Bracci, N. | bioRxiv Science

bioRxiv2025

MERS-CoV antagonizes PKR activation by inhibiting its condensation at viral replication complexes

Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic virus that antagonizes innate immune responses, including the protein kinase R (PKR) pathway. Here, we examine the process of PKR activation in response to an immunostimulatory MERS-CoV mutant encoding an inactive endoribonuclease U and a deletion of accessory protein NS4a. We show that PKR condenses and activates on viral dsRNA proximal to viral double-membrane vesicles (DMVs). Condensates composed of activated PKR disassociate from dsRNA and dissolve, releasing activated PKR molecules into the cytosol where they phosphorylate eIF2 to initiate the integrated stress response. MERS-CoV NS4a protein prevents PKR activation by condensing on dsRNA and occluding PKR binding. Lastly, PKR condensation coincided with its activation in response to Zika virus. These findings establish a comprehensive model for PKR activation in response to positive-strand RNA viruses that replicate within membrane-associated complexes and elucidate how MERS-CoV antagonizes this crucial antiviral pathway. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/670656v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@130c4b1org.highwire.dtl.DTLVardef@c16a1borg.highwire.dtl.DTLVardef@1d4023org.highwire.dtl.DTLVardef@5dbaea_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTSO_LIPKR condenses on viral dsRNA exposed at membrane-associated replication complexes C_LIO_LIPKR condensation initiates autophosphorylation of PKR C_LIO_LIActivated p-PKR molecules disassociate from dsRNA and re-localize to the cytosol C_LIO_LIMERS-CoV NS4a inhibits PKR activation by occluding PKR condensation on dsRNA. C_LI

immunology↗

bioRxiv2023

SARS-CoV-2 nsp15 endoribonuclease antagonizes dsRNA-induced antiviral signaling

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 has caused millions of deaths since emerging in 2019. Innate immune antagonism by lethal CoVs such as SARS-CoV-2 is crucial for optimal replication and pathogenesis. The conserved nonstructural protein 15 (nsp15) endoribonuclease (EndoU) limits activation of double-stranded (ds)RNA-induced pathways, including interferon (IFN) signaling, protein kinase R (PKR), and oligoadenylate synthetase/ribonuclease L (OAS/RNase L) during diverse CoV infections including murine coronavirus and Middle East respiratory syndrome (MERS)-CoV. To determine how nsp15 functions during SARS-CoV-2 infection, we constructed a mutant recombinant SARS-CoV-2 (nsp15mut) expressing a catalytically inactive nsp15. Infection with SARS-CoV-2 nsp15 mut led to increased activation of the IFN signaling and PKR pathways in lung-derived epithelial cell lines and primary nasal epithelial air-liquid interface (ALI) cultures as well as significant attenuation of replication in ALI cultures compared to wild-type (WT) virus. This replication defect was rescued when IFN signaling was inhibited with the Janus activated kinase (JAK) inhibitor ruxolitinib. Finally, to assess nsp15 function in the context of minimal (MERS-CoV) or moderate (SARS-CoV-2) innate immune induction, we compared infections with SARS-CoV-2 nsp15mut and previously described MERS-CoV nsp15 mutants. Inactivation of nsp15 had a more dramatic impact on MERS-CoV replication than SARS-CoV-2 in both Calu3 cells and nasal ALI cultures suggesting that SARS-CoV-2 can better tolerate innate immune responses. Taken together, SARS-CoV-2 nsp15 is a potent inhibitor of dsRNA-induced innate immune response and its antagonism of IFN signaling is necessary for optimal viral replication in primary nasal ALI culture. SIGNIFICANCESevere acute respiratory syndrome coronavirus (SARS-CoV)-2 causes a spectrum of respiratory disease ranging from asymptomatic infections to severe pneumonia and death. Innate immune responses during SARS-CoV-2 infection have been associated with clinical disease severity, with robust early interferon responses in the nasal epithelium reported to be protective. Thus, elucidating mechanisms through which SARS-CoV-2 induces and antagonizes host innate immune responses is crucial to understanding viral pathogenesis. CoVs encode various innate immune antagonists, including the conserved nonstructural protein 15 (nsp15) which contains an endoribonuclease (EndoU) domain. We demonstrate that SARS-CoV-2 EndoU is a crucial interferon antagonist, by providing further evidence for the role of the conserved CoV nsp15 in antagonizing innate immune activation, thereby optimizing CoV replication.

microbiology↗