The SARS-CoV-2 accessory factor ORF7a downregulates MHC class I surface expression

  1. Shuxuan Zheng
  2. Hendrik de Buhr
  3. Patrique Praest
  4. Anouk Evers
  5. Ingrid Brak-Boer
  6. Mariëlle van Grinsven
  7. Ylenia Longo
  8. Liset de Vries
  9. Wilco Nijenhuis
  10. Lukas C. Kapitein
  11. Jeffrey M. Beekman
  12. Monique Nijhuis
  13. Ingo Drexler
  14. Emmanuel J. H. J. Wiertz
  15. Robert Jan Lebbink

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Abstract

The pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in over 500 million infections and more than six million deaths worldwide. Although the viral genomes of SARS-CoV-1 and SARS-CoV-2 share high sequence homology, the clinical and pathological features of COVID-19 differ profoundly from those of SARS. It is apparent that changes in viral genes contribute to the increased transmissibility of SARS-CoV-2 and pathology of COVID-19.

Cytotoxic T lymphocytes play a key role in the elimination of virus-infected cells, mediated by recognition of virus-derived peptides that are presented on MHC class I molecules. Here, we show that SARS-CoV-2 can interfere with antigen presentation thereby evading immune surveillance. SARS-CoV-2 infection of monkey and human cell lines resulted in reduced cell-surface expression of MHC class I molecules. We identified a single viral gene product, the accessory factor open reading frame 7a (ORF7a), that mediates this effect. ORF7a interacts with HLA class I molecules in the ER, resulting in ER retention or impaired HLA heavy chain (HC) trafficking to the Golgi. Ultimately, these actions result in reduced HLA class I surface expression on infected cells. Whereas ORF7a from SARS-CoV-2 reduces surface HLA class I levels, the homologous ORF7a from the 2002 pandemic SARS-CoV-1 did not, suggesting that SARS-CoV-2 ORF7a acquired the ability to downregulate HLA-I during evolution of the virus. We identified a single amino acid in the SARS-CoV-1 ORF7a luminal domain that, upon mutating to the corresponding SARS-CoV-2 ORF7a sequence, induced a gain-of-function in HLA surface downregulation. By abrogating HLA class I antigen presentation via ORF7a, SARS-CoV-2 may evade host immune responses by inhibiting anti-viral cytotoxic T cell activity, thereby contributing to the pathology of COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2022.05.29.493850: (What is this?)

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

    Table 1: Rigor

    Ethicsnot detected.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The antibodies used were: PE-conjugated W6/32 (Serotec MCA81PE, 1:10) [11], anti-CD9-FITC (BD Pharmingen 555371, 1:40), anti-CD46-FITC (BD Pharmingen 555949, 1:20), anti-CD49b-PE (BD Pharmingen 555669, 1:20), anti-CD58-PE (BD Pharmingen 555921, 1:30), anti-CDw119-PE (BD Pharmingen 558934, 1:20).
    anti-CD9-FITC (BD Pharmingen 555371
    suggested: None
    The primary antibody used for Spike staining was human anti-SARS-CoV-2 Spike (1:682, REGN #10987), which was kindly provided by Wentao Li and Berend Jan Bosch (Utrecht University, Utrecht, The Netherlands).
    anti-SARS-CoV-2
    suggested: None
    The goat anti-human IgM+ IgG (H+L) (1:160, Jackson #109-116-127) antibody was used as secondary antibody Inhibition of proteasome and p97: For proteasome inhibition, we employed 20 μM MG132 (Sigma-Aldrich, Zwijndrecht, NL, C2211-5MG) and for p97 inhibition 4 μM CB-5083 (HY-12861; MCE) for 4h each.
    The goat anti-human IgM+ IgG
    suggested: None
    anti-human IgM+ IgG
    suggested: None
    The primary antibodies used for immunoblotting were: HC10, monoclonal mouse anti-SARS-CoV-2 ORF7a (Genetex, 632602, 1:1000), polyclonal rabbit anti-SARS-CoV-2 ORF8a (Genetex, 135591, 1:1000), monoclonal transferrin receptor antibody (H68.4, Invitrogen, 1:1000) and monoclonal StrepII (C23.21, purified in our lab).
    HC10
    suggested: (Hidde L. Ploegh Cat# HC10, RRID:AB_2728622)
    anti-SARS-CoV-2 ORF7a (Genetex, 632602
    suggested: None
    anti-SARS-CoV-2 ORF8a (Genetex, 135591
    suggested: None
    Secondary antibodies used were goat anti-mouse IgG-HRP (115-035-174, Jackson ImmunoResearch Europe Ltd, 1:10000) and mouse anti-rabbit IgG-HRP (211-032-171, Jackson ImmunoResearch Europe Ltd, 1:10000).
    anti-mouse IgG-HRP
    suggested: (Jackson ImmunoResearch Labs Cat# 115-035-174, RRID:AB_2338512)
    anti-rabbit IgG-HRP
    suggested: None
    For StrepII IP 25 μl Streptactin Sepharose® High Performance beads (GE Healthcare, GE28-9355-99) and for HLA-I IP 25 μl Protein G Sepharose® 4 Fast Flow (GE Healthcare, GE17-0618-01) were employed with the HC10 antibody for HLA-IP overnight at 4°C.
    GE28-9355-99
    suggested: None
    The following antibodies were used: mouse anti-SARS-CoV-2 ORF7a (Genetex, 632602, 1:1000) and mouse anti-W6/32 (own production from hybridoma, 1:1000).
    anti-W6/32
    suggested: None
    Secondary antibodies used were goat anti-mouse IgG2a cross-adsorbed secondary antibody, Alexa Fluor 594 (Thermo Fisher, A-21135, 1:600) and goat anti-mouse IgG1 cross-adsorbed secondary antibody, Alexa Fluor 647 (Thermo Fisher, A-21240, 1:600), together with DAPI (Sigma-Aldrich, 1:1000).
    anti-mouse IgG2a
    suggested: (Thermo Fisher Scientific Cat# A-21135, RRID:AB_2535774)
    anti-mouse IgG1
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and viruses: HEK-293T and HK-1 cells were maintained in Roswell Park Memorial Institute medium (RPMI 1640; Life Technologies) supplemented with 5% FCS (Sigma), 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin.
    HEK-293T
    suggested: None
    HK-1
    suggested: RRID:CVCL_7047)
    The MelJuSo, Huh7 and A549-ACE2-TMPRSS2 cells were maintained in Dulbecco’s modified Eagle medium (DMEM; Life Technologies) supplemented with 5% FCS (Sigma), 2 mM L-glutamine, 100 U/mL penicillin, and 100 mg/mL streptomycin.
    Huh7
    suggested: None
    All viruses were propagated and titrated on Vero E6 cells using the tissue culture infective dose 50 (TCID50) endpoint dilution method.
    Vero E6
    suggested: None
    SARS-CoV-2 infections: SARS-CoV-2 viruses (see section ‘cell lines and viruses’) propagated in Vero E6 cells were used to infect Vero E6 or A549-ACE2-TMPRSS2 cells.
    A549-ACE2-TMPRSS2
    suggested: None
    Recombinant DNA
    SentencesResources
    Plasmids: The plasmids of the SARS-CoV-2 cDNA library as cloned in the pLVX-EF1alpha-IRES-Puro (Takara/Clontech) vector were a kind gift from Prof. Nevan Krogan (University of California San Francisco, USA) [10].
    pLVX-EF1alpha-IRES-Puro
    suggested: None
    Cell lines expressing NSP1 and NSP14 did not survive transduction and subsequent antibiotic selection and were, therefore, excluded from the analysis For follow-up studies, we cloned a T2A-mAmetrine cassette in frame downstream of the PuroR gene in the pLV-CMV-IRES-PuroR vector.
    pLV-CMV-IRES-PuroR
    suggested: None
    For the pLV-CMV-IRES-PuroR-T2A-mAmetrine vectors, lentiviruses were produced using standard lentiviral production protocols with third-generation packaging vectors.
    pLV-CMV-IRES-PuroR-T2A-mAmetrine
    suggested: None
    Software and Algorithms
    SentencesResources
    Cells were subjected to flow cytometry (BD FACS Canto II) and the data was analyzed with FlowJo (BD Biosciences) software.
    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: 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.
    • 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.

    Results from scite Reference Check: We found no unreliable references.

    About SciScore

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  2. Version published to 10.1101/2022.05.29.493850v1 on bioRxiv