Hydroxychloroquine-mediated inhibition of SARS-CoV-2 entry is attenuated by TMPRSS2

  1. Tianling Ou
  2. Huihui Mou
  3. Lizhou Zhang
  4. Amrita Ojha
  5. Hyeryun Choe
  6. Michael Farzan

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Abstract

Hydroxychloroquine, used to treat malaria and some autoimmune disorders, potently inhibits viral infection of SARS coronavirus (SARS-CoV-1) and SARS-CoV-2 in cell-culture studies. However, human clinical trials of hydroxychloroquine failed to establish its usefulness as treatment for COVID-19. This compound is known to interfere with endosomal acidification necessary to the proteolytic activity of cathepsins. Following receptor binding and endocytosis, cathepsin L can cleave the SARS-CoV-1 and SARS-CoV-2 spike (S) proteins, thereby activating membrane fusion for cell entry. The plasma membrane-associated protease TMPRSS2 can similarly cleave these S proteins and activate viral entry at the cell surface. Here we show that the SARS-CoV-2 entry process is more dependent than that of SARS-CoV-1 on TMPRSS2 expression. This difference can be reversed when the furin-cleavage site of the SARS-CoV-2 S protein is ablated. We also show that hydroxychloroquine efficiently blocks viral entry mediated by cathepsin L, but not by TMPRSS2, and that a combination of hydroxychloroquine and a clinically-tested TMPRSS2 inhibitor prevents SARS-CoV-2 infection more potently than either drug alone. These studies identify functional differences between SARS-CoV-1 and -2 entry processes, and provide a mechanistic explanation for the limited in vivo utility of hydroxychloroquine as a treatment for COVID-19.

Author Summary

The novel pathogenic coronavirus SARS-CoV-2 causes COVID-19 and remains a threat to global public health. Chloroquine and hydroxychloroquine have been shown to prevent viral infection in cell-culture systems, but human clinical trials did not observe a significant improvement in COVID-19 patients treated with these compounds. Here we show that hydroxychloroquine interferes with only one of two somewhat redundant pathways by which the SARS-CoV-2 spike (S) protein is activated to mediate infection. The first pathway is dependent on the endosomal protease cathepsin L and sensitive to hydroxychloroquine, whereas the second pathway is dependent on TMPRSS2, which is unaffected by this compound. We further show that SARS-CoV-2 is more reliant than SARS coronavirus (SARS-CoV-1) on the TMPRSS2 pathway, and that this difference is due to a furin cleavage site present in the SARS-CoV-2 S protein. Finally, we show that combinations of hydroxychloroquine and a clinically tested TMPRSS2 inhibitor work together to effectively inhibit SARS-CoV-2 entry. Thus TMPRSS2 expression on physiologically relevant SARS-CoV-2 target cells may bypass the antiviral activities of hydroxychloroquine, and explain its lack of in vivo efficacy.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2020.07.22.216150: (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
    hACE2 expression was confirmed by immunofluorescence staining using mouse monoclonal antibody against c-Myc antibody 9E10 (Thermo Fisher) and Goat-anti-mouse FITC (Jackson ImmunoResearch Laboratories, Inc).
    c-Myc
    suggested: None
    Goat-anti-mouse FITC
    suggested: None
    To measure surface TMPRSS2 expression, cells were detached by 1mM EDTA in PBS and then stained by 4 ug/ml of anti-Flag M2 antibody (Sigma, F1804) and 2 ug/ml of Goat anti-mouse IgG (H+L) conjugated with Alexa 647 (Jackson ImmunoResearch Laboratories, Inc, Cat#
    anti-Flag
    suggested: (Sigma-Aldrich Cat# F1804, RRID:AB_262044)
    anti-mouse IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Briefly, HEK293T cells were co-transfected with three plasmids, pMLV-gag-pol, pCAGGS-VSV-G and pQCXIP-myc-hACE2-c9, and the medium was refreshed after overnight incubation of transfection mix.
    HEK293T
    suggested: None
    The parental 293T cells were transduced with generated MLV virus, and the 293T-hACE2 cell lines were selected and maintained with medium containing puromycin (Sigma).
    293T-hACE2
    suggested: None
    HEK293T/ACE2/TMPRSS2 stable cell line was also constructed by transducing 293T-hACE2 cell line with MLV pseudovirus made by cotransfection of pMLV-gag-pol, pQCXIB-TMPRSS2-Flag and pCAGGS-VSV-G at 3:2:1 ratio into 293T cells.
    HEK293T/ACE2/TMPRSS2
    suggested: None
    293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    For the 293T-ACE2 stable cell line, 3 μg/mL of puromycin was added to the growth medium to maintain expression of ACE2.
    293T-ACE2
    suggested: RRID:CVCL_YZ65)
    Pseudovirus infection: HEK293T-ACE2 cells were seeded at 30% density in poly-lysine pre-coated 96-well plates 12-15 hours prior to transfection.
    HEK293T-ACE2
    suggested: None
    Software and Algorithms
    SentencesResources
    Statistical analysis: Data expressed as mean values ± S.D. or S.E.M, and all statistical analysis was performed in GraphPad Prism 7.0 software.
    GraphPad Prism
    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: We found the following clinical trial numbers in your paper:

    IdentifierStatusTitle
    NCT04338906WithdrawnCombination Therapy With Camostat Mesilate + Hydroxychloroqu…

    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 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.
    • No funding statement was detected.
    • 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. ScreenIT

    SciScore for 10.1101/2020.07.22.216150: (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
    hACE2 expression was confirmed by immunofluorescence staining using mouse monoclonal antibody against c-Myc antibody 9E10 (Thermo Fisher) and Goat-anti-mouse FITC (Jackson ImmunoResearch Laboratories, Inc).
    c-Myc
    suggested: None
          <div style="margin-bottom:8px">
        <div><b>Goat-anti-mouse FITC</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">To measure surface TMPRSS2 expression, cells were detached by 1mM EDTA in PBS and then stained by 4 ug/ml of anti-Flag M2 antibody (Sigma, F1804) and 2 ug/ml of Goat anti-mouse IgG (H+L) conjugated with Alexa 647 (Jackson ImmunoResearch Laboratories, Inc, Cat# 115-606-146).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-Flag</b></div>
        <div>suggested: (Sigma-Aldrich Cat# F1804, <a href="https://scicrunch.org/resources/Any/search?q=AB_262044">AB_262044</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-mouse IgG</b></div>
        <div>suggested: (Jackson ImmunoResearch Labs Cat# 115-606-146, <a href="https://scicrunch.org/resources/Any/search?q=AB_2338930">AB_2338930</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">To demonstrate the effect of different amount of TMPRSS2 on antiviral activities of hydroxychloroquine, both transient (high) and stable (low) TMPRSS2 HEK293T-ACE2 cells were used as targets.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK293T-ACE2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For HEK293TACE2 cells transiently expressing the control plasmids (Fig 3A)</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK293TACE2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">These IC50 values are consistent with a previous cell culture study, using replicative SARS-CoV-2 on Vero cells, which do not express TMPRSS2 [10].</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero</b></div>
        <div>suggested: CLS Cat# 605372/p622_VERO, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0059">CVCL_0059</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Thus the potent inhibition of SARS-CoV-2 by hydroxychloroquine in Vero E6 cells, where TMPRSS2 is largely absent, overestimated its potency by 10- to 40-fold, depending on TMPRSS2 expression (Fig 3A).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Vero E6</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Briefly, HEK293T cells were cotransfected with three plasmids, pMLV-gag-pol, pCAGGS-VSV-G and pQCXIP-myc-hACE2-c9, and the medium was refreshed after overnight incubation of transfection mix.</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">The parental 293T cells were transduced with generated MLV virus, and the 293T-hACE2 cell lines were selected and maintained with medium containing puromycin (Sigma).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293T-hACE2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">HEK293T/ACE2/TMPRSS2 stable cell line was also constructed by transducing 293T-hACE2 cell line with MLV pseudovirus made by cotransfection of pMLV-gag-pol, pQCXIB-TMPRSS2-Flag and pCAGGS-VSV-G at 3:2:1 ratio into 293T cells.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK293T/ACE2/TMPRSS2</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>293T</b></div>
        <div>suggested: KCB Cat# KCB 200744YJ, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0063">CVCL_0063</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the 293T-ACE2 stable cell line, 3 μg/mL of puromycin was added to the growth medium to maintain expression of ACE2.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293T-ACE2</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">Statistical analysis Data expressed as mean values ± S.D. or S.E.M, and all statistical analysis was performed in GraphPad Prism 7.0 software.</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>
    </td></tr></table>

    Data from additional tools added to each annotation on a weekly basis.

    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.

  3. Version published to 10.1101/2020.07.22.216150 on bioRxiv