Kallikrein 13: a new player in coronaviral infections

  1. Aleksandra Milewska
  2. Katherine Falkowski
  3. Magdalena Kalinska
  4. Ewa Bielecka
  5. Antonina Naskalska
  6. Pawel Mak
  7. Adam Lesner
  8. Marek Ochman
  9. Maciej Urlik
  10. Jan Potempa
  11. Tomasz Kantyka
  12. Krzysztof Pyrc

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Abstract

Human coronavirus HKU1 (HCoV-HKU1) is associated with respiratory disease and is prevalent worldwide, but in vitro model for virus replication is lacking. Interaction between the coronaviral spike (S) protein and its receptor is the major determinant of virus tissue and host specificity, but virus entry is a complex process requiring a concerted action of multiple cellular elements. Here, we show that KLK13 is required for the infection of the human respiratory epithelium and is sufficient to mediate the entry of HCoV-HKU1 to non-permissive RD cells. We also demonstrated HCoV-HKU1 S protein cleavage by KLK13 in the S1/S2 region, proving that KLK13 is the priming enzyme for this virus. Summarizing, we show for the first time that protease distribution and specificity predetermines the tissue and cell specificity of the virus and may also regulate interspecies transmission. It is also of importance that presented data may be relevant for the emerging coronaviruses, including SARS-CoV-2 and may help to understand the differences in their zoonotic potential.

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    SciScore for 10.1101/2020.03.01.971499: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementThe study was approved by the Bioethical Committee of the Medical University of Silesia in Katowice , Poland ( approval no: KNW/0022/KB1/17/10 dated 16.02.2010) .Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variablenot detected.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Due to the lack of KLK13 specific antibodies , we verified its presence based on RT-PCR ( Fig . 4A) .
    KLK13
    suggested: None
    A mouse monoclonal anti-TMPRSS2 antibody ( clone P5H9-A3; 1:500 dilution; Sigma-Aldrich , Poland) , followed by incubation with a horseradish peroxidase-labeled anti-mouse IgG ( 65 ng/ml; Dako , Denmark ) diluted in 5 % skimmed milk / TBS-Tween ( 0.1 % ) .
    anti-TMPRSS2
    suggested: (Santa Cruz Biotechnology Cat# sc-101847, AB_2205599)
          <div style="margin-bottom:8px">
        <div><b>anti-mouse IgG</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">A horseradish peroxidase-labeled anti-His tag antibody ( 1:25000 dilution; Sigma-Aldrich , Poland ) diluted in 5 % skimmed milk / TBS-Tween ( 0.1 % ) was used to detect the His-tagged HmuY proteins .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-His tag antibody</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-His tag</b></div>
        <div>suggested: None</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">Subsequently , we transduced RD_ctrl , RD_KLK13 and RD_TMPRSS2 cells with HIV particles pseudotyped with HCoV-HKU1 S glycoprotein ( S-HKU1) , control VSV G protein ( VSV-G ) or lacking the fusion protein ( ΔEnv) .</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>RD_TMPRSS2</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">This may be one of the factors limiting the HCoV-HKU1 replication in RD_KLK13 cells , as only minimal replication is observable.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>RD_KLK13</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">RD cells grown in 90 % confluency were infected with HCoV-HKU1 ( 108 RNA copies per ml ) in Dulbecco’s MEM ( Thermo Fisher Scientific , Poland</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>RD</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Lentivirus production and transduction 293T cells were seeded on 10 cm2 dishes , cultured for 24 h at 37°C with 5 % CO2 and transfected with psPAX , pMD2G and third transfer plasmid ( pWPI/KLK13 , pLKO.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>293T</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr></table>

    Results from Barzooka: We also 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 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).

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  2. Version published to 10.1101/2020.03.01.971499v1 on bioRxiv