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

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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. 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

    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)
    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.
    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.
    suggested: None
    Experimental Models: Cell Lines
    A549ACE2 cells: ACE expressing A549 cells were generated as previously described1.
    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.
    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.
    suggested: None
    SARS-CoV-2 infections: SARS-CoV-2 (USA-WA1/2020 strain) was obtained from BEI and propagated in Vero-E6 cells.
    suggested: None
    For RNA-sequencing analysis for HEK-293T cell experiments, GRCh38 assembly was used.
    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.
    suggested: None
    Software and Algorithms
    For ATAC-sequencing analysis, alignments were performed with Bowtie2 (2.1.0)4 using the Hg38 genome using a ChIP-seq pipeline (
    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 (
    suggested: (Bowtie 2, RRID:SCR_016368)
    Orf8 reads were mapped using NGS plot.
    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”.
    suggested: (Trimmomatic, RRID:SCR_011848)
    suggested: (RefSeq, RRID:SCR_003496)
    Alignments with a mapping quality below 5 (using samtools) and duplicated reads were removed.
    suggested: (SAMTOOLS, RRID:SCR_002105)
    STAR was used to generate bam files for subsequent TDF file generation using 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.
    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).
    suggested: (GENCODE, RRID:SCR_014966)
    suggested: (featureCounts, RRID:SCR_012919)
    Data normalization and differential gene expression analysis was performed using DESeq2 R package (v1.26.0).
    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.
    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.
    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.
    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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