The SARS-CoV-2 Spike mutation D614G increases entry fitness across a range of ACE2 levels, directly outcompetes the wild type, and is preferentially incorporated into trimers

  1. William A. Michaud
  2. Genevieve M. Boland
  3. S. Alireza Rabi

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Abstract

Early in the current pandemic, the D614G mutation arose in the Spike protein of SARS-CoV-2 and quickly became the dominant variant globally. Mounting evidence suggests D614G enhances viral entry. Here we use a direct competition assay with single-cycle viruses to show that D614G outcompetes the wildtype. We developed a cell line with inducible ACE2 expression to confirm that D614G more efficiently enters cells with ACE2 levels spanning the different primary cells targeted by SARS-CoV-2. Using a new assay for crosslinking and directly extracting Spike trimers from the pseudovirus surface, we found an increase in trimerization efficiency and viral incorporation of D614G protomers. Our findings suggest that D614G increases infection of cells expressing a wide range of ACE2, and informs the mechanism underlying enhanced entry. The tools developed here can be broadly applied to study other Spike variants and SARS-CoV-2 entry, to inform functional studies of viral evolution and vaccine development.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.25.267500: (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
    PE)- conjugated mouse anti-human IgG1 secondary antibody (BD Biosciences, 555787) on ice for 30 minutes.
    anti-human IgG1
    suggested: None
    To select for the inducible cell colonies that express ACE2 at a higher level, we then stained with 2 μg of anti-ACE2 goat IgG antibody (R&D Systems, AF933) per million cells followed by a FITC-conjugated secondary antibody (Santa Cruz Biotechnology, sc-2356).
    anti-ACE2 goat IgG
    suggested: (Thermo Fisher Scientific Cat# PA5-47488, RRID:AB_2606505)
    Magnetic beads with covalently bound anti-FLAG (Sigma, M8823) or anti-HA (ThermoFisher Scientific, 88838) antibodies were used for immunoprecipitation of the crosslinked trimers using their C-terminal tags.
    anti-FLAG
    suggested: (Sigma-Aldrich Cat# M8823, RRID:AB_2637089)
    anti-HA
    suggested: None
    Antibodies: In addition to the antibodies named in the previous sections, the following antibodies were used for western blot analysis: Rabbit anti-GAPDH (2118) and rabbit anti-HA (3724) were purchased from Cell Signaling and used at 1/1000 dilution.
    anti-GAPDH
    suggested: (Cell Signaling Technology Cat# 2118, RRID:AB_561053)
    anti-HA ( 3724 )
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Inducible cell line production: HEK293T cells were plated at ~50% confluence in a 6-well plate one day before transfection.
    HEK293T
    suggested: None
    For the in vitro competition assay, we added 5.0 ng p24 equivalent of the RFP-expressing SD614-pseudotyped virus to each well of a flat-bottom 96-well plate containing 30,000 HEK-ACE2 cells.
    HEK-ACE2
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmids and viral constructs: The psPAX2 plasmid was a gift from Didier Trono (Addgene plasmid # 12260; http://n2t.net/addgene:12260; RRID:Addgene_12260) and is a lentiviral packaging vector used for second- and third-generation lentiviral production systems.
    detected: RRID:Addgene_12260)
    The pSin-DsRed-IRES-Puro and the corresponding GFP-expressing vector were made using pSin-EF-Sox2-Puro, which was a gift from James Thomson (Addgene plasmid # 16577; http://n2t.net/addgene:16577; RRID:Addgene_16577) 16.
    detected: RRID:Addgene_16577)
    The inducible ACE2 vector, PB-Tet-hAce2, was made from the XLone-GFP parental plasmid which was a gift from Xiaojun Lian 18 (Addgene plasmid # 96930; http://n2t.net/addgene:96930; RRID:Addgene_96930).
    detected: RRID:Addgene_96930)
    The ace2 gene was amplified from a plasmid gifted by Hyeryun Choe 14 (Addgene plasmid # 1786; http://n2t.net/addgene:1786; RRID:Addgene_1786) using CoV-39 and CoV-40 primers.
    detected: RRID:Addgene_1786)

    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:
    One limitation of our findings is the use of in vitro infectivity assays. Although we sought to improve the system’s physiological relevance by utilizing cells with variable and controllable expression levels of ACE2, our results are limited to pseudotyped lentiviruses and thus may differ from physiological settings. Furthermore, similar to other groups 7,9, in order to increase the efficiency of the Spike protein expression on the surface of the virus-producing cells, and therefore, the overall infectivity, we modified the Spike protein by deleting the C-terminal 19 amino acids and instead inserted HA or FLAG peptide tags. This modification modifies the cognate signal peptide on the Spike and preferentially targets the nascent Spike proteins to the cell’s surface []. Therefore, the experiments looking at the Spike protein’s packaging in the virions could be affected by this artificial trafficking. However, we synthesized both the D614 and G614 Spike variants with similar C-terminus modifications, and therefore, the comparison between the amount of these two proteins may still reflect their relative amount in the unmodified SARS-CoV-2. Finally, while our system can be used to investigate the relative amount of each Spike variant in the chimeric trimers, further work is needed to determine if the different Spike trimers have different three-dimensional configurations. In addition to characterizing the fitness advantage of the D614G mutation of the Spike protein and the mechani...

    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

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  2. Version published to 10.1101/2020.08.25.267500v1 on bioRxiv