The ORF8 protein of SARS-CoV-2 mediates immune evasion through down-regulating MHC-Ι

  1. Yiwen Zhang
  2. Yingshi Chen
  3. Yuzhuang Li
  4. Feng Huang
  5. Baohong Luo
  6. Yaochang Yuan
  7. Baijin Xia
  8. Xiancai Ma
  9. Tao Yang
  10. Fei Yu
  11. Jun Liu
  12. Bingfeng Liu
  13. Zheng Song
  14. Jingliang Chen
  15. Shumei Yan
  16. Liyang Wu
  17. Ting Pan
  18. Xu Zhang
  19. Rong Li
  20. Wenjing Huang
  21. Xin He
  22. Fei Xiao
  23. Junsong Zhang
  24. Hui Zhang

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Abstract

We report that SARS-CoV-2 utilizes its ORF8 protein as a unique mechanism to alter the expression of surface MHC-Ι expression to evade immune surveillance. Our study is significant for providing an understanding of the pathogenesis of SARS-CoV-2 and will provide additional perspective to the intensive ongoing investigation into the mechanism and function of T cell antiviral immunity in COVID-19.

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  1. Version published to 10.1073/pnas.2024202118
  2. ScreenIT

    SciScore for 10.1101/2020.05.24.111823: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIRB: Ethics statement and patient cohort: This research was approved by the Ethics Review Board of The Fifth Affiliated Hospital of Sun Yat-sen University and Sun Yat-Sen University.
    Consent: The given written informed consent with approval of the Ethics Committees were accomplished before the study.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variableInfection with authentic SARS-CoV-2: A SARS-CoV-2 strain named as hCoV-19/CHN/SYSU-IHV/2020 strain (Accession ID on GISAID: EPI_ISL_444969) was recently isolated by our lab from a female who was infected at Guangzhou by an Africa-traveler in April 2020 (manuscript submitted to Antimicrobial Agents and Chemotherapy).
    Cell Line AuthenticationAuthentication: Theses cell lines conducted authentication through short tandem repeat profiling, karyotyping and cytochrome c oxidase I testing.
    Contamination: Test for bacterial and fungal contamination was carried out by using current United States Pharmacopeia methods for viral testing adhering to the United States Code of Federal Regulation (9 CFR 113.53) guidelines, while mycoplasma testing was carried out by direct culture and Hoechst DNA staining and Limulus amoebocyte lysate assay to measure endotoxin values.

    Table 2: Resources

    Antibodies
    SentencesResources
    Primary antibodies used in IF assay include anti-GM130 (CST), anti-Calnexin (Proteintech), anti-Rab5 (CST), anti-HA (MBL), anti-Lamp1 (CST), and anti-HLA-A2 (abcam).
    anti-GM130
    suggested: None
    anti-Calnexin ( Proteintech)
    suggested: None
    anti-Rab5
    suggested: None
    anti-HA
    suggested: None
    anti-Lamp1
    suggested: None
    anti-HLA-A2
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: HEK293T, Huh7 and Vero E6 cell lines was obtained from ATCC.
    Huh7
    suggested: CLS Cat# 300156/p7178_HuH7, RRID:CVCL_0336)
    Vero E6
    suggested: RRID:CVCL_XD71)
    Lysosome isolation: For lysosome isolation experiments, HEK293T cells were transfected with indicated plasmids.
    HEK293T
    suggested: NCBI_Iran Cat# C498, RRID:CVCL_0063)
    Software and Algorithms
    SentencesResources
    The sequence alignment of complete genome sequences was performed using MAFFT software with default parameters44.
    MAFFT
    suggested: (MAFFT, RRID:SCR_011811)
    The protein alignments were created by Clustal Omega software using default parameters conducted in MEGA X45.
    Clustal Omega
    suggested: (Clustal Omega, RRID:SCR_001591)
    MEGA
    suggested: (Mega BLAST, RRID:SCR_011920)
    The pairwise sequence identities were calculated using BioEdit software.
    BioEdit
    suggested: (BioEdit, RRID:SCR_007361)
    The similarity analysis based on the genome sequence was performed using SimPlot software46.
    SimPlot
    suggested: None
    Plasmids: The DNA sequences of SARS-CoV-2 structural proteins and ORFs tagged with HA were chemically-synthesized in GENEWIZ (Suzhou, China) and inserted into pcDNA3.1 vector.
    GENEWIZ
    suggested: (GENEWIZ, RRID:SCR_003177)
    Samples were scanned with Zeiss LSM880 confocal microscopy and analyzed with Imaris.
    Imaris
    suggested: (Imaris, RRID:SCR_007370)
    The proteins were then visualized with ProteoSilver Plus Silver Stain Kit (Sigma Aldrich) according to the manufacturer’s instructions.
    ProteoSilver Plus
    suggested: None
    Gene Ontology Consortium) using DAVID Bioinformatics Resources, observing correlation between two replicate experiments.
    DAVID
    suggested: (DAVID, RRID:SCR_001881)
    Spots were then counted using an S6 ultra immunoscan reader (Cellular Technology Ltd.), and the number of IFN-γ positive T cells was calculated by ImmunoSpot 5.1.34 software (Cellular Technology Ltd.).
    ImmunoSpot
    suggested: (ImmunoSpot Software for Analyzing ELISPOT Assays, RRID:SCR_011082)
    Statistical significance performed using GraphPad Prism 6.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Flow cytometry results were analyzed using FlowJo software (Tree Star Inc.). P < 0.05 indicates a statistically significance difference. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on pages 40 and 39. 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.

    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.

  3. ScreenIT

    SciScore for 10.1101/2020.05.24.111823: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementMaterial and Methods Ethics statement and patient cohort This research was approved by the Ethics Review Board of The Fifth Affiliated Hospital of Sun Yat-sen University and Sun Yat-Sen University.Randomizationnot detected.Blindingnot detected.Power Analysisnot detected.Sex as a biological variableInfection with authentic SARS-CoV-2 A SARS-CoV-2 strain named as hCoV-19/CHN/SYSU-IHV/2020 strain (Accession ID on GISAID: EPI_ISL_444969) was recently isolated by our lab from a female who was infected at Guangzhou by an Africa-traveler in April 2020 (manuscript submitted to Antimicrobial Agents and Chemotherapy).Cell Line AuthenticationTheses cell lines conducted authentication through short tandem repeat profiling, karyotyping and cytochrome c oxidase I testing.

    Table 2: Resources

    Antibodies
    SentencesResources
    The following antibodies were used: anti-HLA-A2 (BB7.2), anti-HLA-A,B,C (W6/32), anti-human β2-microglobulin (2M2), and anti-CD8a (53-6.7).
    anti-HLA-A2
    suggested: (Miltenyi Biotec Cat# 130-099-536, AB_2652042)
          <div style="margin-bottom:8px">
        <div><b>anti-HLA-A</b></div>
        <div>suggested: (GenWay Biotech Inc. Cat# 20-783-72736-0.2 mg, <a href="https://scicrunch.org/resources/Any/search?q=AB_1981519">AB_1981519</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-human β2-microglobulin</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-CD8a</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Primary antibodies used in IF assay include anti-GM130 (CST), anti-Calnexin (Proteintech), anti-Rab5 (CST), anti-HA (MBL), anti-Lamp1 (CST), and anti-HLA-A2 (abcam).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>anti-GM130</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-Calnexin</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-Rab5</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-HA</b></div>
        <div>suggested: None</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>anti-Lamp1</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">Furthermore, the MHC- I molecules on various cell lines including human fetal colon cell line FHC, human bronchial epithelial cell line HBE, and human liver cell line Huh7 were also significantly downregulated by ORF8 (Fig.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Huh7</b></div>
        <div>suggested: CLS Cat# 300156/p7178_HuH7, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0336">CVCL_0336</a></div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Importantly, we also found that the expression of MHC-1 on the infected 293T cells was significantly decreased (Fig. 1I).</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><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Cell lines HEK293T, Huh7 and Vero E6 cell lines was obtained from ATCC.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>HEK293T</b></div>
        <div>suggested: KCB Cat# KCB 200744YJ, <a href="https://scicrunch.org/resources/Any/search?q=CVCL_0063">CVCL_0063</a></div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>Vero E6</b></div>
        <div>suggested: <a href="https://scicrunch.org/resources/Any/search?q=CVCL_XD71">CVCL_XD71</a></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">The sequence alignment of complete genome sequences was performed using MAFFT software with default parameters 44.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>MAFFT</b></div>
        <div>suggested: (MAFFT, <a href="https://scicrunch.org/resources/Any/search?q=SCR_011811">SCR_011811</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The protein alignments were created by Clustal Omega software using default parameters conducted in MEGA X45.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Clustal Omega</b></div>
        <div>suggested: (Clustal Omega, <a href="https://scicrunch.org/resources/Any/search?q=SCR_001591">SCR_001591</a>)</div>
      </div>
    
      <div style="margin-bottom:8px">
        <div><b>MEGA</b></div>
        <div>suggested: (Mega BLAST, <a href="https://scicrunch.org/resources/Any/search?q=SCR_011920">SCR_011920</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The pairwise sequence identities were calculated using BioEdit software.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>BioEdit</b></div>
        <div>suggested: (BioEdit, <a href="https://scicrunch.org/resources/Any/search?q=SCR_007361">SCR_007361</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The similarity analysis based on the genome sequence was performed using SimPlot software46.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>SimPlot</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Plasmids The DNA sequences of SARS-CoV-2 structural proteins and ORFs tagged with HA were chemically-synthesized in GENEWIZ (Suzhou, China) and inserted into pcDNA3.1 vector.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>GENEWIZ</b></div>
        <div>suggested: (GENEWIZ, <a href="https://scicrunch.org/resources/Any/search?q=SCR_003177">SCR_003177</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Samples were scanned with Zeiss LSM880 confocal microscopy and analyzed with Imaris.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>Imaris</b></div>
        <div>suggested: (Imaris, <a href="https://scicrunch.org/resources/Any/search?q=SCR_007370">SCR_007370</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">The proteins were then visualized with ProteoSilver Plus Silver Stain Kit (Sigma Aldrich) according to the manufacturer’s instructions.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>ProteoSilver Plus</b></div>
        <div>suggested: None</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Functional pathways representative of each gene signature was analyzed for enrichment in gene categories from the Gene Ontology Biological Processes (GO-BP) database (Gene Ontology Consortium) using DAVID Bioinformatics Resources, observing correlation between two replicate experiments.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>DAVID</b></div>
        <div>suggested: (DAVID, <a href="https://scicrunch.org/resources/Any/search?q=SCR_001881">SCR_001881</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Spots were then counted using an S6 ultra immunoscan reader (Cellular Technology Ltd.), and the number of IFN-γ positive T cells was calculated by ImmunoSpot 5.1.34 software (Cellular Technology Ltd.).</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>ImmunoSpot</b></div>
        <div>suggested: (ImmunoSpot Software for Analyzing ELISPOT Assays, <a href="https://scicrunch.org/resources/Any/search?q=SCR_011082">SCR_011082</a>)</div>
      </div>
    </td></tr><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Statistical significance performed using GraphPad Prism 6.</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><tr><td style="min-width:100px;vertical-align:top;border-bottom:1px solid lightgray">Flow cytometry results were analyzed using FlowJo software (Tree Star Inc.). P < 0.05 indicates a statistically significance difference. * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001.</td><td style="min-width:100px;border-bottom:1px solid lightgray">
      <div style="margin-bottom:8px">
        <div><b>FlowJo</b></div>
        <div>suggested: (FlowJo, <a href="https://scicrunch.org/resources/Any/search?q=SCR_008520">SCR_008520</a>)</div>
      </div>
    </td></tr></table>

    Results from OddPub: Thank you for sharing your data.

    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.

  4. Version published to 10.1101/2020.05.24.111823 on bioRxiv