Coronaviruses are adept at evading host antiviral pathways induced by viral double-stranded RNA, including interferon (IFN) signaling, oligoadenylate synthetase–ribonuclease L (OAS-RNase L), and protein kinase R (PKR). While dysregulated or inadequate IFN responses have been associated with severe coronavirus infection, the extent to which the recently emerged SARS-CoV-2 activates or antagonizes these pathways is relatively unknown. We found that SARS-CoV-2 infects patient-derived nasal epithelial cells, present at the initial site of infection; induced pluripotent stem cell-derived alveolar type 2 cells (iAT2), the major cell type infected in the lung; and cardiomyocytes (iCM), consistent with cardiovascular consequences of COVID-19 disease. Robust activation of IFN or OAS-RNase L is not observed in these cell types, whereas PKR activation is evident in iAT2 and iCM. In SARS-CoV-2–infected Calu-3 and A549ACE2 lung-derived cell lines, IFN induction remains relatively weak; however, activation of OAS-RNase L and PKR is observed. This is in contrast to Middle East respiratory syndrome (MERS)-CoV, which effectively inhibits IFN signaling and OAS-RNase L and PKR pathways, but is similar to mutant MERS-CoV lacking innate immune antagonists. Remarkably, OAS-RNase L and PKR are activated in MAVS knockout A549ACE2 cells, demonstrating that SARS-CoV-2 can induce these host antiviral pathways despite minimal IFN production. Moreover, increased replication and cytopathic effect in RNASEL knockout A549ACE2 cells implicates OAS-RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 fails to antagonize these host defense pathways, which contrasts with other coronaviruses, the IFN signaling response is generally weak. These host–virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.

SummaryCoronaviruses are adept at evading and/or antagonizing double-stranded RNA-induced host antiviral pathways, including interferon signaling, OAS-RNase L and PKR while robust cytokine responses characterize severe coronavirus disease. Knowledge of how newly emerged SARS-CoV-2 interacts with these pathways is minimal. SARS-CoV-2 readily infects patient-derived nasal epithelial cells and induced pluripotent stem cell-derived alveolar type 2 cells(iAT2) and cardiomyocytes(iCM). Robust activation of interferons or RNase L is not observed, while PKR activation is evident in iAT2 and iCM. In SARS-CoV-2 infected Calu-3 and A549ACE2 lung derived cell lines, activation of all pathways is observed, similar to a mutant MERS-CoV lacking innate immune antagonists. Moreover, increased replication in RNASEL knockout A549ACE2 cells, implicates RNase L in restricting SARS-CoV-2. Finally, while SARS-CoV-2 is less adept at antagonizing these host defense pathways compared to other coronaviruses, the innate immune response is still generally weak. These host-virus interactions may contribute to the unique pathogenesis of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and caused the devastating global pandemic of coronavirus disease 2019 (COVID-19), in part because of its ability to effectively suppress host cell responses 1 – 3 . In rare cases, viral proteins dampen antiviral responses by mimicking critical regions of human histone proteins 4 – 8 , particularly those containing post-translational modifications required for transcriptional regulation 9 – 11 . Recent work has demonstrated that SARS-CoV-2 markedly disrupts host cell epigenetic regulation 12 – 14 . 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.

DEAD-box proteins (DBPs) are required in gene expression to facilitate changes to ribonucleoprotein complexes, but the cellular mechanisms and regulation of DBPs are not fully defined. Gle1 is a multifunctional regulator of DBPs with roles in mRNA export and translation. In translation, Gle1 modulates Ded1, a DBP required for initiation. However, overexpression causes defects, suggesting that Ded1 can promote or repress translation in different contexts. Here we show that expression suppresses the repressive effects of , and Gle1 counteracts Ded1 in translation assays Furthermore, Ded1 and Gle1 both affect assembly of pre-initiation complexes. Through mutation analysis and binding assays, we show that Gle1 inhibits Ded1 by reducing its affinity for RNA. Our results are consistent with a model wherein active Ded1 promotes translation, but inactive or excess Ded1 leads to translation repression. Gle1 can inhibit either role of Ded1, positioning it as a gatekeeper to optimize Ded1 activity to the appropriate level for translation. This study suggests a paradigm for finely controlling the activity of DEAD-box proteins to optimize their function in RNA-based processes. It also positions the versatile regulator Gle1 as a potential node for the coordination of different steps of gene expression.

Ded1 is a DEAD-box RNA helicase with essential roles in translation initiation. It binds to the eukaryotic initiation factor 4F (eIF4F) complex and promotes 48S preinitiation complex assembly and start-site scanning of 5′ untranslated regions of mRNAs. Most prior studies of Ded1 cellular function were conducted in steady-state conditions during nutrient-rich growth. In this work, however, we examine its role in the translational response during target of rapamycin (TOR)C1 inhibition and identify a novel function of Ded1 as a translation repressor. We show that C-terminal mutants of DED1 are defective in down-regulating translation following TORC1 inhibition using rapamycin. Furthermore, following TORC1 inhibition, eIF4G1 normally dissociates from translation complexes and is degraded, and this process is attenuated in mutant cells. Mapping of the functional requirements for Ded1 in this translational response indicates that Ded1 enzymatic activity and interaction with eIF4G1 are required, while homo-oligomerization may be dispensable. Our results are consistent with a model wherein Ded1 stalls translation and specifically removes eIF4G1 from translation preinitiation complexes, thus removing eIF4G1 from the translating mRNA pool and leading to the codegradation of both proteins. Shared features among DED1 orthologues suggest that this role is conserved and may be implicated in pathologies such as oncogenesis.