ACE2-expressing A549 cells were generated as previously described3. A549ACE2 cells were grown in RPMI-1640 with 10% FBS and 1% penicillin-streptomycin and were maintained free of mycoplasma. Cells were infected at an MOI of 1 and fixed or lysed at 24 or 48 h after infection.
HEK293T cells were obtained from the American Type Culture Collection (ATCC), cultured in DMEM (with 4.5 g L–1 glucose, l-glutamine and sodium pyruvate) supplemented with 10% FBS (Sigma-Aldrich, F2442-500ML) and 1% penicillin-streptomycin (Gibco, 15140122) and maintained free of mycoplasma. Calcium phosphate transfection was used to introduce plasmid DNA encoding GFP, ORF8 and mutant ORF8 into HEK293T cells. For immunocytochemistry experiments, cells were plated on poly(d-lysine)-coated coverslips. Cells were washed 24 h after transfection with culture medium and fixed or pelleted and flash frozen 48 h after transfection. Cells were fixed using 4% paraformaldehyde (PFA) in PBS for 8 min. To pellet cells, cells were detached from the culture plate using TrypLE Express (Gibco, 12605010) dissociation reagent, spun down for 5 min at 180 g and flash frozen in liquid nitrogen.
Generation of human-derived induced alveolar epithelial type II-like (iAT2) cells was performed as described44. To maintain a stable and pure culture of the iAT2 cell line, SFTPCtdTomato+ cells were sorted and serially passaged every 14 d. Cells were grown in organoid format using 90% Matrigel with a cell density of 400 cells per µl. Cells were fed using CK+DCI medium + Rock inhibitor for the first 48 h after splitting and then changed to K+DCI medium for 5 d followed by CK+DCI medium for 7 d. Every 14 d, alveolosphere organoids were passaged, organoids were released from Matrigel using 2 mg ml–1 Dispase for 1 h at 37 °C and single cells were then generated using 0.05% trypsin for 15 min at 37 °C. Cell number and viability were assessed using Trypan blue, and cells were finally passaged to new Matrigel drops left to polymerize for 30 min at 37 °C in a 5% CO2 incubator, after which cells in solidified Matrigel were fed according to plate format.
For the generation of two-dimensional (2D) alveolar cells for virus infection, when alveolosphere organoids were passaged, cells were plated on precoated 1:30 Matrigel plates at a cell density of 125,000 cells per cm2 using CK+DCI medium + Rock inhibitor for the first 48 h, and the medium was then changed to CK+DCI medium. Seventy-two hours after cell plating, cells were infected with SARS-CoV-2 virus using an MOI of 1 for 48 h.
Cell line validation and testing
Cell lines were authenticated as previously described3. HEK293T and Vero E6 cells were obtained from ATCC at the onset of this project. All cell lines used were confirmed to be negative for mycoplasma and are retested twice annually.
The ORF8 expression plasmid was obtained from Addgene, pLVX-EF1alpha-SARS-CoV-2-orf8-2xStrep-IRES-Puro (Addgene plasmid 141390). ORF8 deletion constructs were produced on the ORF8 backbone using Pfu Turbo HotStart DNA polymerase (Agilent, 600322-51), and primers were created using the DNA-based primer design feature of the online PrimerX tool. Constructs were verified by Sanger sequencing.
SARS-CoV-2 (USA-WA1/2020 strain) was obtained from BEI and propagated in Vero E6 cells. The genome RNA was sequenced and found to be identical to GenBank MN985325.1. Mutant viruses were generated using the cDNA reverse genetics system as previously described42.
Cells were infected with wild-type or mutant SARS-CoV-2 at an MOI of 1 PFU per cell (A549ACE2) or 5 PFU per cell (iAT2) as previously described3. Virus was added to cells for 1 h at 37 °C and was then removed and replaced with medium. Cells were lysed at 48 h after infection and RNA was isolated. All infections and virus manipulations were conducted in a Biosafety Level 3 (BSL3) laboratory using appropriate protective equipment and protocols.
Viral growth kinetics and plaque assays
Growth kinetics analysis and plaque assays were performed as previously described3. In brief, at the indicated time points, 200 µl of supernatant was collected from cells and stored at −80 °C for titration of infectious virus. Samples were diluted in serum-free DMEM and adsorbed onto Vero E6 cells at 37 °C for 1 h before a liquid overlay was added (DMEM with 2% FBS, 1× sodium pyruvate and 0.1% agarose). After 3 d, the overlay was removed and cells were fixed with 4% PFA and stained with crystal violet for plaque visualization and counting. All plaque assays were performed in biological triplicate and technical duplicate.
Viral genome quantification by qRT–PCR
RNA collection, qRT–PCR and viral genome quantification were performed as previously described3. In brief, at the indicated time points, infected cells were lysed using RLT Plus Buffer, genomic DNA was removed and RNA was extracted using the Qiagen RNeasy Mini kit (Qiagen, 74134). cDNA was generated using a High-Capacity cDNA Reverse Transcriptase kit (Applied Biosystems, 4368814). cDNA was amplified using specific qRT–PCR primers targeting viral NSP12 (forward, 5′-GGTAACTGGTATGATTTCG-3′; reverse, 5′-CTGGTCAAGGTTAATATAGG-3′), iQ SYBR Green Supermix (Bio-Rad, 1708880) and the QuantStudio 3 PCR system (Thermo Fisher). Quantification of SARS-CoV-2 genome copies was performed using a standard curve generated by serially diluting a known concentration of the pcDNA6B-nCoV NSP12-FLAG construct encoding the RDRP gene (a gift from G. Stark, Cleveland Clinic) after digestion with XhoI. Genome copy numbers were determined using standard curve analysis in QuantStudio 3 software, and copy numbers per microgram of RNA were calculated using the cDNA reaction volumes and input RNA for the cDNA reactions.
Pelleted cells were briefly thawed on ice. Buffer 1 (15 mM Tris-HCl (pH 7.5), 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 1 mM CaCl2 and 0.25 M sucrose with 1 mM PMSF, 1 mM DTT and a Complete Protease Inhibitor cocktail tablet added immediately before use) was added to the pellet at roughly five times the volume of the pellet and gently pipetted up and down to dissociate the pellet. Samples were incubated on ice for 5 min, followed by addition of an equal volume of buffer 1 with 0.4% NP-40 to the sample. Samples were then mixed by inversion for 5 min at 4 °C. Samples were spun at 200 g for 10 min in a prechilled centrifuge to pellet nuclei. The supernatant (cytoplasmic fraction) was transferred to a new tube. Pellets were resuspended gently in 0.5 ml buffer 1 to wash the nuclei and then pelleted again with the supernatant discarded. Nuclear pellet solubilization buffer (150 mM NaCl, 50 mM Tris-HCl (pH 8.0), 1% NP-40 and 5 mM MgCl2 with 1 mM PMSF, 1 mM DTT and Benzonase enzyme at 250 U µl–1 added shortly before use) was added to the pellet at half the volume of buffer 1 used. Samples were then incubated at room temperature in a thermoshaker until the pellet was fully dissolved. The amount of Benzonase enzyme was doubled in samples with undissolved material left after 20 min. Samples were then centrifuged at 13,000 r.p.m. for 20 min at 4 °C. Supernatant (nuclei fraction) was collected. Sample concentrations were determined by BCA assay, and samples were boiled in a western loading buffer for 10 min before analysis by western blotting.
Chromatin sequential salt extraction
Salt extractions were performed as described51. In brief, a 2× RIPA solution was made (100 mM Tris (pH 8.0), 2% NP-40 and 0.5% sodium deoxycholate) and mixed with varying concentrations of a 5 M NaCl solution to generate RIPA containing 0, 100, 200, 300, 400 and 500 mM NaCl. Pelleted cells were resuspended in buffer A with protease inhibitors (0.3 M sucrose, 60 mM KCl, 60 mM Tris (pH 8.0), 2 mM EDTA and 0.5% NP-40) and rotated at 4 °C for 10 min. Nuclei were pelleted by centrifugation at 6,000 g for 5 min at 4 °C. Supernatant was removed and saved, and 200 µl of RIPA with 0 mM NaCl and protease inhibitors was added to the sample. Samples were mixed by pipetting 15 times and incubated on ice for 3 min before centrifuging at 6,500 g for 3 min at 4 °C. Supernatant was saved and the RIPA steps were repeated for all NaCl concentrations. Samples were then boiled and sonicated before analysis by western blotting.
HEK293T cells were stained and sorted to isolate transfected cells using the same method as described below. Sorted cells were resuspended in cold lysis buffer (10 µl per 10,000 cells; 10 mM Tris-Cl (pH 7.5), 10 mM NaCl, 3 mM MgCl2, 0.1% (vol/vol) NP-40, 0.1% (vol/vol) Tween-20 and 0.01% (vol/vol) digitonin) and washed in wash buffer (10 mM Tris-Cl (pH 7.5), 10 mM NaCl, 3 mM MgCl2 and 0.1% (vol/vol) Tween-20). Transposition was performed with Tagment DNA TDE1 (Illumina, 15027865). Transposition reactions were cleaned with AMPure XP beads (Beckman, A63880), and libraries were generated by PCR with NEBNext High-Fidelity 2× PCR Master Mix (NEB, M0541). Library size was confirmed on a Bioanalyzer before sequencing on the NextSeq 550 platform (40-bp read length, paired end).
Infected A549ACE cells were fixed before collection for ATAC-seq. The protocol was performed as above except with 0.05% Igepal CA-630 added to the lysis buffer. In addition, after the transposase reaction, a reverse cross-linking solution was added (with a final concentration of 50 mM Tris-Cl, 1 mM EDTA, 1% SDS, 0.2 M NaCl and 5 ng ml–1 proteinase K) up to 200 μl. The mixture was incubated at 65 °C with shaking at 1,000 r.p.m. in a heat block overnight and then purified as above.
For ATAC-seq analysis, alignments were performed with Bowtie2 (2.1.0)52 using the hg38 genome with the pipeline at https://github.com/shenlab-sinai/chip-seq_preprocess. Reads were mapped using NGS plot. For HEK293T cell ATAC-seq, genes with high, intermediate, low and no expression were defined by DESeq2 normalized basemean values from HEK293T cell RNA-seq data with under 2 basemean as non-expressing genes and the remaining genes binned into three groups for low, intermediate and high expression. For A549ACE cell ATAC-seq, three biological replicates each with 2–3 technical replicates were performed. Ten million reads from each individual technical replicate were subsetted (SAMtools v1.9, seed 1) and merged, and each condition was then merged across biological replicates. For average profile plots, each condition was downsampled to 40 million reads and plotted against all genes identified by DESeq2 as expressed over 1 from A549ACE RNA-seq data.
For ORF8 ChIP–seq, 2 d after transfection, cells were fixed for 5 min with 1% PFA in PBS and the reaction was then quenched with 2.5 M glycine. Cells were washed twice, collected in PBS with protease and phosphatase inhibitors and then pelleted at 1,200 r.p.m. for 5 min. Cells were then rotated in lysis buffer 1 (50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40 and 0.25% Triton X-100) for 10 min at 4 °C and spun at 1,350 g for 5 min at 4 °C to isolate nuclei. Supernatant was discarded and cells were resuspended in lysis buffer 2 (10 mM Tris-HCl (pH 8), 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA) to lyse nuclei. Samples were rotated for 10 min at room temperature and were spun again at 1,350 g for 5 min at 4 °C. The supernatant was discarded and the pellet was resuspended in lysis buffer 3 (10 mM Tris-HCl (pH 8), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% EDTA and 0.5% N-lauroylsarcosine). Lysates were sonicated on a Covaris sonicator for 40 min (200 cycles per burst). Triton X-100 was added to reach a final concentration of 1%, and lysates were spun at 20,000 g for 10 min at 4 °C. Strep-Tactin magnetic beads (MagStrep type 3 XT beads; IBA, 2-4090-002) were added to the lysates overnight with rotation at 4 °C. Beads were then washed with a low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris (pH 8) and 150 mM NaCl), a high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris (pH 8) and 500 mM NaCl), a LiCl wash buffer (150 mM LiCl, 1% NP-40, 1% sodium deoxycholate, 1 mM EDTA and 10 mM Tris (pH 8)) and then TE with 50 mM NaCl. Chromatin was eluted from beads for 30 min with shaking at room temperature in 55 µl BXT elution buffer (IBA, 2-1042-025) followed by the addition of 150 µl elution buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA and 1% SDS) for 30 min at 65 °C. Samples were removed from beads and cross-linking was reversed by further incubating chromatin overnight at 65 °C. RNA was digested with RNase for 1 h at 37 °C, and protein was digested with proteinase K for 30 min at 55 °C. DNA was then purified with the Zymo PCR purification kit. The Illumina TruSeq ChIP purification kit was used to prepare samples for sequencing on an Illumina NextSeq 500 instrument (42-bp read length, paired end).
For ORF8 ChIP–seq analysis, alignments were performed with Bowtie2 (2.1.0)52 using the hg38 genome with a ChIP–seq pipeline (https://github.com/shenlab-sinai/chip-seq_preprocess). ORF8 reads were mapped using NGS plot. For comparison with histone modification ChIP–seq datasets, ENCODE and 4D nucleome data were used for H3K9ac (experiment ENCSR000ASV), lamin (4DN experiment set 4DNES24XA7U8), H3K9me3 (experiments ENCSR000FCJ and ENCSR179BUC), H3K9me2 (experiment ENCSR55LYM) and H3K27me3 (experiment ENCSR000AKD). To define ORF8-enriched regions, HiddenDomains was used for each of two ORF8 ChIP–seq experiments normalized to input. Output files were merged with bedtools (v2.18.1) intersect to select the subset of enriched regions found in both replicates. DiffBind (3.4.11) was used to examine H3K27me3 enrichment within ORF8-enriched regions. The Deeptools (3.3.0) plotEnrichment tool was used to count percentages of reads of histone modification ENCODE ChIP–seq datasets that were within ORF8-enriched regions. ngs.plot.r (2.63) was used to generate plots of ORF8 enrichment within genomic regions of interest.
For histone PTM ChIP–seq, 4–10 million cells were resuspended in 1 ml of lysis buffer 1 (50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40 and 0.25% Triton X-100) and rotated at 4 °C for 10 min, followed by centrifugation and removal of supernatant. Cells were then resuspended in 1 ml of lysis buffer 2 (10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA and 0.5 mM EGTA) and rotated for 10 min at 4 °C, followed by centrifugation and removal of supernatant. Cells were then resuspended in 1 ml of lysis buffer 3 (10 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate and 0.5% N-lauroylsarcosine) and rotated again for 10 min at 4 °C. Cells were then sonicated with a Covaris S220 sonicator for 35 min (peak incident power, 140; duty factor, 5%; cycles per burst, 200). This was followed by addition of 110 µl Triton X-100 and centrifugation at maximum speed (20,000 g) for 15 min at 4 °C to clear the lysate. The lysate chromatin concentration was then equalized according to DNA content (as measured with a Qubit fluorometer). Following this, 5% of equivalently treated chromatin from Camponotus floridanus pupae was added to all samples according to chromatin concentration, and 50 µl of lysate was saved as input shearing control. Then, 250 µl of equalized lysate was added to washed, antibody-conjugated Protein A/G Dynabeads (2 µg of antibody conjugated to 15 µl of Protein A/G Dynabeads, resuspended in 50 µl per immunoprecipitation), and immunoprecipitations were rotated overnight at 4 °C in a final volume of 300 µl. The following day, immunoprecipitations were washed five times in RIPA wash buffer (50 mM HEPES-KOH (pH 7.5), 500 mM LiCl, 1 mM EDTA, 1% NP-40 and 0.7% sodium deoxycholate) and once in TE (pH 8.0). Washes were followed by two elutions into 75 µl of elution buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA and 1% SDS) at 65 °C for 45 min with shaking (1,100 r.p.m.). DNA was purified by phenol:chloroform:isoamyl alcohol (25:24:1) extraction followed by ethanol precipitation. Pelleted DNA was resuspended in 25 µl TE. Libraries for sequencing were prepared using the NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645), as described by the manufacturer but using half the volume for all reagents and starting material. For PCR amplification, the optimal number of PCR cycles was determined using a qPCR side reaction with 10% of the adaptor-ligated, size-selected DNA. Seven to ten cycles of PCR were used for histone PTM libraries and 5 cycles were used for input controls. Samples were sequenced on a NextSeq 500 instrument (42-bp read length, paired end).
For analysis of histone PTM ChIP–seq data, reads were demultiplexed using bcl2fastq2 (Illumina) with the options ‘–mask-short-adapter-reads 20 –minimum-trimmed-read-length 20 –no-lane-splitting –barcode-mismatches 0’. Reads were trimmed using TRIMMOMATIC53 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 (v2.2.6)52 with the option ‘–sensitive-local’. Alignments with a mapping quality below 5 (using SAMtools) and duplicated reads were removed. Peaks were called using MACS2 (v188.8.131.5260309)54 with the options ‘–call-summits –nomodel –B’. Differential ChIP peaks were called using DiffBind55 with the options ‘bFullLibrarySize=FALSE, bSubControl=TRUE, bTagwise=FALSE’ for dba.analyze(). For DiffBind testing, the DESeq2 algorithm with blocking was used, and ChIP replicate was used as the blocking factor while testing for differences between mock and infected samples. For ChIP signal tracks, individual replicate tracks were produced for RPM and fold enrichment over input control, merged and averaged.
To account for potential global differences in histone PTM abundance that would otherwise be missed by more standard quantile normalization-type approaches, high-quality deduplicated read counts were produced for both human- and C. floridanus-mapping reads, resulting in proportions of reads mapping to the exogenous genome for each histone PTM. Input controls were also treated in this way to account for potential differences in initial spike-in addition between samples. For each histone PTM, the proportion of spike-in reads was normalized by the appropriate input control value. Because spike-ins should be inversely proportional to target chromatin concentration, a ratio of SARS-CoV-2/mock values was produced for each histone PTM × replicate, and for SARS-CoV-2 samples resulting signal values were divided by this ratio. This resulted in per-base-pair signal values adjusted by the degree of global difference in a given histone PTM’s level between sample types.
All antibodies are described in Supplementary Table 6.
RNA was extracted using a Qiagen RNA purification kit. Samples were prepared for sequencing using the Illumina TruSeq purification kit and sequenced on an Illumina NextSeq 500 instrument (75-bp read length, single read). Library size was confirmed on a Bioanalyzer before sequencing on the NextSeq 550 platform (single end, 75 cycles).
For RNA-seq analysis for SARS-CoV-2 infection experiments, a reference genome for alignment was built by concatenating the human (GRCh38 assembly) and SARS-CoV-2 (WA-CDC-WA1/2020 assembly; MN985325.1) genomes. For RNA-seq analysis for HEK293T cell experiments, the GRCh38 assembly was used. For all RNA-seq, reads were aligned using STAR (v2.6.1a) with default parameters and only uniquely mapped reads were retained for downstream analysis. TDF files were generated using IGVtools. Reads were counted towards human genes (GENCODE v35) and SARS-CoV-2 genes (WA-CDC-WA1/2020 assembly; MN985325.1) using featureCounts (v1.6.2). Low-count genes were filtered out so that only genes with counts per million (CPM) values greater than 1 in at least three samples were used. Data normalization and differential gene expression analysis were performed using the DESeq2 R package (v1.26.0). We defined genes as significant using a false discovery rate (FDR) cut-off of 0.05 and 1.5× fold change. GO enrichment analysis for differentially expressed genes was implemented with the clusterProfiler R package (v3.14.3), using the human genome annotation record in the org.Hs.eg.db R package (v3.10.0) and a Benjamini–Hochberg-adjusted P value of 0.05 as the cut-off.
Anti-Strep tag affinity purification, whole-cell lysate and cytoplasmic HLA-A2 co-immunoprecipitation
Protein and binding partners were purified with affinity Strep tag purification. For ORF8 PTM analysis and mass spectrometry binding partner analysis, whole-cell lysates were prepared as described below. Frozen cell pellets were thawed briefly and suspended in lysis buffer (immunoprecipitation (IP) buffer (50 mM Tris-HCl (pH 7.5) at 4 °C, 150 mM NaCl, 1 mM EDTA and 10 mM sodium butyrate) supplemented with 0.5% Nonidet P 40 Substitute (NP-40; Fluka Analytical) and cOmplete mini EDTA-free protease and PhosSTOP phosphatase inhibitor cocktails (Roche)). Samples were incubated on a tube rotator for 30 min at 4 °C. Debris was pelleted by centrifugation at 13,000 g for 15 min at 4 °C. Lysates were then incubated with Strep-Tactin magnetic beads (40 µl; MagStrep type 3 XT beads; IBA, 2-4090-002) for 2 h with rotation at 4 °C. Beads were washed three times with 1 ml wash buffer (IP buffer supplemented with 0.05% NP-40) and then once with 1 ml IP buffer. Strep-tagged ORF8 complexes were eluted from beads in BXT buffer (IBA, 2-1042-025) with shaking at 1,100 r.p.m. for 30 min.
Anti-Strep tag affinity purification for chromatin binding partners
Cells were rotated in lysis buffer 1 (50 mM HEPES-KOH (pH 7.5), 140 mM NaCl, 10 mM sodium butyrate, 1 mM EDTA, 10% glycerol, 0.5% NP-40 and 0.25% Triton X-100) supplemented with 0.5% Nonidet P 40 Substitute (NP-40; Fluka Analytical) and cOmplete mini EDTA-free protease and PhosSTOP phosphatase inhibitor cocktails (Roche) for 10 min at 4 °C and spun at 1,350 g for 5 min at 4 °C to isolate nuclei. Supernatant was discarded and cells were resuspended in lysis buffer 2 (10 mM Tris-HCl (pH 8), 200 mM NaCl, 10 mM sodium butyrate, 1 mM EDTA and 0.5 mM EGTA) to lyse nuclei. Cells were rotated for 10 min at room temperature and were spun again at 1,350 g for 5 min at 4 °C. The supernatant was discarded and the chromatin pellet was resuspended in lysis buffer 3 (10 mM Tris-HCl (pH 8), 100 mM NaCl, 10 mM sodium butyrate, 1 mM EDTA, 0.5 mM EGTA, 0.1% EDTA and 0.5% N-lauroylsarcosine). Lysates were sonicated using a tip sonicator with three 5-s bursts at 50% power with chilling on ice between bursts. After sonication, lysates were brought to a concentration of 1% Triton X-100 to disrupt lamina protein interactions. Debris was pelleted by centrifugation at 16,000 g at 4 °C, and the supernatant was incubated with Strep-Tactin magnetic beads (40 µl; MagStrep type 3 XT beads; Iba, 2-4090-002) for 2 h with rotation at 4 °C. Beads were washed three times with 1 ml wash buffer (IP buffer supplemented with 0.05% NP-40) and then once with 1 ml IP buffer. Strep-tagged ORF8 complexes were eluted from beads in BXT buffer (IBA, 2-1042-025) with shaking at 1,100 r.p.m. for 30 min. To analyse relative ORF8 construct levels in cytoplasmic versus chromatin fractions by western blotting, samples were taken from lysis buffer 1 and lysis buffer 3, respectively.
Chromatin pellet lysate was obtained as described above for chromatin protein immunoprecipitation. Lysates were combined with antibody-conjugated Protein A Dynabeads (15 µg of antibody conjugated to 100 µl of Dynabeads) and rotated overnight at 4 °C. The following day, beads were washed three times with 1 ml wash buffer (IP buffer supplemented with 0.05% NP-40) and then once with 1 ml IP buffer. Chromatin protein complexes were eluted from beads in elution buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA and 1% SDS) for 30 min with shaking at 65 °C.
All antibodies are described in Supplementary Table 6.
Fluorescence immunocytochemistry of HEK293T cells and A549ACE2 cells
Cells were fixed in 4% PFA for 10 min and washed with PBS. Fixed cells were permeabilized using 0.5% Triton X-100 in PBS for 20 min. Cells were blocked in blocking solution (PBS with 3% BSA, 2% serum and 0.1% Triton X-100) for at least 1 h and stained with designated primary antibody overnight at 4 °C. The following day, cell coverslips were washed with PBS and incubated with secondary antibody for 1 h at room temperature. For detection of Strep-tagged ORF8, Strep-Tactin DY-488 (IBA, 2-1562-050; 1:500) was added to the secondary antibody solution. Nuclei were stained with DAPI (1:1,000 in PBS) for 10 min with washing in PBS. Coverslips were mounted onto microscope slides using ProLong Gold antifade reagent (Thermo Fisher).
Fluorescence immunocytochemistry analysis of lamin B1, lamin A/C and H3K9me2
HEK293T cells were fixed with 2% PFA (Electron Microscopy Sciences, 15710) for 8 min at room temperature and washed three times with DPBS (Gibco, 14190-136). Cells were permeabilized with 0.25% Triton X-100 (Thermo Fisher, 28314) for 10 min, washed three times with DPBS for 5 min each wash and blocked in 1% BSA (Sigma, A4503) in PBST (DPBS with 0.05% Tween-20, pH 7.4 (Thermo Fisher, 28320)) for 60 min. Cells were incubated with primary antibody diluted in blocking buffer for 1 h, washed three times with PBST for 5 min each wash and incubated with secondary antibody diluted in blocking buffer for 60 min. Cells were washed twice with PBST and once with PBS for 5 min each wash and were then mounted on a slide using Duolink In Situ Mounting Medium with DAPI (Sigma, DUO82040-5ML). All procedures were performed at room temperature.
Immunohistological staining of patient lung tissue
Formalin-fixed, paraffin-embedded slides were obtained from Penn’s Pathology Clinical Service Center. Slides were deparaffinized and rehydrated as follows: incubation for 10 min with xylene (twice), 10 min with 100% ethanol (twice), 5 min with 95% ethanol, 5 min with 70% ethanol, 5 min with 50% ethanol and then running distilled water. Slides were then processed using heat-induced epitope retrieval (HIER). Slides were incubated in hot sodium citrate buffer (10 mM sodium citrate and 0.05% Tween-20, pH 6.0), placed in a pressure cooker and heated in a water bath for 25 min with high pressure settings. Slides were cooled at room temperature and washed twice in TBS. Membranes were permeabilized in TBS with 0.4% Triton X-100 for 20 min. Slides were then incubated in blocking solution (TBS with 10% goat serum, 1% BSA and 0.025% Triton X-100) for 2 h. Slides were incubated in mouse primary antibody solution containing anti-SARS-CoV-2 nucleocapsid and rabbit anti-H3K9me3 antibody solution overnight at 4 °C. The following day, slides were washed with TBS and incubated in secondary antibody solution. Nuclei were stained with DAPI (5 µg ml–1) in TBS for 10 min followed by washing with TBS. Coverslips were mounted with ProLong Gold antifade reagent (Thermo Fisher). All antibodies are described in Supplementary Table 6.
Fluorescence immunocytochemistry of ORF8 and histone PTMs
Cells were imaged on an upright Leica DM 6000, TCS SP8 laser scanning confocal microscope with 405-nm, 488-nm, 552-nm and 638-nm lasers. The microscope uses two HyD detectors and three PMT detectors. The objective used was a ×63 HC PL APO CS2 oil objective with an NA of 1.40. Type F immersion liquid (Leica) was used for oil objectives. Images were 175.91 × 171.91 µm2, 1,024 × 1,024 pixels and 16 bits per pixel. For PTM quantification, HEK293T cells and human lung tissue were imaged at a single z plane and A549 cells were imaged with a z stack through the nucleus.
Fluorescence immunocytochemistry analysis of lamin B1, lamin A/C and H3K9me2
All confocal immunofluorescence images were acquired using a Leica SP8 laser scanning confocal system with a ×63/1.40-NA HC PL APO CS2 objective and HyD detectors in standard mode with 100% gain. For comparison of lamin A/C and lamin B1 signal intensities between mock and ORF8-positive cells, single-plane confocal images were acquired. All images were acquired with the same microscope settings (zoom, laser power, gain, etc.). For analysis of the organization of H3K9me2-marked chromatin at the nuclear lamina, three-dimensional (3D) images of the middle z plane of the nucleus were taken as z stacks using 0.1-μm intervals with a range of 1 μm per nucleus. Confocal 3D images were deconvoluted with Huygens Professional software using the microscope parameters, standard PSF and automatic settings for background estimation.
Images were analysed using ImageJ software (version 2.0.0-rc-69/1.52p, build 269a0ad53f). Single-z-plane images of HEK293T cells and human lung tissue and summed z stacks through A549 nuclei were used for PTM quantification. Regions of interest (ROIs) of in-focus nuclei were semi-automatically defined using the DAPI channel and the ‘analyze particles’ functionality with manual corrections. HEK293T histone PTMs were quantified in transfected cells and non-transfected neighbouring cells using mean grey values. Signal for Strep-tagged ORF8 constructs (Strep-Tactin-488) and GFP was used to define transfected cells, and the HEK293T histone PTM levels in transfected cells were relativized to the histone PTM levels in non-transfected neighbouring cells. Histone PTMs were quantified in A549 cells and human lung tissue using integrated density values. dsRNA and SARS-CoV-2 nucleocapsid signal was used to define infected A549 cells and human lung cells, respectively. The total fluorescence intensity of the lamin A/C and lamin B1 signal was measured from the whole nuclei of mock and ORF8-positive cells. Analysis of the peripheral heterochromatin organization was performed as a comparison of a fraction of H3K9me2-marked chromatin at the nuclear lamina/periphery of mock and ORF8-positive cells. A fraction of H3K9me2 signal at the nuclear lamina/periphery was measured using lamin B signal as a mask or DAPI signal to create a mask of a 0.6-μm-thick nuclear peripheral zone.
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. SARS-CoV-2 protein sequences were obtained from protein sequences published for the first Wuhan isolate56.
HEK293T cell pellets were gently resuspended in 1 ml FACS buffer (Ca2+/Mg2+-free PBS with 2% BSA) and pelleted at 500 g for 5 min at 4 °C; the supernatant was removed. Cells transfected with ORF8 construct and non-transfected control cells were then gently resuspended in 1 ml FACS buffer with a 1:500 dilution of Strep-Tactin DY-488 and rotated at 4 °C for 1 h, protected from light. Cells were then washed twice in 1 ml FACS buffer, resuspended in 1 ml FACS buffer and filtered through a 35-µm mesh into FACS tubes. A BD Influx cell sorter was used to analyse cells. Strep-Tactin DY-488 and GFP were excited with a 488-nm laser and signal was collected with a 530/40-nm detector. Excluding doublets and cell debris, cells were gated on the Strep-Tactin DY-488 signal, where thresholds were set using non-transfected control cells such that <1% of control cells were considered positive for Strep-Tactin DY-488. Strep-Tactin DY-488-positives cells were collected in FACS buffer and pelleted for subsequent experiments. The FACS gating strategy and cell numbers isolated are shown in Supplementary Fig. 2.
Transfected cells were isolated by FACS as described above. Sorted cells were pelleted, resuspended in 1 ml cold H2SO4 and rotated overnight at 4 °C. Following the overnight incubation, cells were pelleted at maximum speed and the supernatant was transferred to a fresh tube. Trichloroacetic acid was added to 25% by volume, and the cells were left on ice at 4 °C overnight. Cells were again pelleted at maximum speed, and the supernatant was discarded. Prechilled acetone was then used to gently wash the pellet twice. Following the second wash, the tubes were left to air dry before the pellet was resuspended in water. Samples were then broken up by alternating 10 min of sonication and 30 min of shaking at 50 °C until pellets were fully dissolved.
Histone PTM analysis by quantitative mass spectrometry
Purification of histones was validated by SDS–PAGE followed by Coomassie staining demonstrating sufficient enrichment. A BCA assay (Thermo Fisher) was performed for protein estimation using the manufacturer’s instructions, and 20 µg of histone was used for chemical derivatization and digestion as described previously57. In brief, unmodified lysines were derivatized twice with a 1:3 ratio of acetonitrile to proprionic anhydride. Histones were then digested with trypsin in a 1:20 enzyme to protein ratio at 37 °C overnight. Digested histones with newly formed N termini were derivatized twice as done previously. Finally, histones were dried with a vacuum concentrator. The dried samples were reconstituted in 0.1% trifluoroacetic acid (TFA) and desalted with the C18 micro spin column (Harvard Apparatus). The column was prepared with 200 μl of 100% acetonitrile and equilibrated with 200 μl of loading buffer with 0.1% TFA. Peptides were loaded onto the column, washed with loading buffer and eluted with 200 μl of 70% acetonitrile in 0.1% formic acid. All steps for loading, washing and elution were carried out with benchtop centrifugation (300 g for 2 min). The eluted peptides were then dried in a centrifugal vacuum concentrator.
Dried histone peptides were reconstituted in 0.1% formic acid. A synthetic library of 93 heavy labelled and derivatized peptides containing commonly measured histone PTMs58 was spiked into the endogenous samples to a final concentration of approximately 100 ng µl–1 for endogenous peptides and 100 fmol µl–1 for each heavy labelled synthetic analyte. For each analysis, 1 µl of sample was injected onto the column for data-independent analysis on a Q-Exactive HF instrument (Thermo Scientific) attached to an Ultimate 3000 nano-UPLC system and Nanospray Flex ion source (Thermo Scientific). Using aqueous solution of 0.1% formic acid as buffer A and organic solution of 80% acetonitrile and 0.1% formic acid as buffer B, peptides were separated on a 63-min gradient at 400 nl min–1 starting at 4% buffer B and increasing to 32% buffer B over 58 min and then increasing to 98% buffer B over 5 min. The column was then washed at 98% buffer B over 5 min and equilibrated to 3% buffer B. Data-independent acquisition was performed with the following settings. A full MS1 scan from 300 to 950 m/z was acquired with a resolution of 60,000, an automatic gain control (AGC) target of 3 × 106 and a maximum injection time of 55 ms. Then, a series of 25 MS2 scans was acquired across the same mass range with sequential isolation windows of 24 m/z with a collision energy of 28, a resolution of 30,000, an AGC target of 1 × 106 and a maximum injection time of 55 ms. Data analysis and manual inspection using the synthetic library as a reference were performed with Skyline (MacCoss Lab). Ratios were generated using R Studio and statistical analysis was carried out in Excel as in previous histone analysis.
Trypsin and chymotrypsin digestion of ORF8 for identification of ORF8 modifications
The gel band containing ORF8 was destained with 50 mM ammonium bicarbonate with 50% acetonitrile. The band was then reduced in 10 mM DTT in 50 mM ammonium bicarbonate for 30 min at 55 °C. Next, the band was alkylated with 100 mM iodoacetamide in 50 mM ammonium bicarbonate at room temperature for 30 min in the dark. Protein was then digested by incubation with chymotrypsin or trypsin at an approximately 1:20 enzyme to protein ratio at 37 °C overnight. Following digestion, the supernatant was collected. To extract additional peptides from the gel, 150 μl of 50% acetonitrile and 1% TFA was added and samples were incubated with constant shaking for 30 min. The supernatant was collected and 100 μl of acetonitrile was added followed by incubation with constant shaking for 10 min. The final supernatant was collected. All three supernatants were combined and dried. The dried samples were then desalted as described above.
ORF8 versus control immunoprecipitation for identification of binding partners
ORF8 immunoprecipitation elutants were reduced and alkylated as described above. Proteins were then digested and desalted with mini S-Trap (Protifi) following the manufacturer’s instructions. In brief, 25 μl of elutant was combined with 25 μl of 10% SDS to a final SDS concentration of 5% after alkylation. Samples were then acidified with phosphoric acid and precipitated by adding 90% methanol in 100 mM triethylammonium bicarbonate (TEAB) in a 6:1 (vol/vol) ratio. Protein was then added to the trap with benchtop centrifugation (4,000 g for 1 min), washed and digested with trypsin at a 1:10 enzyme to protein ratio at 37 °C overnight. Following digestion, peptides were eluted from the trap with 40 μl of 100 mM TEAB, 40 μl of 0.2% formic acid and 40 μl of 50% acetonitrile in 0.2% formic acid. Combined elutant volumes were then dried.
Chymotrypsin LC–MS/MS and LC–PRM-MS analysis
Dried peptides were reconstituted with 0.1% formic acid, and 2 µg of each sample was injected. Chymotrypsin-digested ORF8 samples were analysed on a Q-Exactive (Thermo Scientific) coupled to an Easy nLC 1000 UHPLC system and Nanospray Flex ion source (Thermo Scientific). The LC instrument was equipped with a 75 µm × 20 cm column packed in house using Reprosil-Pur C18 AQ (2.4 µm; Dr. Maisch). Using the same column and buffer conditions as described previously, peptides were separated on an 85-min gradient at 400 nl min–1 starting at 3% buffer B and increasing to 32% buffer B over 79 min and then increasing to 50% buffer B over 5 min and finally increasing to 90% buffer B over 1 min. The column was then washed at 90% buffer B over 5 min and equilibrated to 3% buffer B. Data-dependent acquisition was performed with dynamic exclusion of 40 s. A full MS1 scan from 350 to 1,200 m/z was acquired with a resolution of 70,000, an AGC target of 1 × 106 and a maximum injection time of 50 ms. Then, a series of MS2 scans was acquired for the top 15 precursors with a charge state of 2–7, a collision energy of 28 and an isolation window of 2.0 m/z. Each MS2 scan was acquired with a resolution of 17,500, an AGC target of 2 × 105 and a maximum injection time of 50 ms. A database search was performed using the human SwissProt sequence and ORF8 sequence with Proteome Discoverer 2.3 or 2.4 (Thermo Scientific) using the following search criteria: carboxyamidomethylation at cysteine residues as a fixed modification; oxidation at methionine and acetylation at lysine as variable modifications; two maximum allowed missed cleavages; precursor MS tolerance of 10 ppm; a 0.02-Da MS/MS. An unscheduled parallel reaction-monitoring method59 was developed to identify or validate 45 possible modified and unmodified peptide targets of ORF8. Peptides were separated with the same LC gradient conditions. A full MS1 scan from 300 to 900 m/z was acquired with a resolution of 70,000, an AGC target of 1 × 106 and a maximum injection time of 50 ms. Then, a series of MS2 scans was acquired with a loop count of 23 precursors, a collision energy of 28 and an isolation window of 1.2 m/z. Each MS2 scan was acquired with a resolution of 17,500, an AGC target of 1 × 106 and a maximum injection time of 100 ms. Data analysis and manual inspection were performed with Skyline60 (MacCoss Lab) and IPSA61.
Trypsin ORF8 LC–MS/MS and LC–PRM/MS analysis and IP LC–MS/MS analysis
Dried peptides were reconstituted with 0.1% formic acid, and 2 µg of each sample was injected. Data-dependent acquisition runs were analysed on a Q-Exactive HF or HF-X (Thermo Scientific) attached to an Ultimate 3000 nano UPLC system and Nanospray Flex Ion Source (Thermo Scientific). Using the same column and buffer conditions as described above, peptides were separated on a 112-min gradient at 400 nl min–1 starting at 5% buffer B, increasing to 35% buffer B over 104 min and then increasing to 60% buffer B over 8 min. The column was then washed at 95% buffer B for 5 min and equilibrated to 5% buffer B. Data-dependent acquisition was performed with dynamic exclusion of 45 s. A full MS1 scan from 380 to 1,200 m/z was acquired with a resolution of 120,000, an AGC target of 3 × 106 and a maximum injection time of 32 ms. Then, a series of MS2 scans were acquired for the top 20 precursors with a charge state of 2–5, a collision energy of 28 and an isolation window of 1.2 m/z. Each MS2 scan was acquired with a resolution of 30,000, an AGC target of 1 × 106 and a maximum injection time of 32 ms (HF) or 55 ms (HFX). A database search was performed using the human SwissProt sequence and ORF8 sequence with Proteome Discoverer 2.3 or 2.4 (Thermo Scientific) with the following search criteria: carboxyamidomethylation at cysteine residues as a fixed modification; oxidation at methionine and acetylation at lysine as variable modifications; two maximum allowed missed cleavages; precursor MS1 tolerance of 10 ppm; a 0.02-Da MS2 tolerance. An unscheduled parallel reaction-monitoring method59 was developed to identify 16 possible modified and unmodified peptide targets of ORF8. Peptides were separated with the same LC gradient conditions. A full MS1 scan from 350 to 950 m/z was acquired with a resolution of 120,000, an AGC target of 3 × 106 and a maximum injection time of 100 ms. Then, a series of MS2 scans were acquired with a loop count of 16 precursors, a collision energy of 28 and an isolation window of 1.2 m/z. Each MS2 scan was acquired with a resolution of 30,000, an AGC target of 1 × 106 and a maximum injection time of 100 ms. Data analysis and manual inspection were performed with Skyline60 (MacCoss Lab) and IPSA61.
Statistics and reproducibility
Box-and-whisker plots show the median as the centre line, box limits for upper and lower quartiles, whiskers for 1.5× the interquartile range and points for outliers. ANOVA testing was performed and plots were generated with R. Bonferroni corrections were applied for multiple comparisons. Fiji was used for image analysis. Imaging and analysis were performed with the experimenter blinded to the experimental condition whenever possible. In some instances, such as for patient tissue imaging, analysis required targeted selection, imaging and analysis of infected cells compared with uninfected cells. This required the experimenter to be aware of cell infection status while imaging. However, in these cases, the measurement of interest (such as staining for a histone modification) was not viewed before choosing fields to avoid biasing selection.
Images are representative of multiple replicates as follows:
Figure 1b: >5 independent experiments.
Figure 1c: two independent experiments.
Figure 1d: three independent experiments.
Figure 1g: five independent samples from two separate runs of FACS sorting.
Figure 2h: two shown of four independent samples from one FACS sort.
Figure 3f: three independent samples per condition from one infection.
Extended Data Fig. 2a: three independent experiments.
Extended Data Fig. 2b,c: >5 independent experiments.
Extended Data Fig. 2d: two independent experiments.
Extended Data Fig. 2e: two independent experiments.
Extended Data Fig. 3b: two independent experiments.
Extended Data Fig. 4a: lamin and histone H3, three independent experiments; HP1α and KAT2A, two independent experiments.
Extended Data Fig. 4b: two independent experiments.
Extended Data Fig. 4c: two independent experiments.
Extended Data Fig. 4d: one independent experiment, repeating previously published data.
Extended Data Fig. 4e: two independent experiments.
Extended Data Fig. 4f: two independent experiments.
Extended Data Fig. 6b: three shown of five independent samples from two runs of FACS sorting.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.