SARS-CoV-2 ORF8 encoded protein contains a histone mimic, disrupts chromatin regulation, and enhances replication

  1. John Kee
  2. Samuel Thudium
  3. David Renner
  4. Karl Glastad
  5. Katherine Palozola
  6. Zhen Zhang
  7. Yize Li
  8. Joseph Cesare
  9. Yemin Lan
  10. Rachel Truitt
  11. Fabian L. Cardenas-Diaz
  12. Darrell N. Kotton
  13. Konstantinos D. Alysandratos
  14. Xianwen Zhang
  15. Xuping Xie
  16. Pei-Yong Shi
  17. Wenli Yang
  18. Edward Morrisey
  19. Benjamin A. Garcia
  20. Shelley L. Berger
  21. Susan R. Weiss
  22. Erica Korb

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Abstract

SARS-CoV-2 emerged in China at the end of 2019 and caused the global pandemic of COVID-19, a disease with high morbidity and mortality. While our understanding of this new virus is rapidly increasing, gaps remain in our understanding of how SARS-CoV-2 can effectively suppress host cell antiviral responses. Recent work on other viruses has demonstrated a novel mechanism through which viral proteins can mimic critical regions of human histone proteins. Histone proteins are responsible for governing genome accessibility and their precise regulation is critical for a cell’s ability to control transcription and respond to viral threats. Here, we show that the protein encoded by ORF8 (Orf8) in SARS-CoV-2 functions as a histone mimic of the ARKS motif in histone 3. Orf8 is associated with chromatin, binds to numerous histone-associated proteins, and is itself acetylated within the histone mimic site. Orf8 expression in cells disrupts multiple critical histone post-translational modifications (PTMs) including H3K9ac, H3K9me3, and H3K27me3 and promotes chromatin compaction while Orf8 lacking the histone mimic motif does not. Further, SARS-CoV-2 infection in human cell lines and postmortem patient lung tissue cause these same disruptions to chromatin. However, deletion of the Orf8 gene from SARS-CoV-2 largely blocks its ability to disrupt host-cell chromatin indicating that Orf8 is responsible for these effects. Finally, deletion of the ORF8 gene affects the host-cell transcriptional response to SARS-CoV-2 infection in multiple cell types and decreases the replication of SARS-CoV-2 in human induced pluripotent stem cell-derived lung alveolar type 2 (iAT2) pulmonary cells. These findings demonstrate a novel function for the poorly understood ORF8-encoded protein and a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation. Finally, this work provides a molecular basis for the finding that SARS-CoV-2 lacking ORF8 is associated with decreased severity of COVID-19.

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  1. ScreenIT

    SciScore for 10.1101/2021.11.10.468057: (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 AnalysisLysates were sonicated using a tip sonicator with 3, 5 second bursts, at 70% power with chilling on ice between bursts.
    Cell Line AuthenticationContamination: A549-ACE cells were grown in RPMI1640 media with 10% FBS and 1% Pen/Strep and maintained free of mycoplasma.

    Table 2: Resources

    Antibodies
    SentencesResources
    250uL of equalized lysate were then added to washed, antibody-conjugated protein A/G Dynabeads (2ug antibody conjugated to 15uL/15uL A/G dynabeads, resuspended in 50uL per IP) and IPs were rotated overnight at 4C in a final volume of 300uL.
    antibody-conjugated protein
    suggested: None
    Slides were incubated in mouse primary antibody solution of anti SARS-CoV-2 nucleocapsid and rabbit anti-H3K9me3 antibody solution overnight at 4°C.
    anti SARS-CoV-2 nucleocapsid
    suggested: (BioLegend Cat# 946101, RRID:AB_2892515)
    anti-H3K9me3
    suggested: None
    The cells were then gently resuspended in 1 mL FACS buffer with a 1:500 dilution of anti-streptactin antibody and rotated at 4°C for 1 hour, protected from light.
    anti-streptactin
    suggested: None
    Data analysis and manual inspection was performed with Skyline10 (MacCoss Lab) and IPSA11. Antibodies: Data analysis and availability: Box and whisker plots show center line median, box limits for upper and lower quartiles, whiskers for 1.5x interquartile range, and points are outliers.
    IPSA11
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    A549ACE2 cells: ACE expressing A549 cells were generated as previously described1.
    A549
    suggested: NCI-DTP Cat# A549, RRID:CVCL_0023)
    A549-ACE cells were grown in RPMI1640 media with 10% FBS and 1% Pen/Strep and maintained free of mycoplasma.
    A549-ACE
    suggested: None
    HEK cells: HEK293T cells were cultured in DMEM (with 4.5 g/L glucose, L-glutamine and sodium pyruvate), 10% fetal bovine serum (Sigma Aldrich F2442-500ML), and 1% Penicillin-Streptomycin (Gibco 15140122) and maintained free of mycoplasma.
    HEK293T
    suggested: None
    SARS-CoV-2 infections: SARS-CoV-2 (USA-WA1/2020 strain) was obtained from BEI and propagated in Vero-E6 cells.
    Vero-E6
    suggested: None
    For RNA-sequencing analysis for HEK-293T cell experiments, GRCh38 assembly was used.
    HEK-293T
    suggested: None
    For PTM quantification, HEK cells and human lung tissue were imaged at a single z-plane and A549 cells were imaged with a z-stack through the nucleus.
    HEK
    suggested: None
    Software and Algorithms
    SentencesResources
    For ATAC-sequencing analysis, alignments were performed with Bowtie2 (2.1.0)4 using the Hg38 genome using a ChIP-seq pipeline (https://github.com/shenlab-sinai/chip-seq_preprocess).
    ChIP-seq
    suggested: (ChIP-seq, RRID:SCR_001237)
    For Orf8 ChIP-sequencing analysis, alignments were performed with Bowtie2 (2.1.0)4 using the Hg38 genome using a ChIP-seq pipeline (
    Bowtie2
    suggested: (Bowtie 2, RRID:SCR_016368)
    Orf8 reads were mapped using NGS plot.
    NGS
    suggested: (PM4NGS, RRID:SCR_019164)
    Reads were trimmed using TRIMMOMATIC (Bolger et al., 2014) with the options “ILLUMINACLIP:[adapter.fa]:2:30:10 LEADING:5 TRAILING:5 SLIDINGWINDOW:4:15 MINLEN:15”, and aligned to a hybrid hg38+C. floridanus (v7.5, RefSeq) genome assembly using bowtie2 v2.2.64 with the option “--sensitive-local”.
    TRIMMOMATIC
    suggested: (Trimmomatic, RRID:SCR_011848)
    RefSeq
    suggested: (RefSeq, RRID:SCR_003496)
    Alignments with a mapping quality below 5 (using samtools) and duplicated reads were removed.
    samtools
    suggested: (SAMTOOLS, RRID:SCR_002105)
    STAR was used to generate bam files for subsequent TDF file generation using IGVtools.
    IGVtools
    suggested: None
    For all RNA-sequencing, reads were aligned using STAR (v2.6.1a) with default parameters and only uniquely mapped reads were retained for downstream analysis.
    STAR
    suggested: (STAR, RRID:SCR_004463)
    Reads were counted towards human genes (GENCODE v35) and SARS-CoV-2 genes(WA-CDC-WA1/2020 MN985325.1 assembly) using featureCounts (v1.6.2).
    GENCODE
    suggested: (GENCODE, RRID:SCR_014966)
    featureCounts
    suggested: (featureCounts, RRID:SCR_012919)
    Data normalization and differential gene expression analysis was performed using DESeq2 R package (v1.26.0).
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    GO enrichment analysis for differentially expressed genes was implemented using clusterProfiler R package (v3.14.3), using human genome annotation record in org.
    clusterProfiler
    suggested: (clusterProfiler, RRID:SCR_016884)
    Image analysis: Images were analyzed using Image J software.
    Image J
    suggested: (ImageJ, RRID:SCR_003070)
    Protein alignment: To identify potential histone mimicry SARS-CoV-2 protein sequences were aligned to human histone protein sequences (H2A, H2B, H3.1,H3.2 H4, H2A.X, H2A.Z, macroH2A, and H3.3) using Multiple Sequence Comparison by Log-Expectation (MUSCLE) with default settings.
    MUSCLE
    suggested: (MUSCLE, RRID:SCR_011812)
    A database search was performed using the human SwisProt sequence and Orf8 sequence with Proteome Discoverer 2.4 (Thermo Scientific) with the following search criteria: carboxyamidomethylation at cysteine residues as a fixed modification; oxidation at methionine, acetylation at lysine, mono-, di-, and tri-methylation at lysine residues as variable modifications; two maximum allowed missed cleavage; 10 ppm precursor MS tolerance; a 0.2 Da MS/MS.
    Proteome Discoverer
    suggested: (Proteome Discoverer, RRID:SCR_014477)
    Fiji was used for image analysis.
    Fiji
    suggested: (Fiji, RRID:SCR_002285)

    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: Please consider improving the rainbow (“jet”) colormap(s) used on page 23. At least one figure is not accessible to readers with colorblindness and/or is not true to the data, i.e. not perceptually uniform.

    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

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  2. Version published to 10.1101/2021.11.10.468057v1 on bioRxiv