJ, SCR_003070)Luciferase activity was measured 72 h after infection and the percent neutralization was calculated using GraphPad Prism 6.0 software (GraphPad Software, San Diego, CA, USA). GraphPadsuggested: (GraphPad Prism, SCR_002798)Statistical analyses of the data were performed by using GraphPad Prism version 6.0 software. GraphPad Prismsuggested: (GraphPad Prism, SCR_002798)Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.
Results from OddPub: We did not find a statement about open data. We also did not find a statement about open code. Researchers are encouraged to share open data when possible (see Nature blog).
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