SARS-CoV-2 NSP14 MTase activity is critical for inducing canonical NF-KB activation

Upon SARS-CoV-2 infection, patients with severe forms of COVID-19 often suffer from a dysregulated immune response and hyperinflammation. Aberrant expression of cytokines and chemokines is associated with strong activation of the immunoregulatory transcription factor NF-KB, which can be directly induced by the SARS-CoV-2 protein NSP14. Here, we use NSP14 mutants and generated cells with host factor knockouts (KOs) in the NF-KB signaling pathways to characterize the molecular mechanism of NSP14-induced NF-KB activation. We demonstrate that full-length NSP14 requires methyltransferase (MTase) activity to drive NF-KB induction. NSP14 WT, but not an MTase-defective mutant, is poorly expressed and inherent post-translational instability is mediated by proteasomal degradation. Binding of SARS-CoV-2 NSP10 or addition of the co-factor S-adenosylmethionine (SAM) stabilizes NSP14 and augments its potential to activate NF-KB. Using CRISPR/Cas9-engineered KO cells, we demonstrate that NSP14 stimulation of canonical NF-KB activation relies on NF-KB factor p65/RELA downstream of the NEMO/IKK complex, while c-Rel or non-canonical RelB are not required to induce NF-KB transcriptional activity. However, NSP14 over -expression is unable to induce canonical IKB kinase 6 (IKK6)/NF-KB signaling and in co-immunoprecipitation assays we do not detect stable associations between NSP14 and NEMO or p65, suggesting that NSP14 activates NF-KB indirectly through its methyltransferase activity. Taken together, our data provide a framework how NSP14 can augment basal NF-KB activation, which may enhance cytokine expression in SARS-CoV-2 infected cells.