SARS-CoV-2 variants of concern have acquired mutations associated with an increased spike cleavage

  1. Alba Escalera
  2. Ana S. Gonzalez-Reiche
  3. Sadaf Aslam
  4. Ignacio Mena
  5. Rebecca L. Pearl
  6. Manon Laporte
  7. Andrea Fossati
  8. Raveen Rathnasinghe
  9. Hala Alshammary
  10. Adriana van de Guchte
  11. Mehdi Bouhaddou
  12. Thomas Kehrer
  13. Lorena Zuliani-Alvarez
  14. David A. Meekins
  15. Velmurugan Balaraman
  16. Chester McDowell
  17. Jürgen A. Richt
  18. Goran Bajic
  19. Emilia Mia Sordillo
  20. Nevan Krogan
  21. Viviana Simon
  22. Randy A. Albrecht
  23. Harm van Bakel
  24. Adolfo Garcia-Sastre
  25. Teresa Aydillo

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Abstract

For efficient cell entry and membrane fusion, SARS-CoV-2 spike (S) protein needs to be cleaved at two different sites, S1/S2 and S2’ by different cellular proteases such as furin and TMPRSS2. Polymorphisms in the S protein can affect cleavage, viral transmission, and pathogenesis. Here, we investigated the role of arising S polymorphisms in vitro and in vivo to understand the emergence of SARS-CoV-2 variants. First, we showed that the S:655Y is selected after in vivo replication in the mink model. This mutation is present in the Gamma Variant Of Concern (VOC) but it also occurred sporadically in early SARS-CoV-2 human isolates. To better understand the impact of this polymorphism, we analyzed the in vitro properties of a panel of SARS-CoV-2 isolates containing S:655Y in different lineage backgrounds. Results demonstrated that this mutation enhances viral replication and spike protein cleavage. Viral competition experiments using hamsters infected with WA1 and WA1-655Y isolates showed that the variant with 655Y became dominant in both direct infected and direct contact animals. Finally, we investigated the cleavage efficiency and fusogenic properties of the spike protein of selected VOCs containing different mutations in their spike proteins. Results showed that all VOCs have evolved to acquire an increased spike cleavage and fusogenic capacity despite having different sets of mutations in the S protein. Our study demonstrates that the S:655Y is an important adaptative mutation that increases viral cell entry, transmission, and host susceptibility. Moreover, SARS-COV-2 VOCs showed a convergent evolution that promotes the S protein processing.

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    SciScore for 10.1101/2021.08.05.455290: (What is this?)

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

    Table 1: Rigor

    EthicsField Sample Permit: Human SARS-CoV-2: nasopharyngeal swab specimens were collected as part of the routine SARS-CoV-2 surveillance conducted by the Mount Sinai Pathogen Surveillance program (IRB approved, HS#13-00981).
    Euthanasia Agents: Anesthetized hamsters were euthanized by intracardiac injection of sodium pentobarbital (Sleepaway - Zoetis) euthanasia solution.
    IACUC: All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Icahn School of Medicine at Mount Sinai (ISMMS)
    Sex as a biological variableHamster infections: Ten female golden Syrian hamster of approximately 4-weeks-old were placed in pairs in five different cages.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line AuthenticationContamination: , non-essential amino acids, penicillin (100 UI/mL), streptomycin (100 UI/mL) (Corning) and normocin (100 ug/mL) (InvivoGen) to prevent mycoplasma infection.

    Table 2: Resources

    Antibodies
    SentencesResources
    Anti-mouse secondary IgG-HRP antibody (abcam, 6823) was used at a dilution 1:5000 to detect SARS-CoV-2 Spike protein and anti-rabbit secondary IgG-HRP antibody (Kindle Biosciences, R1006) at 1:2000 to detect SARS-CoV-2 nucleocapsid.
    secondary IgG-HRP
    suggested: None
    anti-rabbit secondary IgG-HRP
    suggested: None
    After 1-hour, anti-mouse SARS-CoV-2-NP antibody (1C7C7, kindly provided by Dr. Moran) was added at a dilution of 1:1000 in 1% milk-TBST and incubated for 1 hour at RT.
    anti-mouse SARS-CoV-2-NP
    suggested: None
    Then, cells were washed two times in PBS and stained with goat anti-mouse secondary IgG-HRP antibody (abcam, 6823) at a dilution of 1:5000 in 1% milk-TBST and incubated for 1 hour at RT.
    anti-mouse secondary IgG-HRP
    suggested: None
    After 24 post-infection, cells were fixed with 10% methanol-free formaldehyde and incubated with primary antibodies against spike KL-S-3A7 (43) and nucleoprotein polyclonal anti-serum (44) diluted in 3% bovine serum albumin (BSA) for 1 hour at RT.
    anti-serum
    suggested: None
    Then, cells were washed and stained with secondary antibodies anti-mouse Alexa Fluor-488 (ThermoFisher; A21202) and anti-Rabbit Alexa Fluor 568 (ThermoFisher; A11011) in 5% BSA for 1h at RT.
    anti-mouse
    suggested: (Molecular Probes Cat# A-21202, RRID:AB_141607)
    anti-Rabbit
    suggested: (Molecular Probes Cat# A-11011, RRID:AB_143157)
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: VeroE6 and Caco-2 cell lines were originally purchased from the American Type Culture Collection (ATCC)
    Caco-2
    suggested: CLS Cat# 300137/p1665_CaCo-2, RRID:CVCL_0025)
    Specimens were selected for viral culture on Vero-E6 cells based on the complete viral genome sequence information (31).
    Vero-E6
    suggested: None
    Viral transport media was incubated with VeroE6 cells until cytopathic effect was observed and supernatants from infected cells were used for plaque purification of clonal population as previously described (42).
    VeroE6
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Briefly, ten-fold serial dilutions were performed in infection media for SARS-CoV-2 and inoculated onto confluent VeroE6 or VeroE6-TMPRSS2 cell monolayer.
    VeroE6-TMPRSS2
    suggested: JCRB Cat# JCRB1819, RRID:CVCL_YQ49)
    Software and Algorithms
    SentencesResources
    Primary antibodies against SARS-CoV-2 Spike S2 protein (Abcam; ab6823) and nucleocapsid (Novus Biologicals; NB100-56576) were purchased from the indicated suppliers and used at a dilution of 1:3000 and 1:2000 respectively.
    Novus Biologicals
    suggested: (Novus Biologicals, RRID:SCR_004286)
    Oxford Nanopore sequencing: The frequency of variants at position S:655 in the hamster samples was further confirmed with Oxford Nanopore (ONT) sequencing in a MinION Mk1C instrument.
    MinION
    suggested: (MinION, RRID:SCR_017985)
    Sequencing data acquisition was done with the ONT software MinKNOW v4.3.7.
    MinKNOW
    suggested: None
    The Genome assembly was done with the Artic pipeline (artic-network/artic-ncov2019) with default parameters, where reads were aligned to the reference genome Wuhan-Hu-1 (MN908947.3) using minimap2 (2.17-r941), consensus variants were called with Nanopolish (0.13.2).
    Nanopolish
    suggested: (Nanopolish, RRID:SCR_016157)

    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.

    About SciScore

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  2. Version published to 10.1101/2021.08.05.455290v1 on bioRxiv