Histone mimicry by the SARS-CoV-2 protein disrupts the epigenetic regulation of host cells

Histone mimicry by the SARS-CoV-2 protein disrupts the epigenetic regulation of host cells

In a recent study published in Nature Researchers showed that a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) protein acts as a histone mimic to disrupt epigenetic regulation of host cells.

Studie: SARS-CoV-2 stört die epigenetische Regulation des Wirts durch Histon-Mimikry. Bildquelle: PHOTOCREO Michal Bednarek/Shutterstock

Current evidence suggests that infection with SARS-CoV-2 suppresses innate immune responses and disrupts epigenetic regulation. However, how this happens remains unknown. In rare cases, other virulent viruses can interfere with epigenetic regulation by mimicking host proteins, particularly histones. Histones package DNA into complex structures and regulate access to the genome.

Histones are subject to a spectrum of post-translational modifications (PTMs) that are dynamically regulated to control gene expression. Histone mimicry allows viruses to disrupt the ability of cells to respond to infection and regulate gene expression. Nevertheless, histone mimicry by coronaviruses (CoVs) has not yet been validated.

The study and results

The present study investigated whether SARS-CoV-2 employs histone mimicry to affect chromatin regulation and infection response. First, the researchers conducted a bioinformatics comparison of SARS-CoV-2 proteins with human histones. There was an identical six-residue match between amino acids 50–55 of open reading frame 8 (ORF8) and the critical regions in the N-terminus of the H3 histone.

These residues lie in a disordered region on the surface of the monomeric ORF8. Interestingly, the ARKS sequence was found within this motif, which is present in two different locations in the H3 tail. Proteomic characterization revealed that DNA methyltransferase 1 (DNMT1) is the binding partner of ORF8.

Next, the intracellular localization of ORF8 was examined to determine whether it functions as a histone mimic. HEK293T cells were transfected with a Strep-tagged construct encoding ORF8, which was visualized using a fluorescent probe. Immunofluorescence showed that ORF8 was typically localized in the cytoplasm and nuclear periphery.

However, cell fractionation showed that it was located in both the nucleus and the cytoplasm. In addition, the team found that ORF8 colocalized with lamins B1 and A/C in transfected cells. The expression pattern was confirmed in the SARS-CoV-2-infected A549 cell line expressing angiotensin-converting enzyme 2 (ACE2). Next, chromatin binding was assessed using increasing salt concentrations. ORF8 dissociated from the chromatin fraction at similar salt concentrations as lamins and histones.

In contrast, deletion of the ARKSAP motif in ORF8 (ORF8ΔARKSAP) enabled dissociation at lower salt concentrations, suggesting that the putative histone mimic motif influences the strength of association between ORF8 and chromatin. Chromatin immunoprecipitation with sequencing (ChIP-seq) demonstrated the enrichment of ORF8 within specific genomic regions, particularly those associated with the H3K27me3 modification.

ORF8 was also found to associate with lysine acetyltransferase 2A (KAT2A). ORF8ΔARKSAP was not associated with chromatin proteins, suggesting that the ARKSAP motif enhances the association of ORF8 with chromatin proteins. A targeted mass spectrometric analysis was performed to investigate whether the histone mimic site is modified similarly to histones. This identified a lysine residue in the proposed mimic site that was acetylated similarly to H3 histone.

Furthermore, ORF8 expression caused a significant decrease in KAT2A abundance, while nuclear lamina proteins and lamina-associated heterochromatin levels remained unchanged or increased slightly. Next, the team observed that histone PTMs associated with transcriptional repression were increased in transfected HEK293T cells expressing ORF8, while those associated with active gene expression were depleted. In particular, those within the ARKS motif of H3 were highly disrupted.

The transposase-accessible chromatin assay using high-throughput sequencing showed that ORF8 decreased chromatin accessibility, but not ORF8ΔARKSAP. RNA sequencing was performed to define differentially expressed genes (DEGs) in transfected cells. ORF8 and ORF8ΔARKSAP shared a subset of DEGs, but the presence of the histone mimic motif resulted in less dynamic changes in gene expression.

Genes that were downregulated in response to ORF8 compared to ORF8ΔARKSAP had better chromatin accessibility and higher basal levels of H3K9ac modification than upregulated genes. Next, a mutant SARS-CoV-2 lacking ORF8 (SARS-CoV-2ΔORF8) was generated; A549ACE2 cells were infected with this mutant or SARS-CoV-2 to compare viral genome levels and viral particle production.

There were no differences in virus titers or genome copy number at 24 hours, and only minor changes were evident at 48 hours. SARS-CoV-2 infection caused a strong increase in repressive histone PTMs (H3K9me3 and H3K27me3). Conversely, this effect was attenuated in the absence of ORF8.

Further experiments with a mutant SARS-CoV-2 in which the ARKSAP motif was deleted from ORF8 (SARS-CoV-2ΔARKSAP) showed a significant attenuation of the effects of infection on H3K9ac and chromatin accessibility, replicating the effects of ORF8 deletion. Infection with wild-type SARS-CoV-2 reduced KAT2A expression, whereas SARS-CoV-2ΔARKSAP or SARS-CoV-2ΔORF8 infection did not.

Finally, induced pluripotent stem cell (iPSC)-derived type II lung alveolar cells (iAT2) were infected with wild-type and mutant SARS-CoV-2 cells. The genome copy number of mutant viruses was reduced 48 hours after infection, suggesting that ORF8, particularly the ARKSAP motif, affects virus replication. The virus particles produced by the ΔORF8 mutant were fewer than those of the wild-type virus. In contrast, the ΔARKSAP mutant appeared to be similar to wild-type SARS-CoV-2, suggesting that ORF8 has an ARKSAP-independent function of viral particle generation.

Conclusions

The study showed that ORF8 of SARS-CoV-2 contains an ARKS motif and that expression of ORF8 disrupts the regulation of histone PTMs. The researchers found the association of ORF8 with chromatin-associated proteins, nuclear lamina and histones. Similar to histones, ORF8 undergoes acetylation within the histone mimic motif.

Deletion of ORF8 resulted in a reduction in virus replication in iAT2 cells, whereas the viral genome copy number was explicitly affected by loss of the histone mimic motif. Overall, the researchers identified a new case of histone mimicry during SARS-CoV-2 infection and described mechanisms by which the virus disrupts host cell chromatin regulation.

Reference:

• Kee J, Thudium S, Renner DM, et al. (2022). SARS-CoV-2 stört die epigenetische Regulation des Wirts durch Histon-Mimikry. Natur. doi: 10.1038/s41586-022-05282-z https://www.nature.com/articles/s41586-022-05282-z