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SARS-CoV-2 disrupts host epigenetic regulation via histone mimicry - Nature.com

SARS-CoV-2 disrupts host epigenetic regulation via histone mimicry - Nature.com

SARS-CoV-2 disrupts host epigenetic regulation via histone mimicry - Nature.com
Oct 05, 2022 35 mins, 42 secs

In rare cases, viral proteins dampen antiviral responses by mimicking critical regions of human histone proteins4,5,6,7,8, particularly those containing post-translational modifications required for transcriptional regulation9,10,11.

However, how SARS-CoV-2 controls the host cell epigenome and whether it uses histone mimicry to do so remain unclear.

Here we show that the SARS-CoV-2 protein encoded by ORF8 (ORF8) functions as a histone mimic of the ARKS motifs in histone H3 to disrupt host cell epigenetic regulation.

ORF8 is associated with chromatin, disrupts regulation of critical histone post-translational modifications and promotes chromatin compaction.

Deletion of either the ORF8 gene or the histone mimic site attenuates the ability of SARS-CoV-2 to disrupt host cell chromatin, affects the transcriptional response to infection and attenuates viral genome copy number.

These findings demonstrate a new function of ORF8 and a mechanism through which SARS-CoV-2 disrupts host cell epigenetic regulation.

Further, this work provides a molecular basis for the finding that SARS-CoV-2 lacking ORF8 is associated with decreased severity of COVID-19.

Histone proteins are modified by a wide range of post-translational modifications (PTMs) that are dynamically regulated to control gene expression9,10,11.

Histone mimicry allows viruses to disrupt the host cell’s ability to regulate gene expression and respond to infection effectively.

However, no validated cases of histone mimicry have previously been reported within coronaviruses.

Although SARS-CoV-2 probably uses many mechanisms to interfere with host cell functions, we examined whether it uses histone mimicry to disrupt chromatin regulation and the transcriptional response to infection.

To determine whether histone mimicry is used by SARS-CoV-2, we first performed a bioinformatic comparison of all SARS-CoV-2 viral proteins18 with all human histone proteins (Extended Data Fig. 1a,b).

Most SARS-CoV-2 proteins are highly similar to those in the coronavirus strain that caused the previous major SARS-CoV outbreak with the notable exception of the proteins encoded by ORF3b and ORF8, of which ORF8 is the most divergent in SARS-CoV-2 (refs 19,20).

Notably, we detected an identical match between amino acids 50–55 of the protein encoded by ORF8 and critical regions within the histone H3 N-terminal tail (Fig. 1a).

Furthermore, ORF8 aligns to a longer sequential set of amino acids (six residues) than in any previously described and validated case of histone mimicry4,5,6,7,21 or a putative histone mimic in the SARS-CoV-2 envelope protein22,23 (Extended Data Fig. 1c,d).

On the basis of a crystal structure of ORF8, these residues are located in a disordered region on the surface of the protein in an ORF8 monomer24.

Most compellingly, the motif contains the ‘ARKS’ sequence, which is found at two distinct sites in the histone H3 tail (Fig. 1a) and is well established as one of the most critical regulatory regions within H3.

This amino acid stretch is absent from the previous SARS-CoV ORF8-encoded protein both before and after a deletion generated ORF8a and ORF8b25 but is present in bat SARS-CoV-2 and variants of concern (Extended Data Fig. 1e,f).

ORF8 is highly expressed during infection26,27, with ORF8 transcript expressed at higher levels than histone H3 and ORF8 protein expressed at over 20% above the level of the most abundant histone H3 protein within 24 h of infection28 (Extended Data Fig. 1g,h).

Finally, proteomic characterization of SARS-CoV-2 protein binding partners indicates that ORF8 binds DNA methyltransferase 1 (DNMT1)22,29.

a, ORF8 contains an ARKS motif at amino acid 50 that matches the histone H3 tail.

b, Lamin A/C staining of HEK293T cells transfected to express Strep–ORF8.

c, ORF8 and lamin A/C staining of SARS-CoV-2-infected A549ACE2 cells at MOI = 1, 48 h after infection.

d, Sequential salt extraction of HEK293T cells expressing ORF8 or ORF8ΔARKSAP.

g, ORF8 expression results in decreased levels of KAT2A.

To determine whether ORF8 functions as a histone mimic, we began by examining its intracellular localization.

We transfected HEK293T cells with a construct encoding Strep-tagged ORF8 and visualized ORF8 with a Strep-Tactin-conjugated fluorescent probe.

Although ORF8 localization was variable in appearance, ORF8 was typically located in the cytoplasm and at the periphery of the nucleus when using immunofluorescence (Fig. 1b), as previously reported30, and in both the cytoplasm and nucleus when using cell fractionation (Extended Data Fig. 2a).

Given the observed expression pattern, we next asked whether ORF8 colocalizes with lamin proteins.

We found that ORF8 colocalized with lamin B1 and lamin A/C in cells transfected to express ORF8 (Fig. 1b and Extended Data Fig. 2b,c).

Next, we infected an A549 lung epithelial-derived cell line expressing the ACE2 receptor (A549ACE2) with SARS-CoV-2, stained cells with an antiserum specific to ORF8 (Extended Data Fig. 2d,e) and confirmed a similar expression pattern in infected cells (Fig. 1c).

Notably, while other functions have been proposed for ORF8 (refs 30,31,32,33,34,35,36,37), a potential role for ORF8 in the nucleus of host cells and specifically in regulating chromatin has not been explored.

We found that ORF8 dissociated from the chromatin fraction at salt concentrations similar to those at which lamin and histone proteins dissociate (Fig. 1d).

By contrast, ORF8 with a deletion of the ARKSAP motif (ORF8ΔARKSAP) dissociated at lower salt concentrations and was present at lower levels in the chromatin fraction in comparison to ORF8 with this motif (Fig. 1d and Extended Data Fig. 2f,g), indicating that the putative histone mimic site affects the strength of ORF8’s association with chromatin.

Although ORF8 did not have clearly defined peaks, ORF8 immunoprecipitation showed enrichment over input (Fig. 1e) and ORF8 was enriched within specific genomic regions, particularly those associated with H3K27me3 (Extended Data Fig. 2h–k).

We found that ORF8 co-immunoprecipitated with lamin B1, histone H3 and HP1α, a protein associated with both lamin proteins and histones (Extended Data Fig. 3a).

Reciprocal co-immunoprecipitation for lamin B1 and histone H3 confirmed ORF8 binding (Extended Data Fig. 3b).

Next, we tested whether ORF8 also co-immunoprecipitates with the histone-modifying enzymes that target the ARKS motif within histone H3.

We found that ORF8 was associated with the histone acetyltransferase KAT2A (also known as GCN5), which targets H3K9 (Fig. 1f).

Although both ORF8 and ORF8ΔARKSAP immunoprecipitated with a previously established cytoplasmic binding partner, HLA-A2 (ref. 30), we did not detect ORF8ΔARKSAP association with chromatin proteins, indicating that the ARKSAP motif strengthens ORF8’s association with chromatin proteins (Extended Data Fig. 3c,d).

Further, ORF8 did not bind to BRD4, which preferentially binds acetylated histone H4 (Extended Data Fig. 3e).

However, the transcription factor SP2 was detected and confirmed to bind to ORF8 by co-immunoprecipitation (Extended Data Fig. 3f).

On the basis of the observation that ORF8 associates with KAT2A, we used targeted mass spectrometry to determine whether the proposed ORF8 histone mimic site is modified similarly to histones.

Using a bottom–up approach, ORF8 was purified from cells, reduced, alkylated and digested.

High-resolution mass spectrometry differentiated the precursor from the trimethylated peptide and matched all product ions within a mass error of 10 ppm (Fig. 1f and Extended Data Fig. 3g).

This demonstrates that ORF8 is acetylated on the lysine within the proposed ARKS histone mimic site, similarly to histone H3.

Notably, presence of acetylated lysine within the ARKSAP motif is probably incompatible with dimerization of ORF8, which involves a hydrogen-bond interaction at this residue24, and thus suggests that ORF8 can exist as a monomer within cells.

ORF8 expression resulted in a marked decrease in the abundance of KAT2A (Fig. 1g), whereas levels of nuclear lamina proteins and lamina-associated heterochromatin were unchanged or slightly increased (Extended Data Fig. 3h–l).

These findings suggest that not only does ORF8 associate with proteins such as acetyltransferases, but it probably also is modified by them similarly to histone H3 and induces their degradation.

Taken together, these findings demonstrate that ORF8 is well positioned to act as a histone mimic on the basis of its association with chromatin and chromatin-modifying enzymes and its ability to deplete the histone acetyltransferase KAT2A.

We next examined whether ORF8 expression disrupts histone PTMs using an unbiased mass spectrometry approach.

HEK293T cells were transfected with a control plasmid encoding GFP or with a plasmid encoding ORF8 with a Strep tag.

Transfected cells, identified by GFP fluorescence or by a Strep-Tactin-conjugated fluorescent probe, were isolated using fluorescence-activated cell sorting (FACS).

Histones were purified through acid extraction, and bottom–up unbiased mass spectrometry was performed to quantify all detected histone PTMs.

Notably, histone modifications associated with transcriptional repression were increased while numerous histone modifications associated with active gene expression were depleted in cells expressing ORF8 (Fig. 2a).

For example, the peptides containing methylated H3K9 and H3K27, which are associated with transcriptional repression, showed robustly increased abundance in response to ORF8 expression.

Conversely, the peptide containing both H3K9ac and H3K14ac, both of which have a well-established link to active gene expression, showed decreased abundance in response to ORF8 expression.

These data support a role for ORF8 as a putative histone mimic and demonstrate that it is capable of disrupting histone PTM regulation at numerous critical sites within histones.

a, Mass spectrometry analysis of histone PTMs in control (GFP-expressing) or ORF8-expressing HEK293T cells isolated by FACS.

The z score and fold change are shown for modifications that were significantly changed in response to ORF8 expression, were detected in over 1% of the total peptide abundance and have well-established functions (full results shown in Supplementary Table 2).

b–g, Immunofluorescence analysis of HEK293T cells transfected to express GFP or Strep–ORF8 showing that ORF8 expression increases H3K9me3 (b,c) and H3K27me3 (d,e) while decreasing H3K9ac (f,g)1

Conversely, ORF8 with deletion of the histone mimic site ARKSAP (ORF8ΔARKSAP) does not affect these histone PTMs.

n = 614 (GFP), 497 (ORF8) and 170 (ORF8ΔARKSAP) cells for H3K9me3; 616, 550 and 154 cells for H3K27me3; and 666, 568 and 170 cells for H3K9ac compiled from three independent transfections.

h, Western blot analysis of histones isolated from FACS-sorted transfected cells2

i, ATAC-seq of HEK293T cells expressing GFP, ORF8 or ORF8ΔARKSAP isolated by FACS.

We found that cells expressing ORF8 exhibited increased H3K9me3 and H3K27me3 and decreased H3K9ac staining compared with those transfected with control plasmid (Fig. 2b–g).

ORF8 expression did not significantly disrupt H3K27ac, global acetylation, H3S10 phosphorylation, H3K9me2 or lamin B (Extended Data Fig. 4a,b).

Although ORF8ΔARKSAP was expressed at similar levels to ORF8 (Extended Data Fig. 4c), it did not increase H3K9me3 or H3K27me3 and had a non-significant intermediate effect on H3K9ac (Fig. 2b–g).

Next, we examined an acquired mutation in ORF8 commonly found in SARS-CoV-2 strains encoding an S84L substitution (ORF8S84L).

This site is unlikely to affect protein stability31,39 and lies outside the histone mimic region, and the substitution is thus not expected to affect the ability of ORF8 to regulate histone PTMs.

Expression of ORF8S84L also increased H3K9me3 and H3K27me3 levels while decreasing H3K9ac (Extended Data Fig. 4d–f), indicating that, as predicted, this common variant does not alter the histone mimic function of ORF8.

Similarly, a six-residue deletion in another unstructured region of ORF8 with similar amino acid make-up but a different sequence (AGSKSP) as the histone mimic site did not affect the ability of ORF8 to disrupt histone regulation (Extended Data Fig. 4g).

To ensure equal levels of expression of ORF8 and ORF8ΔARKSAP, we isolated transfected cells by FACS (Extended Data Fig. 5a).

Similarly, CUT&Tag sequencing of H3K9ac demonstrated that ORF8, but not ORF8ΔARKSAP, deceased H3K9ac (Extended Data Fig. 5b,c).

Finally, assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) demonstrated that ORF8, but not ORF8ΔARKSAP, decreased chromatin accessibility (Extended Data Fig. 5d and Fig. 2i).

The changes in both H3K9ac and chromatin accessibility were largely global but were particularly evident for genes with intermediate to high expression (Extended Data Fig. 5e–h).

To determine how these chromatin disruptions affect gene expression, we used RNA sequencing (RNA-seq) to define differentially expressed genes in transfected cells (Extended Data Fig. 6a–c).

While ORF8 and ORF8ΔARKSAP shared a subset of differentially expressed genes, the presence of the histone mimic motif resulted in less dynamic gene expression changes.

Distinct gene groups were also differentially expressed between ORF8 and ORF8ΔARKSAP, with ORF8 decreasing gene expression relative to ORF8ΔARKSAP, particularly highly expressed genes (Extended Data Fig. 6d–i and Supplementary Table 3).

Genes that were downregulated in response to ORF8 expression relative to ORF8ΔARKSAP also had higher basal levels of H3K9ac and greater accessibility than genes that were upregulated (Extended Data Fig. 6j,k), suggesting that they may be more sensitive to depletion of H3K9ac.

Together, these results support a model in which ORF8 has multiple functions as previously proposed30,31,32,33,40 and activates a number of gene expression pathways, particularly in the absence of the ARKSAP motif.

However, presence of the ARKSAP motif dampens the host cell transcriptional response and decreases expression of genes with high accessibility and H3K9ac.

Together, these data define a role for ORF8 in disruption of host cell histone PTMs through a new case of histone mimicry of the ARKS motifs in histone H3.

Having shown that ORF8 alone is sufficient to disrupt chromatin regulation, we next examined the effect of ORF8 on histone PTM regulation in the context of viral infection.

We generated a recombinant mutant SARS-CoV-2 virus with a deletion of ORF8 (SARS-CoV-2ΔORF8) using a cDNA reverse genetics system41,42.

We infected A549ACE2 cells with SARS-CoV-2 or SARS-CoV-2ΔORF8 and compared the levels of the viral genomes and infectious virus production in the presence and absence of ORF8.

Because of their overexpression of the ACE2 receptor, these cells are readily and rapidly infected by SARS-CoV-2 and thus provide an ideal system in which to compare the cellular responses to mutant forms of the virus without the confounding factor of different rates of infection.

No differences in genome copy number or viral titre were detected at 24 h, and only subtle differences were observed at 48 h (Extended Data Fig. 7a,b and Fig. 3a,b), allowing for direct comparison of these two viruses at these early time points after infection.We therefore infected A549ACE cells with SARS-CoV-2 or SARS-CoV-2ΔORF8 and used ChIP–seq with spike-in normalization (ChIP-RX) to allow for the detection of global changes in histone PTMs.

We found that SARS-CoV-2 infection resulted in robust increases in H3K9me3 and H3K27me3 compared with mock-infected cells (Extended Data Fig. 7c–e), mirroring the effects of ORF8 expression.

However, deletion of ORF8 substantially attenuated this effect, indicating that the effect of SARS-CoV-2 on repressive histone modifications is partly due to ORF8 expression.

Finally, ChIP-RX indicated that SARS-CoV-2 infection resulted in decreased H3K9ac, and this effect was again attenuated in cells infected with SARS-CoV-2ΔORF8 (Fig. 3d,e).

These data demonstrate that ORF8 contributes to the effects of SARS-CoV-2 infection on chromatin accessibility and histone modifications in host cells.

a,b, Reverse transcription with quantitative PCR (qRT–PCR) analysis of expression of the SARS-CoV-2 gene RDRP (a) and plaque assay analysis of viral titre (b) in A549ACE cells 48 h after infection with wild-type SARS-CoV-2 (SARS-CoV-2WT), SARS-CoV-2ΔARKSAP or SARS-CoV-2ΔORF8 at MOI = 1.

f, Western blot analysis of KAT2A in A549ACE cells following infection with wild-type or mutant SARS-CoV-2 viruses.

g, Post-mortem lung tissue from patients with COVID-19 stained for H3K9me3 and nucleocapsid protein to identify SARS-CoV-2-infected cells. Arrows indicate infected cells.

h, Quantification of H3K9me3 in infected cells compared with neighbouring cells and with control tissue.

n = 12 infected cells and 131 uninfected neighbouring cells from three patients with COVID-19 and 60 cells from three control individuals.

Because it is likely that ORF8 has multiple effects on cellular function, on the basis of both recent puplications30,31,32,33,40 and our mechanistic data, we also sought to determine whether these effects were specifically due to the histone mimic motif.

In A549ACE2 cells, SARS-CoV-2ΔARKSAP replicated similarly to wild-type virus (Fig. 3a,b) but substantially alleviated the effect of infection on chromatin accessibly and H3K9ac, matching the effects of ORF8 deletion (Fig. 3c–e).

Given the robust effects of SARS-CoV-2 on H3K9ac and the ability of ORF8 to deplete KAT2A (Fig. 1g), we also examined the effect of infection on KAT2A levels.

Wild-type SARS-CoV-2 infection reduced KAT2A expression, whereas infection with SARS-CoV-2ΔORF8 or SARS-CoV-2ΔARKSAP did not (Fig. 3f).

These data indicate that ORF8, and specifically the ARKSAP motif within ORF8, contributes to the effects of SARS-CoV-2 on the host cell epigenome.

To ensure that the differences observed in host cell chromatin regulation following SARS-CoV-2 and SARS-CoV-2ΔORF8 infection are not due to any subtle difference in rates of infection between viruses, we sought to further confirm these finding using an approach that is independent of the number of cells infected.

We used immunocytochemistry to stain for histone modifications of interest, using staining for double-stranded RNA (dsRNA) to identify and specifically examine infected cells.

At 24 h after infection, cells infected with SARS-CoV-2 had increased H3K9me3 and H3K27me3 and decreased H3K9ac compared with either mock-infected cells or uninfected neighbouring cells (Extended Data Fig. 8a–f).

We stained tissue for H3K9me3 as well as for SARS-CoV-2 nucleocapsid protein to identify infected cells.

We found that, in all patient samples, infected cells showed increased H3K9me3 staining compared with neighbouring cells within the same tissue, as well as compared with control tissue (Fig. 3g,h and Extended Data Fig. 8g).

While sample availability limits the conclusions that can be drawn from this assay, this finding indicates that histone PTMs are also disrupted in patients with severe COVID-19 disease.

In summary, we found that the effects of SARS-CoV-2 infection on histone PTMs and chromatin compaction require ORF8 expression and mirror the ARKSAP-dependent effects of ORF8.

Next, we examined how the changes in histone PTMs detected through ChIP–seq relate to gene expression using RNA-seq.

All viruses contained similar numbers of reads, and the only difference in SARS-CoV-2 transcript expression was for ORF8 in SARS-CoV-2ΔORF8 (Extended Data Fig. 9a–d).

However, in wild-type virus, ORF8 transcript was highly expressed and more abundant than histone H3-encoding transcripts (Extended Data Fig. 9e).

Interestingly, early in infection, the three viruses tested each disrupted a distinct set of genes, indicating that presence of the histone mimic motif changes the transcriptional response to infection (Fig. 4a–c).

By 48 h after infection, all three viruses made up the vast majority of the mapped reads and resulted in robust changes in gene expression compared with mock-infected cells (Extended Data Fig. 9c,f,g).

The functional groups of genes most induced by infection also differed among the three viruses, indicating distinct host cell responses at early time points (Fig. 4d and Extended Data Fig. 10a).

This is notable given that wild-type SARS-CoV-2 and SARS-CoV-2ΔARKSAP had nearly identical copy numbers and replication rates in A549ACE2 cells (Fig. 3a,b), and thus the different transcriptional responses are unlikely to be due to differences in the number of cells infected or the viral load within infected cells.

Interestingly, direct comparison of SARS-CoV-2ΔORF8 and SARS-CoV-2ΔARKSAP also showed distinct gene expression changes and functional group enrichment (Extended Data Fig. 10b,c), indicating again that ORF8 probably has multiple functions beyond those mediated by the ARKSAP domain.

In addition, gene expression changes in response to infection were correlated with changes in H3K9ac (Extended Data Fig. 10d–f).

Notably, these data further support recent findings indicating that SARS-CoV-2 results in a limited early transcriptional response1,2,43 and demonstrate that the ORF8 ARKSAP domain is linked to changes in gene expression.

a, Differential gene expression analysis by RNA-seq of A549ACE2 cells 24 h after infection with SARS-CoV-2WT, SARS-CoV-2ΔORF8 or SARS-CoV-2ΔARKSAP, compared with mock infection.

e,f, qRT–PCR analysis of expression of the SARS-CoV-2 gene RDRP (e) and plaque assay analysis of viral titre (f) in iAT2 pulmonary cells at 48 h after infection with SARS-CoV-2WT, SARS-CoV-2ΔORF8 or SARS-CoV-2ΔARKSAP at MOI = 1.

Given the robust effects of ORF8 deletion on host cell chromatin regulation and the transcriptional response to infection, we sought to test whether ORF8 mediates the replication of SARS-CoV-2 using a physiologically relevant cell type.

Induced human pluripotent stem cell-derived lung alveolar type II (iAT2) pulmonary cells44 were infected with SARS-CoV-2, SARS-CoV-2ΔORF8 or SARS-CoV-2ΔARKSAP (multiplicity of infection (MOI) = 1).

Notably, we observed that both mutant viruses had decreased genome copy numbers at 48 h after infection in most replicates (Fig. 4e and Supplementary Table 4), suggesting that ORF8, and specifically the ARKSAP domain, affects SARS-CoV-2 genome replication in a host cell.

Taken together, this work presents a link between a specific SARS-CoV-2 protein and the epigenetic disruptions that occur in response to infection and provides a mechanistic explanation for mounting evidence12,13,45 that epigenetic disruptions contribute to the severity of COVID-19.

The work described here identifies a new case of histone mimicry during infection by SARS-CoV-2 and defines a mechanism through which SARS-CoV-2 acts to disrupt host cell chromatin regulation.

We found that the protein encoded by the SARS-CoV-2 ORF8 gene contains an ARKS motif and that ORF8 expression disrupts histone PTM regulation.

ORF8 is associated with chromatin-associated proteins, histones and the nuclear lamina and is itself acetylated within the histone mimic motif similarly to histones.

ORF8 expression disrupts multiple critical histone PTMs and promotes chromatin compaction, whereas ORF8 lacking the histone mimic motif does not.

Further, SARS-CoV-2 infection in human cell lines and post-mortem patient lung tissue causes similar global disruptions to chromatin acting in part through the histone mimic.

In addition, deletion of the ORF8 gene or the sequence encoding the histone mimic affects the host cell transcriptional response to SARS-CoV-2 infection.

Finally, loss of ORF8 decreases the replication of SARS-CoV-2 in human induced pluripotent stem cell-derived iAT2 pulmonary cells while loss of the histone mimic motif specifically affects viral genome copy number.

Notably, the role of ORF8 in chromatin disruption early in infection is not inconsistent with other proposed roles for ORF8 in other cellular compartments or at later stages of infection30,31,32,34,46 and does not preclude other proposed mechanisms of transcriptional disruption in response to SARS-CoV-2 (ref. 23).

In fact, our data point towards a model in which ORF8 has multiple functions, including acting as a histone mimic motif.

The effects of deletion of accessory proteins from SARS-CoV-2 in a transgenic mouse model appear complex, with ORF8 loss causing decreases in replication and viral load but having limited effects on survival47.

However, data from patients with COVID-19 were used to examine a rare 382-nucleotide deletion variation in SARS-CoV-2 isolated in Singapore that results in the loss of a small portion of ORF7B and the majority of the ORF8 gene.

Our findings in human iAT2 pulmonary cells point towards the loss of ORF8 as a possible cause for these differences and provide an epigenetic mechanism underlying the role of ORF8 in promoting SARS-CoV-2 virulence within the patient population.

Cells were infected at an MOI of 1 and fixed or lysed at 24 or 48 h after infection.

Calcium phosphate transfection was used to introduce plasmid DNA encoding GFP, ORF8 and mutant ORF8 into HEK293T cells.

Seventy-two hours after cell plating, cells were infected with SARS-CoV-2 virus using an MOI of 1 for 48 h.

HEK293T and Vero E6 cells were obtained from ATCC at the onset of this project.

The ORF8 expression plasmid was obtained from Addgene, pLVX-EF1alpha-SARS-CoV-2-orf8-2xStrep-IRES-Puro (Addgene plasmid 141390).

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.

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).

HEK293T cells were stained and sorted to isolate transfected cells using the same method as described below.

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 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.

RNA was digested with RNase for 1 h at 37 °C, and protein was digested with proteinase K for 30 min at 55 °C.

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).

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.

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).

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.

Seven to ten cycles of PCR were used for histone PTM libraries and 5 cycles were used for input controls.

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’.

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.

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.

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.

For RNA-seq analysis for HEK293T cell experiments, the GRCh38 assembly was used.

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.

After sonication, lysates were brought to a concentration of 1% Triton X-100 to disrupt lamina protein interactions.

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.

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.

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.

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).

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.

For comparison of lamin A/C and lamin B1 signal intensities between mock and ORF8-positive cells, single-plane confocal images were acquired.

Single-z-plane images of HEK293T cells and human lung tissue and summed z stacks through A549 nuclei were used for PTM quantification.

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.

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.

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 light2

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.

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.

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.

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.

Histones were then digested with trypsin in a 1:20 enzyme to protein ratio at 37 °C overnight.

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.

Ratios were generated using R Studio and statistical analysis was carried out in Excel as in previous histone analysis.

Protein was then digested by incubation with chymotrypsin or trypsin at an approximately 1:20 enzyme to protein ratio at 37 °C overnight.

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.

In some instances, such as for patient tissue imaging, analysis required targeted selection, imaging and analysis of infected cells compared with uninfected cells.

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.

4a: lamin and histone H3, three independent experiments; HP1α and KAT2A, two independent experiments.

5a: exact cell numbers and replicates described in Extended Data Fig.

5c: exact cell numbers and replicates described in Extended Data Fig.

5e: same images as in Extended Data Fig.

10a: exact cell numbers and replicates described in Extended D ata Fig.

10c: exact cell numbers and replicates described in Extended Data Fig.

10e: exact cell numbers and replicates described in Extended Data Fig.

SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial-derived cells and cardiomyocytes.

Suppression of the antiviral response by an influenza histone mimicC

A core viral protein binds host nucleosomes to sequester immune danger signalsD

Adenovirus core protein VII downregulates the DNA damage response on the host genome.

Drawing on disorder: how viruses use histone mimicry to their advantage.

The language of covalent histone modifications.

Translating the histone code.

Methylation of a histone mimic within the histone methyltransferase G9a regulates protein complex assembly.

A SARS-CoV-2 protein interaction map reveals targets for drug repurposing.

Binding of the SARS-CoV-2 envelope E protein to human BRD4 is essential for infection.

Structure of SARS-CoV-2 ORF8, a rapidly evolving immune evasion protein.

ORF8 and ORF3b antibodies are accurate serological markers of early and late SARS-CoV-2 infection.

Accurate diagnosis of COVID-19 by a novel immunogenic secreted SARS-CoV-2 orf8 protein.

Proteomics of SARS-CoV-2-infected host cells reveals therapy targets.

The ORF8 protein of SARS-CoV-2 mediates immune evasion through down-regulating MHC-Ι.

Mutations in SARS-CoV-2 ORF8 altered the bonding network with interferon regulatory factor 3 to evade host immune system.

The ORF8 protein of SARS-CoV-2 induced endoplasmic reticulum stress and mediated immune evasion by antagonizing production of interferon β.

SARS-CoV-2 ORF8 forms intracellular aggregates and inhibits IFNγ-induced antiviral gene expression in human lung epithelial cells.

ORF8 contributes to cytokine storm during SARS-CoV-2 infection by activating IL-17 pathway.

Viral mimicry of interleukin-17A by SARS-CoV-2 ORF8.

Severe acute respiratory syndrome coronavirus 2 ORF8 protein inhibits type I interferon production by targeting HSP90B1 signaling.

The ORF6, ORF8 and nucleocapsid proteins of SARS-CoV-2 inhibit type I interferon signaling pathway.

SARS-CoV-2 accessory protein ORF8 is secreted extracellularly as a glycoprotein homodimer.

Similarities and differences in the conformational stability and reversibility of ORF8, an accessory protein of SARS-CoV-2, and its L84S variant.

Lost in deletion: the enigmatic ORF8 protein of SARS-CoV-2.

SARS-CoV-2 induces double-stranded RNA-mediated innate immune responses in respiratory epithelial-derived cells and cardiomyocytes.

Derivation of self-renewing lung alveolar epithelial type II cells from human pluripotent stem cells.

Structural analysis of SARS-CoV-2 ORF8 protein: pathogenic and therapeutic implications.

Identification of a novel SARS-CoV-2 variant with a truncated protein in ORF8 gene by next generation sequencing.

Complete workflow for analysis of histone post-translational modifications using bottom-up mass spectrometry: from histone extraction to data analysis.

Stable-isotope-labeled histone peptide library for histone post-translational modification and variant quantification by mass spectrometry.

generated cells, samples and DNA constructs and performed analysis

generated iAT2 cells with guidance from W.Y

(a) Alignments performed to identify putative histone mimic sites within the SARS-CoV-2 genome

(b) The number of exact sequential overlapping amino acids found between SARS-CoV-2 proteins and histone proteins

(c) The overlap of Orf8 and H3 compared to other proposed cases of histone mimicry

Protein E is a previously proposed mimic in SARS-CoV-2

G9a is a human protein that mimics H3

(d) Previously proposed histone H3 mimic in SARS-CoV-2 protein E

(g-h) Orf8 transcript (g) and protein (h) expression in SARS-CoV-2 infected Caco-2 cells from published datasets

Subcellular fractionation of HEK293T cells transfected with Strep-Orf8 indicates Orf8 is present in the cytoplasm and nucleus

(b) HEK293T cells expressing Orf8 co-stained with Lamin B and streptactin to detect Orf8

(d) Orf8 antiserum specifically detects Strep-tagged Orf8 by western blot In HEK293T cells transfected with Strep-Orf8

(e) Orf8 antiserum specifically stains infected A549ACE cells 48 h after SARS-CoV-2 infection with no staining observed in mock infection

(g) Cellular fractions generated for use in subsequent chromatin-immunoprecipitation for ChIP-sequencing shows lower ratio of Orf8ΔARKSAP present in chromatin fraction then Orf8

‘-‘ indicates cells that are not expressing Orf8 for negative control IPs performed in parallel

(c) Orf8ΔARKSAP co-immunoprecipitation with LaminB, HP1α, and H3, and KAT2A is not detected

(d) Orf8 and Orf8ΔARKSAP co-immunoprecipitates with MHC1 complex protein HLA-A2

(g) Targeted mass spectrometry analysis of trypsin-digested Orf8 of the peptide containing the proposed histone mimic in Orf8

(h) Representative confocal images of HEK293T cells transfected with orf8 expression construct (green) and stained for Lamin A/C (cyan) and Lamin B1 (red)

(i) Bar graphs show distributions of total Lamin A/C or Lamin B1 signal intensity per cell in Mock and Orf8-expressing cells

n = 129 and 86 cells per sample

(j) Representative confocal images of HEK293T cells transfected with Orf8 expression construct (green) and stained for H3K9me2 (red) and Lamin B (cyan)

(k) Bar graph show fraction of H3K9me2-marked chromatin at the nuclear lamina/periphery of control and Orf8-expressing cells

n ≥ 32 cells per sample

(l) Representative confocal images shown in j of HEK293T cells transfected with Orf8 expression construct (green) and stained for Lamin B1 (red) with Orf8 and Lamin B1 enrichment through the cell plotted along the dotted lines shown in image

(b) H3 serine 10 phosphorylation (H3S10ph), H3K9 dimethylation (H3K9me2), Lamin B and global acetylation (of histones or of non-histone proteins) in GFP or Orf8 expressing cells isolated by FACS

(c) Orf8 constructs tested show similar levels of expression in HEK293T cells

N = 137 (Orf8), 87 (Orf8ΔARKSAP), 120 (S84L) from 4 independent transfections

(d–f) Orf8 with a mutation commonly found in the SARS-CoV-2 genome, Orf8-S84L, shows the same effects on histone PTMs H3K9me3 (d), H3K27me3 (e), and H3K9ac (f)

N = 332 (GFP), 237 (S84L) cells for H3K9me3; 186, 166 cells for H3K27me3; 332, 237 cells for H3K9ac from 2 independent transfections

N = 216 (GFP), 120 (Orf8), 88 (AGSKSP) cells from 2 independent transfections

(a) Expression of WT Orf8 and Orf8ΔARKSAP from FAC sorted cells used for western blot analysis

(b) Expression of WT Orf8 and Orf8ΔARKSAP from FAC sorted cells used for H3K9ac CUT&TAG

(d) Expression of WT Orf8 and Orf8ΔARKSAP from FAC sorted cells used for ATAC-seq

(g) H3K9ac CUT&TAG and ATAC-seq gene tracks of genes relevant to viral responses in HEK 293T cells expression a control plasmid, Orf8, or Orf8ΔARKSAP

(h) Gene track example with limited changes between Orf8 and Orf8ΔARKSAP in H3K9ac CUT&TAG and ATAC-seq

(a) Expression of Orf8WT and Orf8ΔARKSAP from FAC sorted cells used for RNA-seq

(b) Volcano plot of differential gene expression analysis of Orf8WT expressing cells compared to GFP expressing cells

(c) Volcano plot of differential gene expression analysis of Orf8ΔARKSAP expressing cells compared to GFP expressing cells

(d) Overlap of genes down or upregulated by Orf8 and Orf8ΔARKSAP compared to GFP expressing cells

(e) Gene ontology analysis of genes that are downregulated by Orf8 compared to Orf8ΔARKSAP

(f-g) Volcano plot of differential gene expression analysis of Orf8WT expressing cells compared to Orf8ΔARKSAP expressing cells graphed by p value (f) and mean expression (g)

(h) Gene tracks of genes that are induced by Orf8ΔARKSAP but show a dampened response to Orf8

(i) Gene tracks of a gene that is not disrupted by Orf8 expression

(j) H3K9ac CUT&Tag reads at down and upregulated DEGs in Orf8 verses Orf8ΔARKSAP

(k) ATAC-seq reads at down and upregulated DEGs in Orf8 verses Orf8ΔARKSAP

For RNA-seq, N = 2 for GFP and Orf8ΔARKSAP and N = 3 for Orf8WT

(a) qRT-PCR analysis of expression of SARS-CoV-2 gene RdRp analysis of viral titer in A549ACE pulmonary cells at 24 and 48 h after infection with SARS-CoV-2WT or SARS-CoV-2ΔOrf8 at MOI = 1

(b) Plaque assay analysis of viral titer in A549ACE pulmonary cells at 24 and 48 h after infection with SARS-CoV-2WT or SARS-CoV-2ΔOrf8 at MOI = 1

(c-d) ChIP-RX for H3K9me3 (c) and H3K27me3 (d) of A549ACE cells with SARS-CoV-2WT, SARS-CoV-2ΔOrf8, or mock infection at MOI = 1, 48 h after infection

(a,c,e) H3K9me3 (a), H3K27me3 (c) or H3K9ac (e) staining of A549ACE cells 24 h after SARS-CoV-2WT, SARS-CoV-2ΔOrf8, or mock infection at MOI = 1

For H3K9me3 N = 39 (SARS-CoV-2), 35 (SARS-CoV-2ΔOrf8), for H3K27me3 N = 48 (SARS-CoV-2), 37 (SARS-CoV-2ΔOrf8), for H3K9ac N = 94 (SARS-CoV-2), 120 (SARS-CoV-2ΔOrf8) cells normalized to uninfected neighbor cells from 1-2 independent infections

Dotted line indicates relative signal in Mock infected condition

(g) Quantification of H3K9me3 in infected cells and neighboring cells from the same tissue slice for each individual patient sample shown separately, relative to control samples

For sample 1, N = 3 infected and 48 uninfected, sample 2, N = 7 infected and 125 uninfected, sample 3, N = 5 infected and 55 uninfected cells

Box plots centered on median, bounds at 25th and 75th percentile, minimum and maximum defined as median ± 1.5x interquartile range, with whiskers extended to lowest/highest value in range

(b) Levels of SARS-CoV-2 transcripts in cells infected with SARS-CoV-2WT, SARS-CoV-2ΔARKSAP, or SARS-CoV-2ΔOrf8 24 h after infection

(d) Levels of SARS-CoV-2 transcripts in cells infected with SARS-CoV-2WT, SARS-CoV-2ΔOrf8, or SARS-CoV-2ΔARKSAP 48 h after infection

(e) Normalized reads of SARS-CoV-2 Orf8 and human transcripts of histone H3 genes in cells infected with SARS-CoV-2WT or SARS-CoV-2ΔOrf8

(g) Differential gene expression analysis by RNA-seq of A549ACE cells 48 h after SARS-CoV-2WT, SARS-CoV-2ΔOrf8, or SARS-CoV-2ΔARKSAP, compared to mock infection at MOI = 1

Box plots centered on median, bounds at 25th and 75th percentile, minimum and maximum defined as median ± 1.5x interquartile range, with whiskers extended to lowest/highest value in range

Gene ontology analysis of gene expression changes following ORF8 expression

Histone PTM changes with ORF8 expression

Gene ontology analysis of gene expression changes following SARS-CoV-2 infection of A549ACE cells

SARS-CoV-2 disrupts host epigenetic regulation via histone mimicry

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