Multiple sites on SARS‐CoV ‐2 spike protein are susceptible to proteolysis by cathepsins B, K, L, S, and V

  1. Keval Bollavaram
  2. Tiffanie H. Leeman
  3. Maggie W. Lee
  4. Akhil Kulkarni
  5. Sophia G. Upshaw
  6. Jiabei Yang
  7. Hannah Song
  8. Manu O. Platt

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Abstract

SARS‐CoV‐2 is the coronavirus responsible for the COVID‐19 pandemic. Proteases are central to the infection process of SARS‐CoV‐2. Cleavage of the spike protein on the virus's capsid causes the conformational change that leads to membrane fusion and viral entry into the target cell. Since inhibition of one protease, even the dominant protease like TMPRSS2, may not be sufficient to block SARS‐CoV‐2 entry into cells, other proteases that may play an activating role and hydrolyze the spike protein must be identified. We identified amino acid sequences in all regions of spike protein, including the S1/S2 region critical for activation and viral entry, that are susceptible to cleavage by furin and cathepsins B, K, L, S, and V using PACMANS, a computational platform that identifies and ranks preferred sites of proteolytic cleavage on substrates, and verified with molecular docking analysis and immunoblotting to determine if binding of these proteases can occur on the spike protein that were identified as possible cleavage sites. Together, this study highlights cathepsins B, K, L, S, and V for consideration in SARS‐CoV‐2 infection and presents methodologies by which other proteases can be screened to determine a role in viral entry. This highlights additional proteases to be considered in COVID‐19 studies, particularly regarding exacerbated damage in inflammatory preconditions where these proteases are generally upregulated.

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  1. Version published to 10.1002/pro.4073
  2. ScreenIT

    SciScore for 10.1101/2021.02.17.431617: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    No key resources detected.

    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:
    Though we find this study to be useful, there are limitations to be considered. The results presented identify hypothetical protease cleavage sites on SARS-CoV-2-S, and spike protein hydrolysis by cathepsins K, S, and V was verified with recombinant in vitro studies. However, confirmation of the identity of these sequences to confirm they match with PACMANS predictions must be completed with mass spectrometry or site-directed mutagenesis for confirmation. The benefits of the computational approaches applied here are that a process has been presented of PACMANS followed by molecular docking to generate hypotheses for investigating other proteases, even beyond those included in this study. Upregulated plasmin, thrombin, and other serine proteases may be present at the site as well, implicated in the hypercoagulable state induced by SARS-CoV-2 infection48. Another example involves the D614G SARS-CoV-2 spike mutant that has been shown to be a more infectious virus with an enhanced susceptibility of spike protein to furin cleavage, while also altering the dynamics of the spike protein conformation49. This site was not identified in the unbiased approach used in the studies presented here, but if other mutations are identified, they can be input into PACMANS to determine their putative result on activating cleavage. Another study identified a cleavage site on spike protein from SARS-CoV, the virus that caused the SARS outbreak in 2009, at position T6788, but that site also was not ...

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 32. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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.

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  3. Version published to 10.1101/2021.02.17.431617v1 on bioRxiv