SARS-CoV-2 ORF10 hijacking ubiquitination machinery reveals potential unique drug targeting sites.

Viruses often manipulate ubiquitination pathways to facilitate their replication and pathogenesis. CUL2 known as the substrate receptor of cullin-2 RING E3 ligase, is bound by SARS-CoV-2 ORF10 to increase its E3 ligase activity, leading to degradation of IFT46, a protein component of the intraflagellar transport (IFT) complex B. This results in dysfunctional cilia, which explains certain symptoms that are specific to COVID-19. However, the precise molecular mechanism of how ORF10 recognizes CUL2 remains unknown. Here, we determined the crystal structure of CUL2 complexed with the N-terminal extension (NTE) of SARS-CoV-2 ORF10 (2.9 Å). The structure reveals that the ORF10 N-terminal heptapeptide (NTH) mimics the Gly/N-degron to bind CUL2. Mutagenesis studies identified key residues within ORF10 that are key players in its interaction with CUL2 both in ITC assay and cells. In addition, we prove that enhancement of CUL2 activity for IFT46 degradation by which ORF10-mediated correlates with the binding affinity between ORF10 and CUL2. Finally, we used a Global Protein Stability system to show that the NTH of ORF10 mimics the Gly/N-degron motif, thereby binding competitively to CUL2 and inhibiting the degradation of target substrates bearing the Gly/N-degron motif. Overall, this study sheds light on how SARS-CoV-2 ORF10 exploits the ubiquitination machinery for proteasomal degradation, and offers valuable insights for optimizing PROTAC-based drug design based on NTH CUL2 interaction, while pinpointing a promising target for the development of treatments for COVID-19.