Unconventional secretion of unglycosylated ORF8 is critical for the cytokine storm during SARS-CoV-2 infection

  1. Xiaoyuan Lin
  2. Beibei Fu
  3. Yan Xiong
  4. Na Xing
  5. Weiwei Xue
  6. Dong Guo
  7. Mohamed Zaky
  8. Krishna Pavani
  9. Dusan Kunec
  10. Jakob Trimpert
  11. Haibo Wu

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Abstract

Coronavirus disease 2019 is a respiratory infectious disease caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Evidence on the pathogenesis of SARS-CoV-2 is accumulating rapidly. In addition to structural proteins such as Spike and Envelope, the functional roles of non-structural and accessory proteins in regulating viral life cycle and host immune responses remain to be understood. Here, we show that open reading frame 8 (ORF8) acts as messenger for inter-cellular communication between alveolar epithelial cells and macrophages during SARS-CoV-2 infection. Mechanistically, ORF8 is a secretory protein that can be secreted by infected epithelial cells via both conventional and unconventional secretory pathways. Conventionally secreted ORF8 is glycosylated and loses the ability to recognize interleukin 17 receptor A of macrophages, possibly due to the steric hindrance imposed by N-glycosylation at Asn78. However, unconventionally secreted ORF8 does not undergo glycosylation without experiencing the ER-Golgi trafficking, thereby activating the downstream NF-κB signaling pathway and facilitating a burst of cytokine release. Furthermore, we show that ORF8 deletion in SARS-CoV-2 attenuates inflammation and yields less lung lesions in hamsters. Our data collectively highlights a role of ORF8 protein in the development of cytokine storms during SARS-CoV-2 infection.

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  1. Version published to 10.1371/journal.ppat.1011128
  2. Version published to 10.1101/2021.12.03.471057v2 on bioRxiv
  3. ScreenIT

    SciScore for 10.1101/2021.12.03.471057: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All animal experimental procedures were approved by the Animal Ethics Committees of the School of Life Sciences, Chongqing University.
    IRB: All animal study protocols were reviewed and approved by Chongqing University School of Life Sciences review boards for animal studies.
    Sex as a biological variableViral infection: Specific-pathogen-free, ten-week-old male mice were inoculated intranasally with SARS-CoV-2 virus with 4×105 plaque-forming units (PFU).
    Randomizationnot detected.
    BlindingThe investigators were blinded during data collection and analysis.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Blots were probed with the indicated antibodies: anti-ORF8 (NBP3-05720), anti-GAPDH (NBP2-27103)
    anti-ORF8
    suggested: None
    anti-GAPDH
    suggested: None
    ORF8 antibody (NBP3-05720, Novus Biologicals) was coated with blank ELISA plates in carbonate buffer to prepare ELISA kit.
    NBP3-05720
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines and coronavirus: Calu-3 epithelial cells (HTB-55), Jurkat cells (Clone E6-1,
    Jurkat
    suggested: None
    TIB-152), THP-1 cells (TIB-202), Vero E6 cells (CRL-1586) and Sf9 cells (CRL-1711) were purchased from ATCC.
    THP-1
    suggested: None
    Vero E6
    suggested: None
    Sf9
    suggested: None
    Firstly, Calu-3 epithelial cells were infected by SARS-CoV-2 with/without Brefeldin A or Monensin pretreatment.
    Calu-3
    suggested: BCRJ Cat# 0264, RRID:CVCL_0609)
    In brief, Flag-tagged ORF8 and HA-tagged YIF1B were co-transfected into HEK-293FT cells.
    HEK-293FT
    suggested: RRID:CVCL_6911)
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: B6.Cg-Tg(K18-ACE2)2Prlmn/J mice (hACE2) were obtained from The Jackson Laboratory.
    B6.Cg-Tg(K18-ACE2)2Prlmn/J
    suggested: RRID:IMSR_JAX:034860)
    To generate YIF1B-deficient (Yif1b-/-) mice based on K18-ACE2 transgenic background, we designed sgRNAs targeting the Exon 3-5 of Yif1b.
    Yif1b-/-
    suggested: None
    Recombinant DNA
    SentencesResources
    In brief, seven different DNA fragments spanning the entire genome of SARS-CoV-2 (USA_WA1/2020 SARS-CoV-2 sequence, GenBank accession No. MT020880) were synthesized by Beijing Genomics Institute (BGI, Shanghai, China) and cloned into the pUC57 or pCC1 (kindly provided by Dr. Yonghui Zheng) plasmid by standard molecular cloning methods.
    pUC57
    suggested: RRID:Addgene_40306)
    pCC1
    suggested: RRID:Addgene_83007)
    For the generation of SARS-CoV-2 ORF8-N78Q variant, AAT CAA nucleotide substitutions were introduced into a subclone of pUC57-F7 containing the ORF8 gene of the SARS-CoV-2 wild-type infectious clone by overlap-extension PCR.
    pUC57-F7
    suggested: None
    Then, synthesized ORF8 sequence was amplified by PCR and inserted into a pFastBac HT A plasmid (Thermo Fisher Scientific)
    pFastBac HT
    suggested: RRID:Addgene_135554)
    Recombinant pFastBac-ORF8 plasmid was transformed into DH10Bac-competent cells to obtain Bacmid-ORF8, which was transfected into Sf9 cells to produce ORF8 protein.
    pFastBac-ORF8
    suggested: None
    Software and Algorithms
    SentencesResources
    Images were captured followed by quantification using Image J software.
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    Then, the purified YIF1B protein was added into the lipid solution and rotated for 1 hour, followed by incubation with Biobeads SM2 (Bio-Rad Laboratories) to absorb the detergent.
    Bio-Rad Laboratories
    suggested: (Bio-Rad Laboratories, RRID:SCR_008426)

    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

    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.

  4. Version published to 10.1101/2021.12.03.471057v1 on bioRxiv