Ubiquitination of SARS-CoV-2 NSP6 and ORF7a Facilitates NF-kappa B Activation

Patients with severe coronavirus disease 2019 tend to have high levels of proinflammatory cytokines, which eventually lead to cytokine storm and the development of acute respiratory distress syndrome. However, the detailed molecular mechanisms of proinflammatory cytokine production remain unknown. Here, we screened severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genes and found that nonstructural protein 6 (NSP6) and open reading frame 7a (ORF7a) activated the NF-kappa B pathway. NSP6 and ORF7a interacted with transforming growth factor beta-activated kinase 1 (TAK1), and knockout (KO) of TAK1 or NF-kappa B essential modulator (NEMO) abolished NF-kappa B activation by NSP6 and ORF7a. Interestingly, K61 of NSP6 was conjugated to K63-linked polyubiquitin chains by the E3 ubiquitin ligase tripartite motif-containing 13, and this polyubiquitination of NSP6 appeared crucial for recruitment of NEMO to the NSP6-TAK1 complex and NF-kappa B activation. On the other hand, ring finger protein 121 (RNF121) was required for the polyubiquitination of ORF7a. Knockdown of RNF121 significantly decreased ORF7a binding of TAK1 and NEMO, resulting in the suppression of NF-kappa B activation. Taken together, our results provide novel molecular insights into the pathogenesis of SARS-CoV-2 and the host immune response to SARS-CoV-2 infection. IMPORTANCE The detailed molecular basis of the induction of proinflammatory cytokines and chemokines by SARS-CoV-2 is unclear, although such induction is clearly related to the severity of COVID-19. Here, we show that SARS-CoV-2 NSP6 and ORF7a lead to NF-kappa B activation through associations with TAK1. K63-linked polyubiquitination of NSP6 and ORF7a by TRIM13 and RNF121, respectively, appears essential for NF-kappa B activation. These results suggest that inhibition of the NSP6 and ORF7a gene products may reduce the severity of COVID-19 symptoms by decreasing proinflammatory cytokine levels.

Automated summary

This study reveals the molecular mechanisms behind the induction of proinflammatory cytokines by the SARS-CoV-2 virus. The NSP6 and ORF7a proteins activate the NF-kappa B pathway through interactions with TAK1. The polyubiquitination of NSP6 and ORF7a is essential for NF-kappa B activation, with TRIM13 and RNF121 playing key roles in this process. These findings provide novel insights into the pathogenesis of SARS-CoV-2 and the host immune response.