SARS-CoV-2 Spike evolution influences GBP and IFITM sensitivity

  1. Dejan Mesner
  2. Ann-Kathrin Reuschl
  3. Matthew V.X Whelan
  4. Taylor Bronzovich
  5. Tafhima Haider
  6. Lucy G. Thorne
  7. Greg J. Towers
  8. Clare Jolly

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Abstract

SARS-CoV-2 spike requires proteolytic processing for viral entry. The presence of a polybasic furin-cleavage site (FCS) in spike, and evolution towards an optimised FCS by dominant variants of concern (VOCs), are linked to enhanced infectivity and transmission. Here we show that interferon-inducible antiviral restriction factors Guanylate binding proteins (GBP) 2 and 5 interfere with furin-mediated cleavage of SARS-CoV-2 spike and inhibit the infectivity of early-lineage Wuhan-Hu-1, while VOCs Alpha and Delta have evolved to escape restriction. Strikingly, we find Omicron is unique amongst VOCs, being restricted by GBP2/5, and also IFITM1, 2 and 3. Replacing the spike S2 domain in Omicron with Delta shows S2 is the determinant of entry route and IFITM sensitivity. We conclude that VOC evolution under different selective pressures has influenced sensitivity to spike-targeting restriction factors, with Omicron selecting spike changes that not only mediate antibody escape, and altered tropism, but also sensitivity to innate immunity.

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  1. Version published to 10.1101/2022.03.07.481785v2 on bioRxiv
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    SciScore for 10.1101/2022.03.07.481785: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Twenty micrograms of cell lysate and an equal volume of purified virus were separated by SDS-PAGE, transferred to nitrocellulose membrane and membranes probed using the following primary antibodies: anti-SARS-CoV Spike (100% overlap with the SARS-CoV-2 epitope) (Invitrogen, PA1-41165, 1:1000)
    anti-SARS-CoV
    suggested: None
    SARS-CoV-2 epitope
    suggested: None
    PA1-41165
    suggested: (Thermo Fisher Scientific Cat# PA1-41165, RRID:AB_1087210)
    , anti SARS-CoV nucleoprotein (N) monoclonal antibody (clone CR3009, gift from Laura McCoy, 0.5 μg/ml), anti-HA-tag (Biolegend, 16B12, 1:2000)
    anti SARS-CoV nucleoprotein ( N
    suggested: None
    anti-HA-tag
    suggested: None
    Primary antibodies were incubated overnight at 4°C with agitation and detected with fluorescent secondary antibodies: anti-Rabbit IgG (IRDye 800CW, Abcam, ab216773, 1:10,000), anti-Mouse IgG (IRDye 680RD, Abcam, ab216776, 1:10,000) and anti-Human IgG (IRDye 800CW, Licor, 925-68078, 1:10,000), and imaged with an Odyssey Cxl Infrared Imager (Licor).
    anti-Rabbit IgG
    suggested: None
    anti-Mouse IgG
    suggested: None
    ab216776
    suggested: None
    anti-Human IgG
    suggested: (LI-COR Biosciences Cat# 925-68078, RRID:AB_2814915)
    Cells were then washed and permeabilised with Perm buffer (Biolegend) for 5 min at RT and stained with primary antibodies (20 min at RT): anti-HA-tag-APC (Biolegend, 16B12, 1:150), anti-Gag-PE (Beckam Coulter, KC57, 1:250).
    anti-HA-tag-APC
    suggested: None
    anti-Gag-PE
    suggested: None
    KC57
    suggested: (Beckman Coulter Cat# 6604667, RRID:AB_1575989)
    SARS-CoV-2 infection was detected by intracellular staining for nucleoprotein (N) using anti-SARS-CoV N monoclonal antibody (human, clone CR3009, gift from Laura McCoy, 1 μg/ml) and secondary anti-Human IgG-AF488 (Jackson labs, 1:400).
    anti-SARS-CoV N
    suggested: None
    anti-Human IgG-AF488
    suggested: (SouthernBiotech Cat# 2040-30, RRID:AB_2795650)
    For analysis of cell surface spike infection, 293T cells were harvested with 5 mM EDTA (Sigma), washed, stained with Zombie NIR and anti-SARS-CoV-2 Spike antibody from human convalescent sera (NIBSC, 20/130 1:200) on ice for 30 min.
    anti-SARS-CoV-2
    suggested: None
    Primary antibody incubation was carried out for 1h at RT with rabbit anti-HA antibody (H6908, Sigma-Aldrich) to label IFITM1, IFITM2 and IFITM3 and mouse anti-CD63 (IB5, a gift from M. Marsh, UCL) to label endogenous CD63.
    anti-HA
    suggested: (Sigma-Aldrich Cat# H6908, RRID:AB_260070)
    IFITM1
    suggested: None
    IFITM2
    suggested: None
    IFITM3
    suggested: None
    IB5
    suggested: None
    CD63
    suggested: None
    Primary antibodies were detected with secondary anti-rabbit AlexaFluor-488 and anti-mouse AlexaFluor-568 conjugates (Jackson Immuno Research) for 1h.
    anti-rabbit
    suggested: None
    anti-mouse
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells: HEK293T/17 cells (abbreviated herein as 293T cells) were obtained from American Type Culture Collection (ATCC,
    293T
    suggested: KCB Cat# KCB 200744YJ, RRID:CVCL_0063)
    Vero.E6 cells were obtained from the National Institute for Biological Standards and Control (NIBSC).
    Vero.E6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Vero.E6-TMPRSS2 cells (stable cell line expressing TMPRSS2 under G418 selection) were a gift from Mala Maini (UCL).
    Vero.E6-TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    HeLa-TZM-bl cells (expressing luciferase and beta-galactosidase under the control of HIV-1 LTR) were obtained from the Centre for AIDS Reagents (CFAR).
    HeLa-TZM-bl
    suggested: None
    Pseudovirus infection: Target cells were seeded into white 96-well plates 24h before infection (Caco2, Vero.E6 and Vero.E6-TMPRSS2 cells seeded at 1.5×104 cells/well, 293T-ACE2, HeLa-ACE2 and HeLa-TZMbl cells seeded at 1×104 cells/well).
    HeLa-ACE2
    suggested: JCRB Cat# JCRB1845, RRID:CVCL_B3LW)
    HeLa-TZMbl
    suggested: None
    IFITM inhibition assay: Caco2 cells, WT or stably expressing IFITM1/2/3 (described above), were infected with indicated spike PVs and luciferase expression was measured 48 h post-infection as described above.
    Caco2
    suggested: None
    Infection of Caco2-IFITM1/2/3 cells with live SARS-CoV-2 virus was done as described above.
    Caco2-IFITM1/2/3
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmids: SARS-CoV-2 Spike expression vectors were originally synthesised by Genewiz and subcloned into pCDNA3.1+ vector.
    pCDNA3.1+
    suggested: RRID:Addgene_117272)
    Lentiviral backbone packaging plasmid (expressing HIV Gag, Pol, Tat and Rev) p8.91 and the reporter plasmid encoding luciferase gene pCSLW were a gift from Greg Towers (UCL).
    p8.91
    suggested: None
    Pseudovirus production: Spike pseudoviruses (PVs) were made by co-transfection of spike, p8.91 and pCSLW plasmids as described previously (Dicken et al., 2021).
    pCSLW
    suggested: None
    For HIV-1 Env PV, 293T cells were seeded as described above and transfected with 240 ng p8.91, 240 ng pCSLW,120 ng pSVIII-JRFL Env and indicated doses of GBP or empty vector control.
    pCSLW,120
    suggested: None
    pSVIII-JRFL
    suggested: None
    Software and Algorithms
    SentencesResources
    Plasmids: SARS-CoV-2 Spike expression vectors were originally synthesised by Genewiz and subcloned into pCDNA3.1+ vector.
    Genewiz
    suggested: (GENEWIZ, RRID:SCR_003177)
    Scale bars and RGB composite images were created in in FIJI ImageJ software package (Schindelin et al., 2012).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Statistical Analysis: Statistical significance was calculated using Prism 9 (GraphPad Prism) using indicated statistical tests and significance was assumed when P < 0.05.
    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.

    Results from scite Reference Check: We found no unreliable references.

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  3. Version published to 10.1101/2022.03.07.481785v1 on bioRxiv