SARS-CoV-2 ORF9c Is a Membrane-Associated Protein that Suppresses Antiviral Responses in Cells

  1. Ana Dominguez Andres
  2. Yongmei Feng
  3. Alexandre Rosa Campos
  4. Jun Yin
  5. Chih-Cheng Yang
  6. Brian James
  7. Rabi Murad
  8. Hyungsoo Kim
  9. Aniruddha J. Deshpande
  10. David E. Gordon
  11. Nevan Krogan
  12. Raffaella Pippa
  13. Ze’ev A. Ronai

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Abstract

Disrupted antiviral immune responses are associated with severe COVID-19, the disease caused by SAR-CoV-2. Here, we show that the 73-amino-acid protein encoded by ORF9c of the viral genome contains a putative transmembrane domain, interacts with membrane proteins in multiple cellular compartments, and impairs antiviral processes in a lung epithelial cell line. Proteomic, interactome, and transcriptomic analyses, combined with bioinformatic analysis, revealed that expression of only this highly unstable small viral protein impaired interferon signaling, antigen presentation, and complement signaling, while inducing IL-6 signaling. Furthermore, we showed that interfering with ORF9c degradation by either proteasome inhibition or inhibition of the ATPase VCP blunted the effects of ORF9c. Our study indicated that ORF9c enables immune evasion and coordinates cellular changes essential for the SARS-CoV-2 life cycle.

One-sentence summary

SARS-CoV-2 ORF9c is the first human coronavirus protein localized to membrane, suppressing antiviral response, resembling full viral infection.

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  1. ScreenIT

    SciScore for 10.1101/2020.08.18.256776: (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
    , β-Tubulin (abcam ab6046) primary antibodies.
    β-Tubulin
    suggested: (Abcam Cat# ab6046, RRID:AB_2210370)
    Experimental Models: Cell Lines
    SentencesResources
    Cell lines: A549 cells were cultured in Dulbecco’s Modified Eagle’s Medium (Corning) supplemented with 10% Fetal Bovine Serum (Gibco, Life Technologies) and 1% Penicillin/Streptomycin (Corning) and maintained at 37°C in a humidified atmosphere of 5% CO2. Transfection: Ten million A549 cells were plated in p15 dishes and transfected using Jet Prime (Polyplus) with 15 μg of Strep-tagged expression ORF constructs or empty vector.
    A549
    suggested: None
    Software and Algorithms
    SentencesResources
    RNA-Seq sequencing reads were aligned using STAR aligner version 2.7 (53) based on human genome version 38 and Ensemble gene annotation version 84
    STAR
    suggested: (STAR, RRID:SCR_015899)
    Gene differential expression was performed using estimated read counts from RSEM by the R Bioconductor package DESeq2 following generalized linear model based on negative binomial distribution (55)
    RSEM
    suggested: (RSEM, RRID:SCR_013027)
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    Pathway and network analysis: Significant differentially expressed genes and proteins were then analyzed using Ingenuity Pathway Analysis (Qiagen, Redwood City, USA) using Canonical Pathways and Upstream Regulators.
    Ingenuity Pathway Analysis
    suggested: (Ingenuity Pathway Analysis, RRID:SCR_008653)
    Differentially expressed genes and differentially expressed proteins were further analyzed using Metascape for MCODE network analysis (56).
    Metascape
    suggested: (Metascape, RRID:SCR_016620)
    Subcellular locations of proteins were analyzed using DAVID based on Gene Ontology Cellular Component category (57) and with the aid of Protein Atlas Protein localization information.
    DAVID
    suggested: (DAVID, RRID:SCR_001881)
    Sequence analysis of ORF9c protein: Protein sequences similar to SARS-CoV-2 ORF9c were retrieved through NCBI Blastp using the nr database (58)
    Blastp
    suggested: (BLASTP, RRID:SCR_001010)
    Phylogenetic tree was built using PhyML algorithm by 100 times bootstrap, and visualized using Seaview (60) and Geneious version 2020.2.2 (San Diego, CA)
    PhyML
    suggested: (PhyML, RRID:SCR_014629)
    Seaview
    suggested: (SeaView, RRID:SCR_015059)
    Geneious
    suggested: (Geneious, RRID:SCR_010519)
    Differential expression of RNA-Seq was performed using DESeq2 bioconductor package following Negative Binomial Distribution and Wald test.
    bioconductor
    suggested: (Bioconductor, RRID:SCR_006442)
    Public datasets used in this study was processed by Coronascape component of Metascape (56), including Stukalov et al. (39) from BioRxiv, and Blanco-Melo et al. (22).
    BioRxiv
    suggested: (bioRxiv, RRID:SCR_003933)

    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.

    About SciScore

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  2. Version published to 10.1101/2020.08.18.256776 on bioRxiv