SARS-CoV-2 mutations affect proteasome processing to alter CD8 + T cell responses
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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.
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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
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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 dehydrogenasesuggested: NoneGAPDHsuggested: (Millipore Cat# MAB374, RRID:AB_2107445)anti-SARS-CoV-2 nucleocapsid antibody ( 2µg/µlsuggested: NonePrimary antibodies were probed by IRDye 680LT goat anti-mouse (Li-Cor, … 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
Ethics not detected. Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources 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 dehydrogenasesuggested: NoneGAPDHsuggested: (Millipore Cat# MAB374, RRID:AB_2107445)anti-SARS-CoV-2 nucleocapsid antibody ( 2µg/µlsuggested: NonePrimary 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-mousesuggested: (LI-COR Biosciences Cat# 926-68020, RRID:AB_10706161)anti-rabbitsuggested: NoneExperimental Models: Cell Lines Sentences Resources Lentivirus was produced using HEK293T cells, expression constructs were co-transfected with psPAX2 and pMD2. HEK293Tsuggested: RRID:CVCL_HA71)Recombinant DNA Sentences Resources Lentivirus was produced using HEK293T cells, expression constructs were co-transfected with psPAX2 and pMD2. psPAX2suggested: RRID:Addgene_12260)pMD2suggested: NoneSoftware and Algorithms Sentences Resources G using PEIPro PEIProsuggested: NoneData was analysed using FlowJo v10 (BD Biosciences). FlowJosuggested: (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. Fijisuggested: (Fiji, RRID:SCR_002285)Cells were fixed with BD CellFix and run on an Attune NXT Flow Cytometer (Thermofisher). BD CellFixsuggested: NoneData was plotted and analysed in GraphPad Prism v9.2.0. GraphPad Prismsuggested: (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. Skylinesuggested: (Skyline, RRID:SCR_014080)Chromatograms were generated using R software package v4.0.1 and ggplot2 v3.3.2 and ggridges v0.5.2. ggplot2suggested: (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. PRIDEsuggested: (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.
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- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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