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

作者：Marie J. Tofaute, Benjamin Weller, Carina Graß, Hridi Halder, Bushra Dohai, Pascal Falter‐Braun, Daniel Krappmann · 发表于：Bioscience Reports · 年份：2023 · DOI：10.1042/bsr20231418 · 被引用次数：12 · 研究领域：interferon and immune responses、SARS-CoV-2 and COVID-19 Research、RNA regulation and disease

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-κB, 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-κB signaling pathways to characterize the molecular mechanism of NSP14-induced NF-κB activation. We demonstrate that full-length NSP14 requires methyltransferase (MTase) activity to drive NF-κB 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-κB. Using CRISPR/Cas9-engineered KO cells, we demonstrate that NSP14 stimulation of canonical NF-κB activation relies on NF-κB factor p65/RELA downstream of the NEMO/IKK complex, while c-Rel or non-canonical RelB are not required to induce NF-κB transcriptional activity. However, NSP14 overexpression is unable to induce canonical IκB kinase β (IKKβ)/NF-κB signaling and in co-immunoprecipitation assays we do not detect stable associations between NSP14 and NEMO or p65, suggesting that NSP14 activates NF-κB indirectly through its methyltransferase activity. Taken togethe...