Structure-Guided Mutagenesis Alters Deubiquitinating Activity and Attenuates Pathogenesis of a Murine Coronavirus

  1. Xufang Deng
  2. Yafang Chen
  3. Anna M. Mielech
  4. Matthew Hackbart
  5. Kristina R. Kesely
  6. Robert C. Mettelman
  7. Amornrat O’Brien
  8. Mackenzie E. Chapman
  9. Andrew D. Mesecar
  10. Susan C. Baker

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Abstract

Coronaviruses employ a genetic economy by encoding multifunctional proteins that function in viral replication and also modify the host environment to disarm the innate immune response. The coronavirus papain-like protease 2 (PLP2) domain possesses protease activity, which cleaves the viral replicase polyprotein, and also DUB activity (deconjugating ubiquitin/ubiquitin-like molecules from modified substrates) using identical catalytic residues. To separate the DUB activity from the protease activity, we employed a structure-guided mutagenesis approach and identified residues that are important for ubiquitin binding. We found that mutating the ubiquitin-binding residues results in a PLP2 that has reduced DUB activity but retains protease activity. We engineered a recombinant murine coronavirus to express the DUB mutant and showed that the DUB mutant virus activated an earlier type I interferon response in macrophages and exhibited reduced replication in mice. The results of this study demonstrate that PLP2/DUB is an interferon antagonist and a virulence trait of coronaviruses.

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    SciScore for 10.1101/782409: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementThe experimental protocol was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at Loyola University Chicago (IACUC#: 2016-029).Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variableC57BL/6 female mice were purchased from The Jackson Laboratory and maintained in the Comparative Medicine Facility of Loyola University Chicago.Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The membrane was incubated with either polyclonal rabbit anti-GFP antibody (A11122, Life Technologies) for the protease assay, or mouse anti-flag (F3165, Sigma) for the DUB assay.
    anti-GFP
    suggested: (Molecular Probes Cat# A-11122, AB_221569)
          <div style="margin-bottom:8px">
        <div><b>anti-flag ( F3165</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The membrane was then washed three times for 15 minutes in TBST buffer followed by incubation with either secondary donkey anti-rabbit-HRP antibody (711-035-152, Jackson ImmunoResearch) or goat anti-mouseHRP antibody (1010-05, SouthernBiotech).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-rabbit-HRP</b></div>
        <div>suggested: (Kindle Biosciences Cat# R1006, <a href="https://scicrunch.org/resources/Any/search?q=AB_2800464">AB_2800464</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-mouseHRP</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The expression of PLP2, β-actin, and calnexin were probed with mouse anti-V5 antibody (R960, ThermoFisher), mouse anti–β-actin (A00702, Genscript), or mouse anti-calnexin antibody (2433S, Cell Signaling), respectively.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>PLP2</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>β-actin</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-V5</b></div>
        <div>suggested: (Thermo Fisher Scientific Cat# R960CUS, <a href="https://scicrunch.org/resources/Any/search?q=AB_2792973">AB_2792973</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>mouse anti-calnexin antibody</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-calnexin</b></div>
        <div>suggested: (Cell Signaling Technology Cat# 2433, <a href="https://scicrunch.org/resources/Any/search?q=AB_2243887">AB_2243887</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">Cells Human embryonic kidney (HEK) 293T cells were purchased the from American Type Culture Collection (ATCC, # CRL-11268) and maintained in DMEM (#10-017-CV, Corning) containing 10% fetal calf serum (FCS) and supplemented with 1% nonessential amino acids, 1% HEPES, 2% L-glutamine, 1% sodium pyruvate, and 1% penicillin/streptomycin.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>293T</b></div>
        <div>suggested: ATCC Cat# CRL-11268, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_1926">CVCL_1926</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">For the protease activity assay, HEK293T cells were transfected with 25 ng nsp2/3-GFP plasmid and 200 ng pCAGGS-PLP2-V5 expression plasmids (wild-type and mutant).</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;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">Graphs of virus kinetics were generated using Prism software (GraphPad Software).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Prism</b></div>
        <div>suggested: (PRISM, <a href="https://scicrunch.org/resources/Any/search?q=SCR_005375">SCR_005375</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>GraphPad</b></div>
        <div>suggested: (GraphPad Prism, <a href="https://scicrunch.org/resources/Any/search?q=SCR_002798">SCR_002798</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">MH and RCM were supported by NIH T32 Training Grant for Experimental Immunology (#AI007508) and RCM was supported by the Arthur J.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Arthur J</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr></table>

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  2. Version published to 10.1128/jvi.01734-19
  3. Version published to 10.1101/782409v2 on bioRxiv
  4. Version published to 10.1101/782409v1 on bioRxiv