The Spike D614G mutation increases SARS-CoV-2 infection of multiple human cell types

  1. Zharko Daniloski
  2. Tristan X Jordan
  3. Juliana K Ilmain
  4. Xinyi Guo
  5. Gira Bhabha
  6. Benjamin R tenOever
  7. Neville E Sanjana

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Abstract

A novel variant of the SARS-CoV-2 virus carrying a point mutation in the Spike protein (D614G) has recently emerged and rapidly surpassed others in prevalence. This mutation is in linkage disequilibrium with an ORF1b protein variant (P314L), making it difficult to discern the functional significance of the Spike D614G mutation from population genetics alone. Here, we perform site-directed mutagenesis on wild-type human-codon-optimized Spike to introduce the D614G variant. Using multiple human cell lines, including human lung epithelial cells, we found that the lentiviral particles pseudotyped with Spike D614G are more effective at transducing cells than ones pseudotyped with wild-type Spike. The increased transduction with Spike D614G ranged from 1.3- to 2.4-fold in Caco-2 and Calu-3 cells expressing endogenous ACE2 and from 1.5- to 7.7-fold in A549 ACE2 and Huh7.5 ACE2 overexpressing ACE2. Furthermore, trans -complementation of SARS-CoV-2 virus with Spike D614G showed an increased infectivity in human cells. Although there is minimal difference in ACE2 receptor binding between the D614 and G614 Spike variants, the G614 variant is more resistant to proteolytic cleavage, suggesting a possible mechanism for the increased transduction.

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  1. ScreenIT

    SciScore for 10.1101/2020.06.14.151357: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line AuthenticationContamination: Cells were regularly passaged and tested for presence of mycoplasma contamination (MycoAlert Plus Mycoplasma Detection Kit, Lonza)

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary antibody incubations were performed overnight at 4°C using the following antibodies: rabbit anti-GAPDH 14C10 (0.1 μg/mL, Cell Signaling 2118S), mouse anti-rhodopsin antibody clone 1D4 (1 μg/mL, Novus NBP1-47602) which recognizes the C9-tag added to the Spike proteins.
    anti-GAPDH
    suggested: (Cell Signaling Technology Cat# 2118, RRID:AB_561053)
    Following the primary antibody, the blots were incubated with IRDye 680RD donkey anti-rabbit (0.2 μg/mL, LI-COR 926-68073) or with IRDye 800CW donkey anti-mouse (0.2 μg/mL, LI-COR 926-32212) for 1 hour at room temperature.
    anti-rabbit
    suggested: (LI-COR Biosciences Cat# 926-68073, RRID:AB_10954442)
    anti-mouse
    suggested: (LI-COR Biosciences Cat# 926-32212, RRID:AB_621847)
    Spike was immunoprecipitated using 2 μg C9 antibodies (Novus NBP1-47602) per sample and incubated on a rotator at 4°C for at least 4 hours.
    C9
    suggested: None
    Western blotting was performed as described above using mouse anti-rhodopsin antibody clone 1D4 (1 μg/mL, Novus NBP1-47602) which recognizes the C9-tag added to the Spike proteins.
    anti-rhodopsin
    suggested: (Novus Cat# NBP1-47602, RRID:AB_10010560)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture: A549 cells were obtained from ATCC, HEK293FT cells were obtained from Thermo Scientific, and Huh-7.5 and Caco-2 were a kind gift of B. tenOever (Mt. Sinai).
    A549
    suggested: None
    HEK293FT
    suggested: ATCC Cat# PTA-5077, RRID:CVCL_6911)
    Huh-7.5
    suggested: RRID:CVCL_7927)
    Caco-2
    suggested: None
    Briefly, for each virus, a T-225 flask of 80% confluent HEK293T cells (Thermo) was transfected in OptiMEM (Thermo) using 25 μg of the transfer plasmid, 20 μg psPAX2, 22 μg spike plasmid, and 175 μl of linear Polyethylenimine (1 mg/ml) (Polysciences).
    HEK293T
    suggested: None
    ACE2 lentiviral cloning and ACE2 stable cell line overexpression: To generate pLenti-ACE2-Hygro, we amplified human ACE2 (hACE2) from pcDNA3.1-ACE2 (Addgene 1786) and cloned it into a lentiviral transfer pLEX vector carrying the hygromycin resistance gene using Gibson Assembly Master Mix (NEB E2611L).
    ACE2
    suggested: RRID:CVCL_DR94)
    Huh7.5-ACE2 and A549-ACE2 cell lines were generated by lentiviral transduction of ACE2.
    Huh7.5-ACE2
    suggested: None
    A549-ACE2
    suggested: None
    SARS-CoV-2 was propagated in Vero E6 cells in DMEM supplemented with 2% FBS, 4.5 g/L D-glucose, 4 mM L-glutamine, 10 mM
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    Band intensity quantification was performed by first converting Odyssey multichannel TIFFs into 16-bit grayscale image (Fiji) and the then selecting lanes and bands in ImageLab 6.1 (BioRad).
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    For each peptide, we computed the difference in predicted affinity between the D614 and G614 variant using R/RStudio and visualized them using the pheatmap R package.
    pheatmap
    suggested: (pheatmap, RRID:SCR_016418)
    Statistical analysis: Data analysis was performed using R/Rstudio 3.6.1 and GraphPad Prism 8 (GraphPad Software Inc.)
    GraphPad
    suggested: (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: 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 TrialIdentifier: No clinical trial numbers were referenced.

    Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

    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.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  2. Version published to 10.7554/elife.65365 on eLife
  3. Version published to 10.1101/2020.06.14.151357v2 on bioRxiv
  4. ScreenIT

    SciScore for 10.1101/2020.06.14.151357: (What is this?)

    Please note, not all rigor criteria are appropriate for all manuscripts.

    Table 1: Rigor

    Institutional Review Board Statementnot detected.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line AuthenticationCells were regularly passaged and tested for presence of mycoplasma contamination ( MycoAlert Plus Mycoplasma Detection Kit , Lonza)

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary antibody incubations were performed overnight at 4°C using the following antibodies: rabbit anti-GAPDH 14C10 ( 0.1 μg/mL , Cell Signaling 2118S) , mouse anti-rhodopsin antibody clone 1D4 ( 1 μg/mL , Novus NBP1-47602 ) which recognizes the C9-tag added to the Spike proteins .
    anti-GAPDH
    suggested: (Cell Signaling Technology Cat# 2118, AB_561053)
    Following the primary antibody , the blots were incubated with IRDye 680RD donkey anti-rabbit ( 0.2 μg/mL , LI-COR 926-68073 ) or with IRDye 800CW donkey anti-mouse ( 0.2 μg/mL , LI-COR 926-32212 ) for 1 hour at room temperature.
    anti-rabbit
    suggested: (LI-COR Biosciences Cat# 926-68073, AB_10954442)
    Spike was immunoprecipitated using 2 µg C9 antibodies ( Novus NBP1-47602 ) per sample and incubated on a rotator at 4°C for at least 4 hours .
    C9
    suggested: None
    Western blotting was performed as described above using mouse anti-rhodopsin antibody clone 1D4 ( 1 μg/mL , Novus NBP1-47602 ) which recognizes the C9-tag added to the Spike proteins .
    anti-rhodopsin
    suggested: (Novus Cat# NBP1-47602, AB_10010560)
    Following the primary antibody , the blots were incubated with IRDye 800CW donkey anti-mouse ( 0.2 μg/mL , LI-COR 926-32212 ) for 1 hour at room temperature.
    anti-mouse
    suggested: (LI-COR Biosciences Cat# 926-32212, AB_621847)
    Experimental Models: Cell Lines
    SentencesResources
    Cell culture A549 cells were obtained from ATCC , HEK293FT cells were obtained from Thermo Scientific , and Huh-7.5 and Caco-2 were a kind gift of T . Jordan and B . tenOever ( Mt . Sinai) .
    A549
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>HEK293FT</b></div>
        <div>suggested: ATCC Cat# PTA-5077, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_6911">CVCL_6911</a></div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Huh-7.5</b></div>
        <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_7927">CVCL_7927</a></div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Caco-2</b></div>
        <div>suggested: CLS Cat# 300137/p1665_CaCo-2, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0025">CVCL_0025</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly , for each virus , a T-225 flask of 80 % confluent HEK293T cells ( Thermo ) was transfected in OptiMEM ( Thermo ) using 25 µg of the transfer plasmid , 20 µg psPAX2 , 22 µg spike plasmid , and 175 µl of linear Polyethylenimine ( 1 mg/ml ) ( Polysciences) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK293T</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">ACE2 lentiviral cloning and ACE2 stable cell line overexpression To generate pLenti-ACE2-Hygro , we amplified human ACE2 ( hACE2 ) from pcDNA3.1-ACE2 ( Addgene 1786 ) and cloned it into a lentiviral transfer pLEX vector carrying the hygromycin resistance gene using Gibson Assembly Master Mix ( NEB E2611L) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>ACE2</b></div>
        <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_DR94">CVCL_DR94</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Huh7.5-ACE2 and A549-ACE2 cell lines were generated by lentiviral transduction of ACE2 .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Huh7.5-ACE2</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>A549-ACE2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Software and Algorithms</b></td></tr><tr><td style="min-width:100px;text=align:center"><i>Sentences</i></td><td style="min-width:100px;text-align:center"><i>Resources</i></td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Z . D . is supported by an American Heart Association postdoctoral fellowship . N.E . S. is supported by New York University and New York Genome Center startup funds , National Institutes of Health ( NIH)/National</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>American Heart Association</b></div>
        <div>suggested: (American Heart Association, <a href="https://scicrunch.org/resources/Any/search?q=SCR_007210">SCR_007210</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Band intensity quantification was performed by first converting Odyssey multichannel TIFFs into 16-bit grayscale image ( Fiji ) and the then selecting lanes and bands in ImageLab 6.1 ( BioRad) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Fiji</b></div>
        <div>suggested: (Fiji, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002285">SCR_002285</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For each peptide , we computed the difference in predicted affinity between the D614 and G614 variant using R/RStudio and visualized them using the pheatmap R package.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>pheatmap</b></div>
        <div>suggested: (pheatmap, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016418">SCR_016418</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical analysis Data analysis was performed using R/Rstudio 3.6.1 and GraphPad Prism 8 ( GraphPad Software Inc . )</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>GraphPad</b></div>
        <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
      </div>
    </td></tr></table>

    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 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 OddPub: Thank you for sharing your code.

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore is not a substitute for expert review. SciScore checks for the presence and correctness of RRIDs (research resource identifiers) in the manuscript, and detects sentences that appear to be missing RRIDs. SciScore also checks to make sure that rigor criteria are addressed by authors. It does this by detecting sentences that discuss criteria such as blinding or power analysis. SciScore does not guarantee that the rigor criteria that it detects are appropriate for the particular study. Instead it assists authors, editors, and reviewers by drawing attention to sections of the manuscript that contain or should contain various rigor criteria and key resources. For details on the results shown here, including references cited, please follow this link.

  5. Version published to 10.1101/2020.06.14.151357v1 on bioRxiv