SARS-CoV-2 mutations affect proteasome processing to alter CD8 + T cell responses

  1. Dannielle Wellington
  2. Zixi Yin
  3. Zhanru Yu
  4. Raphael Heilig
  5. Simon Davis
  6. Roman Fischer
  7. Suet Ling Felce
  8. Philip Hublitz
  9. Ryan Beveridge
  10. Danning Dong
  11. Guihai Liu
  12. Xuan Yao
  13. Yanchun Peng
  14. Benedikt M Kessler
  15. Tao Dong

Reviewed by

Read the full article

Listed in

Log in to save this article

Abstract

Viral CD8 + epitopes are generated by the cellular turnover of viral proteins, predominantly by the proteasome. Mutations located within viral epitopes can result in escape from memory T cells but the contribution of mutations in flanking regions of epitopes in SARS-CoV-2 has not been investigated. Focusing on two of the most dominant SARS-CoV-2 nucleoprotein CD8 + epitopes, we identified mutations in epitope flanking regions and investigated the contribution of these mutations to antigen processing and T cell activation using SARS-CoV-2 nucleoprotein transduced B cell lines and in vitro proteasomal processing of peptides. We found that decreased NP 9-17 -B*27:05 CD8 + T cell responses to the NP-Q7K mutation correlated with lower epitope surface expression, likely due to a lack of efficient epitope production by the proteasome, suggesting immune escape caused by this mutation. In contrast, NP-P6L and NP-D103N/Y mutations flanking the NP 9-17 -B*27:05 and NP 105-113 -B*07:02 epitopes, respectively, increased CD8 + T cell responses associated with enhanced epitope production by the proteasome. Our results provide evidence that SARS-CoV-2 mutations outside the epitope could have a significant impact on antigen processing and presentation, thereby contributing to escape from immunodominant T cell responses. Alternatively, mutations could enhance antigen processing and efficacy of T cell recognition, opening new avenues for improving future vaccine designs.

One Sentence Summary

Natural mutations in the flanking regions of known immunodominant SARS-CoV-2 nucleoprotein epitopes can decrease CD8 + T cell responses leading to partial escape.

Article activity feed

  1. ScreenIT

    SciScore for 10.1101/2022.04.08.487623: (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
    Anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody clone 6C5 (Merck Millipore, MAB374) was used as control antibody; anti-SARS-CoV-2 nucleocapsid antibody (2µg/µl, Sino Biological, 40143-R001) was used to probe NP expression.
    Anti-glyceraldehyde 3-phosphate dehydrogenase
    suggested: None
    GAPDH
    suggested: (Millipore Cat# MAB374, RRID:AB_2107445)
    anti-SARS-CoV-2 nucleocapsid antibody ( 2µg/µl
    suggested: None
    Primary antibodies were probed by IRDye 680LT goat anti-mouse (Li-Cor, 926-68020) and IRDye 800LT goat anti-rabbit (Li-Cor, 925-32210), and visualized using the iBright FL1000 (Invitrogen).
    anti-mouse
    suggested: (LI-COR Biosciences Cat# 926-68020, RRID:AB_10706161)
    anti-rabbit
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Lentivirus was produced using HEK293T cells, expression constructs were co-transfected with psPAX2 and pMD2.
    HEK293T
    suggested: RRID:CVCL_HA71)
    Recombinant DNA
    SentencesResources
    Lentivirus was produced using HEK293T cells, expression constructs were co-transfected with psPAX2 and pMD2.
    psPAX2
    suggested: RRID:Addgene_12260)
    pMD2
    suggested: None
    Software and Algorithms
    SentencesResources
    G using PEIPro
    PEIPro
    suggested: None
    Data was analysed using FlowJo v10 (BD Biosciences).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    Images from Western blot experiments were analysed by Fiji software for band density and expressed in GraphPad Prism as a percentage of the GAPDH expression.
    Fiji
    suggested: (Fiji, RRID:SCR_002285)
    Cells were fixed with BD CellFix and run on an Attune NXT Flow Cytometer (Thermofisher).
    BD CellFix
    suggested: None
    Data was plotted and analysed in GraphPad Prism v9.2.0.
    GraphPad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Peptide abundance was measured as accumulated ion counts after extraction of the chromatographic peak profiles of the precursor masses using Skyline version 21 and Bruker Compass DataAnalysis Version 5.3.
    Skyline
    suggested: (Skyline, RRID:SCR_014080)
    Chromatograms were generated using R software package v4.0.1 and ggplot2 v3.3.2 and ggridges v0.5.2.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (34) partner repository with the dataset identifier PXD032054.
    PRIDE
    suggested: (Pride-asap, RRID:SCR_012052)

    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.
    • No funding statement was detected.
    • No protocol registration statement was detected.

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

    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.

  2. Version published to 10.1101/2022.04.08.487623v1 on bioRxiv