Inhibition of major histocompatibility complex-I antigen presentation by sarbecovirus ORF7a proteins

  1. Fengwen Zhang
  2. Trinity M. Zang
  3. Eva M. Stevenson
  4. Xiao Lei
  5. Dennis C. Copertino
  6. Talia M. Mota
  7. Julie Boucau
  8. Wilfredo F. Garcia-Beltran
  9. R. Brad Jones
  10. Paul D. Bieniasz

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Abstract

Viruses employ a variety of strategies to escape or counteract immune responses, including depletion of cell surface major histocompatibility complex class I (MHC-I), that would ordinarily present viral peptides to CD8 + cytotoxic T cells. As part of a screen to elucidate biological activities associated with individual severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) viral proteins, we found that ORF7a reduced cell surface MHC-I levels by approximately fivefold. Nevertheless, in cells infected with SARS-CoV-2, surface MHC-I levels were reduced even in the absence of ORF7a, suggesting additional mechanisms of MHC-I down-regulation. ORF7a proteins from a sample of sarbecoviruses varied in their ability to induce MHC-I down-regulation and, unlike SARS-CoV-2, the ORF7a protein from SARS-CoV lacked MHC-I downregulating activity. A single amino acid at position 59 (T/F) that is variable among sarbecovirus ORF7a proteins governed the difference in MHC-I downregulating activity. SARS-CoV-2 ORF7a physically associated with the MHC-I heavy chain and inhibited the presentation of expressed antigen to CD8 + T cells. Specifically, ORF7a prevented the assembly of the MHC-I peptide loading complex and caused retention of MHC-I in the endoplasmic reticulum. The differential ability of ORF7a proteins to function in this way might affect sarbecovirus dissemination and persistence in human populations, particularly those with infection- or vaccine-elicited immunity.

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  1. Version published to 10.1073/pnas.2209042119
  2. ScreenIT

    SciScore for 10.1101/2022.05.25.493467: (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 AuthenticationContamination: All cell lines used in this study were monitored by SYBR® Green real-time PCR RT assay periodically to ensure the absence of retroviral contamination and were stained with DAPI to test for mycoplasma contamination.

    Table 2: Resources

    Antibodies
    SentencesResources
    Antibodies: Primary antibodies, including anti-ORF7a, anti-pan HLA ABC, HLA allele-specific antibodies, and peptide-loading complex antibodies, are listed and described in the Supplementary Information Table.
    Antibodies
    suggested: None
    anti-ORF7a,
    suggested: None
    Secondary antibodies included goat anti-rabbit and goat anti-mouse from Novus conjugated to DyLight 650 or Janelia Fluor 646 and goat anti-mouse from ThermoFisher Scientific conjugated to Alexa Flour 488 for immunofluorescence, or IRDye® 800CW, or IRDye® 680 (LI-COR Biosciences) for Western blot analysis.
    anti-rabbit
    suggested: (GenWay Biotech Inc. Cat# GWB-646B8F, RRID:AB_10260289)
    anti-mouse
    suggested: (Novus Cat# NBP1-72739JF646, RRID:AB_2857337)
    At 48 h post-transduction, cells were detached from plates with 5 mM EDTA in PBS and stained for cell surface HLA-A, B, C expression with anti-HLA-A, B, C antibody conjugated to AF647 (W6/32, Biolegend).
    anti-HLA-A
    suggested: None
    In some experiments, cell surface HLA-A level was determined with antibody against the HLA-A allele (YTH862.2), followed by Goat anti-Rat Alexa Fluro 488.
    anti-Rat
    suggested: None
    The same procedure was done to quantify tetherin downregulation, except that 293T cells stably expressing HA-tagged tetherin were used as target cells, and cells were stained with anti-HA antibody.
    anti-HA
    suggested: None
    After 48 h, cells were washed with PBS (Corning) and harvested using TrypLE (Life Technologies) before staining with Live/Dead Blue dead cell stain (Invitrogen) and a BV510-conjugated pan-HLA class I antibody (clone W6/32, BioLegend).
    pan-HLA class I
    suggested: None
    Intracellular staining was performed in Perm/Wash Buffer with an AF647-conjugated pan-HLA class I antibody (clone W6/32, BioLegend) and a SARS-CoV-2 nucleocapsid antibody (clone A20087H, mouse IgG2b isotype, BioLegend) followed by a secondary antibody stain with PE-Cy7-conjugated anti-mouse IgG2b (BioLegend).
    mouse IgG2b
    suggested: (Bioss Cat# bs-0330R-Cy7, RRID:AB_10894111)
    After lysis on ice for 20 min, followed by centrifugation at 10,000 rpm for 10 min at 4°C, clarified lysates were mixed with 2 μg mouse anti-ORF7a monoclonal antibody (Genetex), mouse anti-HLA-A (Proteintech 66013-1-Ig), or rabbit anti-HLA-B (ThermoFisher Scientific PA5-35345)
    anti-ORF7a
    suggested: (SICGEN Cat# AB0409, RRID:AB_2895426)
    anti-HLA-B
    suggested: (Thermo Fisher Scientific Cat# PA5-35345, RRID:AB_2552655)
    + 0.1 μg/mL each of anti-CD3 (Ultra-LEAF purified anti-human CD3 antibody clone OKT3; BioLegend, San Diego, CA) and anti-CD28 (
    anti-CD3
    suggested: None
    anti-human CD3
    suggested: (STEMCELL Technologies Cat# 10971, RRID:AB_2827806)
    anti-CD28
    suggested: None
    Ultra-LEAF purified Anti-human CD28 antibody clone 28.2; BioLegend), 37°C 5% CO2.
    Anti-human CD28
    suggested: (Fitzgerald Industries International Cat# 10R-CD28aHU, RRID:AB_1283221)
    Cells were then washed in 2% FBS phosphate-buffered saline + 2mM EDTA and surface stained with fluorochrome-conjugated antibodies to CD3-Brilliant Violet 785 clone OKT3, CD8-BV605 clone SK1, MHC-I-PacBlue clone W6/32, (all from BioLegend),
    CD8-BV605
    suggested: None
    Following 10 min permeabilization at room temperature with 0.2% IGEPAL CA-630 (Sigma I3021), 10% goat serum in PBS, cells were probed for 1 hr at RT with primary antibodies in 0.1% Tween20 (Fisher BP337-500), 10% goat serum in PBS as indicated: HLA-A (Sigma H1650-100STS, 0.4 μg/ml), beta-2 microglobulin (Novus NBP2-44471, 0.4 μg/ml) or ORF7a (Bioworld NCP011, 2 μg/ml).
    beta-2 microglobulin
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: The human embryonic kidney HEK-293T cell line, human hepatoma-derived HuH-7.5 cell line, and human alveolar basal epithelial A549 cell line were maintained in DMEM supplemented with 10% fetal bovine serum (Sigma F8067) and gentamicin (Gibco).
    HEK-293T
    suggested: None
    HuH-7.5
    suggested: RRID:CVCL_7927)
    The viral stocks were then used to inoculate105 target cells (293T, U2OS, or HuH7.5) in 12-well plates at an MOI of 0.5.
    293T
    suggested: None
    U2OS
    suggested: None
    Assessment of MHC-I downregulation after live SARS-CoV-2 infection: Viral stocks of USA-WA1/2020 (BEI Resources) and icSARS-CoV-2-mNG (UTMB WRCEVA (32)) were generated by expanding virus on Vero-E6 cells (BEI Resources) and determining viral titers by plaque assay on Vero-E6 cells.
    Vero-E6
    suggested: None
    A549-ACE2 cells (ATCC) were plated at 1 × 106 cells per well in a 6-well plate in DMEM (Corning) supplemented with HEPES (Corning), penicillin/streptomycin (Corning)
    A549-ACE2
    suggested: None
    To measure the sensitivity of MHC-I to Endo H treatment, A549 cells were transduced with lentivirus stocks expressing ORF7a proteins from SARS-CoV, SARS-CoV-2, or bat SARSr CoVs at an MOI of 0.5.
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    Recombinant DNA
    SentencesResources
    After digestion with XhoI and NotI, the purified PCR products were then inserted into the pSCRPSY expression vector.
    pSCRPSY
    suggested: None
    Assessment of MHC-I downregulation by ORF7a: To assess HLA downregulation by SARS-CoV viral proteins, viral stocks were generated by transfecting 5 μg of pSCRPSY-based expression plasmids encoding either no SARS-CoV viral protein (empty vector) or individual SARS-CoV open reading frame, 5 μg of an HIV-1 Gag-Pol expression plasmid (pCRV1/GagPol) and 1 μg of VSV-G expression plasmid in 293T cells in 10-cm dishes using polyethylenimine (PolySciences)
    pSCRPSY-based
    suggested: None
    pCRV1/GagPol
    suggested: None
    VSV-G
    suggested: RRID:Addgene_138479)
    Software and Algorithms
    SentencesResources
    Flow cytometry was performed on a BD Symphony (BD Biosciences, San Jose, CA) and analyses were performed using FlowJo v10.7.1 and GraphPad Prism 9 software. Immunoprecipitation: HEK-293T cells were seeded at 2 X105 in 6-well plates and, on the next day, were inoculated with ORF7a-expressing SCRPSY lentivirus stocks.
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Image analysis was done using ImageJ (Version 2.0.0-rc-59/1.51w).
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)

    Results from OddPub: Thank you for sharing your data.

    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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  3. Version published to 10.1101/2022.05.25.493467v1 on bioRxiv