SARS-CoV-2 endoribonucleaseU and ORF6 work synergistically to antagonize innate immune signaling 2327626

Abstract Introduction Coronaviruses (CoVs) produce dsRNA during genome replication and mRNA synthesis. Upon sensing dsRNA, host cells activate numerous antiviral pathways. CoVs encode multiple proteins that antagonize antiviral responses. The conserved CoV nsp15 contains an endoribonuclease (EndoU) that cleaves viral ssRNA to limit dsRNA accumulation. Similarly, the viral protein, NS6, encoded by open reading frame (ORF)6 is expressed by severe acute respiratory syndrome (SARS)-CoV and SARS-CoV-2 and has been shown to block STAT translocation, but its absence has minimal impact on IFN-stimulated genes (ISGs). A viral genome has limited coding capacity, and ORFs encoding proteins without a significant function are typically mutated or deleted over time. Therefore, we infer that ORF6 plays an essential role in immune evasion, perhaps dependent upon other viral proteins. Methods A recombinant SARS-CoV-2 encoding an inactive EndoU (nsp15mut) and an interrupted ORF6 (ORF6stop) was constructed. Viral kinetics and innate immune induction were assessed in both Calu-3 cells and primary nasal air-liquid interface (ALI) cultures using RT-qPCR and Western blot analysis. Results Replication of SARS-CoV-2 nsp15mut/ORF6stop in nasal ALI cultures was attenuated at both 33 °C and 37 °C compared to WT SARS-CoV-2. A significant increase in type I and III IFN transcripts was also observed in nasal ALI cultures infected with nsp15mut/ORF6stop compared to WT and each single mutant. Robust induction of various ISGs were also observed via Western blot analysis, with a faster induction observed at 37 °C. Conclusion The synergistic relationship between nsp15 and ORF6 is essential for efficient SARS-CoV-2 replication. We hypothesize that since nsp15 functions by reducing dsRNA upstream of ORF6, nsp15 acts as a more potent antagonist leaving little activity for ORF6 to account for. However, when both EndoU activity and NS6 are absent, a robust induction of IFN and ISGs are induced to levels higher than that of the nsp15mut alone. Funding Source This work was supported by National Institutes of Health grants R01 AI140442 (SRW), R01AI169537 (SRW&NAC), RO1A1AI161175 (LM-S); Department of Veterans Affairs Merit Review 1-I01-BX005432-01 (NAC&SRW); the Penn Center for Research on Emerging Viruses (SRW Topic Categories Viral Immunology (VIR)