Sequential ER stress and inflammatory responses are induced by SARS-CoV-2 ORF3 through ERphagy

  1. Xiaolin Zhang
  2. Ziwei Yang
  3. Xubing Long
  4. Qinqin Sun
  5. Fan Wang
  6. Pei-hui Wang
  7. Xiaojuan Li
  8. Ersheng Kuang

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Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections have resulted in a number of severe cases of COVID-19 and deaths worldwide. However, knowledge of SARS-CoV-2 infection, diseases and therapy remains limited, underlining the urgency of fundamental studies and drug development. Studies have shown that induction of autophagy and hijacking of autophagic machinery are essential for infection and replication of SARS-CoV-2; however, the mechanism of this manipulation and function of autophagy during SARS-CoV-2 infection remain unclear. In the present study, we identified ORF3 as an inducer of autophagy and revealed that ORF3 localizes to the ER and induces FAM134B-related ERphagy through the HMGB1-Beclin1 pathway. As a consequence, ORF3 induces ER stress and inflammatory responses through ERphagy and sensitizes cells to ER stress-induced cell death, suggesting that SARS-CoV-2 ORF3 hijacks ERphagy and then harms ER homeostasis to induce inflammatory responses through excessive ER stress. These findings reveal a sequential induction of ERphagy, ER stress and acute inflammatory responses during SARS-CoV-2 infection and provide therapeutic potential for ERphagy and ER stress-related drugs for COVID-19 treatment and prevention.

Importance

SARS-CoV-2 infection and replication require autophagosome-like double-membrane vacuoles. Inhibition of autophagy suppresses viral replication, indicating the essential role of autophagy in SARS-CoV-2 infection. However, how SARS-CoV-2 hijacks autophagy and the function of autophagy in the disease progression remain unknown. Here, we reveal that SARS-CoV-2 ORF3 induces ERphagy and consequently induces ER stress to trigger acute inflammatory responses and enhance sensitivity to ER stress-induced apoptosis. Our studies uncover ERphagy-induced inflammatory responses during SARS-CoV-2 infection and provide a promising therapeutic approach for treating SARS-CoV-2 infection and inflammatory responses in COVID-19 by manipulating autophagy and ER stress.

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  1. ScreenIT

    SciScore for 10.1101/2020.11.17.387902: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board Statementnot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Cells, antibodies and chemicals: HeLa, HEK293, HEK293T and Beclin-1 KO HEK293T (kindly provided by Dr Yang Du, Sun Yat-Sen University) cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS); human adenocarcinoma lung tissue-derived epithelial (A549) cells were cultured in RPMI 1640 (Gibco) medium with 10% FBS.
    HEK293
    suggested: None
    Beclin-1 KO HEK293T
    suggested: None
    A549
    suggested: None
    The following antibodies were used in this study: anti-LC3B (#3868), Beclin-1(#3495), Akt (#4691), p-Akt(Thr308) (#2965), p-Akt(Ser473) (#4060), GRP78/BiP (#3183), CHOP (#2895), p-eIF2α (#3398), eIF2α(#5324), p-p38 MAPK(Thr180/Tyr182) (#4511), p38 MAPK (#8690), p-SAPK/JNK(Thr183/Tyr185) (#4668), SAPK/JNK(#9252), p-c-Jun (Ser73) (#3270), and c-Jun (#9165) were purchased from Cell Signaling Technology (Beverly, MA, USA); anti-p62 (P0067) and Flag (F3165) were purchased from Sigma-Aldrich (St. Louis, MO, USA).;
    anti-LC3B
    suggested: (Cell Signaling Technology Cat# 3868, RRID:AB_2137707)
    Beclin-1
    suggested: (Cell Signaling Technology Cat# 3495, RRID:AB_1903911)
    p-Akt(Thr308
    suggested: None
    p-Akt(Ser473
    suggested: None
    p-eIF2α
    suggested: None
    p-p38 MAPK(Thr180/Tyr182
    suggested: None
    p38 MAPK
    suggested: (Cell Signaling Technology Cat# 8690, RRID:AB_10999090)
    p-SAPK/JNK(Thr183/Tyr185
    suggested: None
    Ser73
    suggested: None
    c-Jun (#9165) were purchased from Cell Signaling Technology (Beverly, MA, USA)
    suggested: None
    anti-p62
    suggested: (Sigma-Aldrich Cat# P0067, RRID:AB_1841064)
    P0067
    suggested: (Sigma-Aldrich Cat# P0067, RRID:AB_1841064)
    Flag (F3165)
    suggested: (Sigma-Aldrich Cat# F3165, RRID:AB_259529)
    After three washes with PBS containing 0.1% Triton X-100, the cells were incubated with Alexa 488- or 555-labeled anti-rabbit IgG antibodies (Invitrogen, Carlsbad, CA) for 1 h.
    anti-rabbit IgG
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cells, antibodies and chemicals: HeLa, HEK293, HEK293T and Beclin-1 KO HEK293T (kindly provided by Dr Yang Du, Sun Yat-Sen University) cells were cultured in DMEM (Gibco) supplemented with 10% fetal bovine serum (FBS); human adenocarcinoma lung tissue-derived epithelial (A549) cells were cultured in RPMI 1640 (Gibco) medium with 10% FBS.
    HeLa
    suggested: None
    HEK293
    suggested: None
    HEK293T
    suggested: None
    A549
    suggested: None
    Experimental Models: Organisms/Strains
    SentencesResources
    The following antibodies were used in this study: anti-LC3B (#3868), Beclin-1(#3495), Akt (#4691), p-Akt(Thr308) (#2965), p-Akt(Ser473) (#4060), GRP78/BiP (#3183), CHOP (#2895), p-eIF2α (#3398), eIF2α(#5324), p-p38 MAPK(Thr180/Tyr182) (#4511), p38 MAPK (#8690), p-SAPK/JNK(Thr183/Tyr185) (#4668), SAPK/JNK(#9252), p-c-Jun (Ser73) (#3270), and c-Jun (#9165) were purchased from Cell Signaling Technology (Beverly, MA, USA); anti-p62 (P0067) and Flag (F3165) were purchased from Sigma-Aldrich (St. Louis, MO, USA).;
    Beclin-1
    suggested: None
    GRP78/BiP
    suggested: None
    CHOP
    suggested: None
    Software and Algorithms
    SentencesResources
    The raw sequencing datasets generated in this study are available on the NCBI Gene Expression Omnibus (GEO) server under the accession number GSE158484.
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)
    GSEA analysis: Gene set enrichment analysis was performed using GSEA software (https://www.gsea-msigdb.org/gsea/index.jsp) with number of permutations = 1000, permutation type = gene_set, enrichment statistic = weighted and metric for ranking genes = Signal2Noise.
    GSEA
    suggested: (SeqGSEA, RRID:SCR_005724)

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

    About SciScore

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  2. Version published to 10.1101/2020.11.17.387902v1 on bioRxiv