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

Abstract

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 (KO) 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 together, our data provide a framework how NSP14 can augment basal NF-κB activation, which may enhance cytokine expression in SARS-CoV-2 infected cells.

The Role of the Nuclear Factor-Kappa B (NF-κB) Pathway in SARS-CoV-2 Infection

“…Specifically, the wild-type NSP14 (NSP14 WT), as opposed to an MTase-defective mutant, exhibits low expression levels, and its inherent post-translational instability is mediated through proteasomal degradation. Furthermore, the binding of SARS-CoV-2 NSP10 or the addition of the co-factor S-adenosylmethionine stabilizes NSP14, enhancing its potential to activate NF-κB [ 121 ]. The NSP14’s stimulation of canonical NF-κB activation relies on NF-κB factor p65/RelA downstream of the NEMO/IKK complex.…”

“…Co-immunoprecipitation assays do not detect stable associations between NSP14 and NEMO or p65, suggesting that NSP14 indirectly activates NF-κB through its methyltransferase activity. The data provide a comprehensive framework elucidating how NSP14 can enhance basal NF-κB activation, potentially contributing to elevated cytokine expression in SARS-CoV-2-infected cells [ 121 ].…”