SARS-CoV-2 spike-protein D614G mutation increases virion spike density and infectivity

  1. Lizhou Zhang
  2. Cody B. Jackson
  3. Huihui Mou
  4. Amrita Ojha
  5. Haiyong Peng
  6. Brian D. Quinlan
  7. Erumbi S. Rangarajan
  8. Andi Pan
  9. Abigail Vanderheiden
  10. Mehul S. Suthar
  11. Wenhui Li
  12. Tina Izard
  13. Christoph Rader
  14. Michael Farzan
  15. Hyeryun Choe

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Abstract

SARS-CoV-2 variants with spike (S)-protein D614G mutations now predominate globally. We therefore compare the properties of the mutated S protein (S G614 ) with the original (S D614 ). We report here pseudoviruses carrying S G614 enter ACE2-expressing cells more efficiently than those with S D614 . This increased entry correlates with less S1-domain shedding and higher S-protein incorporation into the virion. Similar results are obtained with virus-like particles produced with SARS-CoV-2 M, N, E, and S proteins. However, D614G does not alter S-protein binding to ACE2 or neutralization sensitivity of pseudoviruses. Thus, D614G may increase infectivity by assembling more functional S protein into the virion.

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  1. ScreenIT

    SciScore for 10.1101/2020.06.12.148726: (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 Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    1 μg/ml anti-p30 MLV gag antibody (Abcam, ab130757) and 1:10,000 dilution of goat-anti-mouse IgG-HRP polyclonal antibody (Jackson ImmunoResearch, 115-036-062) were used to detect MLV gag protein as an internal control.
    1 μg/ml anti-p30 MLV gag antibody ( Abcam , ab130757 )
    suggested: None
    anti-p30 MLV gag antibody ( Abcam , ab130757 )
    suggested: None
    IgG-HRP
    suggested: None
    As with MLV PV, the S-protein bands were visualized using the anti-Flag M2 antibody, and the N-protein band was detected using pooled convalescent plasma at a 1:500 dilution and 10 ng/ml goat-anti-human IgG antibody conjugated with polymerized HRP (Fitzgerald, 61R-I166AHRP40).
    anti-Flag
    suggested: (Advanced Targeting Systems Cat# AB-450-500, RRID:AB_10585851)
    IgG
    suggested: (Fitzgerald Industries International Cat# 61R-I166AHRP40, RRID:AB_10815602)
    To measure ACE2-binding ability or the level of the S1 domain present on the cell surface, cells were detached two days post transfection with Accutase (Stemcell Technologies Inc.) and incubated with either 1 μg/ml purified hACE2-NN-Ig or anti-Myc antibody (clone 9E10, National Cell Culture Center, Minneapolis, MN), respectively, on ice. hACE2-NN-Ig was previous described and was purified using Protein A-Sepharose CL-4B (GE Healthcare)31.
    anti-Myc
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Infection assays were performed by spinoculating (at 2,100 x g for 30 min at 10°C) PVs onto the Mock- and hACE2-293T cells seeded on multiwell plates.
    hACE2-293T
    suggested: None
    Cell-surface expression and analysis of the S protein: HEK293T cells, approximately 80% confluent in 6-well plates were transfected with 8 μl PEI 40,000
    HEK293T
    suggested: None
    Mock- and hACE2-HEK293T cells on 96-well plates were infected with the preincubation mixes and infection levels were assessed 24 h later by measuring luciferase activity using the Luc-Pair Firefly Luciferase HS Assay Kit (GeneCopoeia)
    hACE2-HEK293T
    suggested: None
    Software and Algorithms
    SentencesResources
    Genotype frequency at residue 614 was calculated using R (R Foundation for Statistical Computing) with the Biostrings package.
    Biostrings
    suggested: (Biostrings, RRID:SCR_016949)
    Logo plots of D614G variation were generated by WebLogo after sequence alignment.
    WebLogo
    suggested: (WEBLOGO, RRID:SCR_010236)
    Statistical analysis: All appropriate data were analyzed with GraphPad Prism 7 (GraphPad Software Inc.).
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    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 10.1038/s41467-020-19808-4
  3. ScreenIT

    SciScore for 10.1101/2020.06.12.148726: (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 Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    To independently confirm that similar number of virions was analyzed, the lower part of the same membrane was blotted with an anti-p30 MLV gag antibody (Fig. 2c).
    anti-p30 MLV gag
    suggested: None
    The M2 antibody used in this experiment binds the Flag tag located at both the N- and C-termini of a protein, but it binds N-terminal Flag tag more efficiently33.
    M2
    suggested: None
    To differentiate these possibilities, we appended the Myc-tag to the N-terminus of the S-protein that is Flag tagged at its C-terminus and repeated the study, this time detecting the S1 domain with an anti-Myc antibody.
    anti-Myc
    suggested: None
    For western blot analyses, 5-10 µl of purified PV, which is equivalent to 0.5-1.0 x 1010 vector genomes, was loaded per lane of the 4-12% Bis-Tris gel (Life Technologies), transferred to the PVDF membrane, and blotted with 1 µg/ml anti-Flag M2 antibody (Sigma-Aldrich, F1804) to detect the S-protein bands.
    anti-Flag
    suggested: (Sigma-Aldrich Cat# F1804, AB_262044)
    1 µg/ml anti-p30 MLV gag antibody (Abcam, ab130757) and 1:10,000 dilution of goat-anti- mouse IgG-HRP polyclonal antibody (Jackson ImmunoResearch, 115-036-062) were used to detect MLV gag protein as an internal control.
    1 µg/ml anti-p30 MLV gag antibody (Abcam, ab130757)
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>anti-p30 MLV gag antibody (Abcam, ab130757)</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>mouse IgG-HRP</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">As with MLV PV, the S-protein bands were visualized using the anti-Flag M2 antibody, and the N-protein band was detected using pooled convalescent plasma at a 1:500 dilution and 10 ng/ml goat-anti-human IgG antibody conjugated with polymerized HRP (Fitzgerald, 61RI166AHRP40).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>IgG</b></div>
        <div>suggested: (Fitzgerald Industries International Cat# 61R-I166AHRP40, <a href="https://scicrunch.org/resources/Any/search?q=AB_10815602">AB_10815602</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;text-align:center; padding-top:4px;" colspan="2"><b>Experimental Models: Cell Lines</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">We observed PVG614 infected hACE2-293T cells with approximately 9-fold higher efficiency than did PVD614 (Fig. 1c,d).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>hACE2-293T</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">MLV PVs were produced by transfecting HEK293T cells at ~60% confluency in T175 flasks using the calciumphosphate method with 70 µg of total DNA.</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">Mock- and hACE2HEK293T cells on 96-well plates were infected with the preincubation mixes and infection levels were assessed 24 h later by measuring luciferase activity using the LucPair Firefly Luciferase HS Assay Kit (GeneCopoeia).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>hACE2HEK293T</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">Genotype frequency at residue 614 was calculated using R (R Foundation for Statistical Computing) with the Biostrings package.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Biostrings</b></div>
        <div>suggested: (Biostrings, <a href="https://scicrunch.org/resources/Any/search?q=SCR_016949">SCR_016949</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Logo plots of D614G variation were generated by WebLogo after sequence alignment.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>WebLogo</b></div>
        <div>suggested: (WEBLOGO, <a href="https://scicrunch.org/resources/Any/search?q=SCR_010236">SCR_010236</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">All appropriate data were analyzed with GraphPad Prism 7 (GraphPad Software Inc.).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>GraphPad Prism</b></div>
        <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
      </div>
    
      <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 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 data.

    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.

  4. Version published to 10.1101/2020.06.12.148726v1 on bioRxiv