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

Abstract

Viruses often manipulate ubiquitination pathways to facilitate their replication and pathogenesis. CUL2^ZYG11B 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^ZYG11B remains unknown. Here, we determined the crystal structure of CUL2^ZYG11B 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^ZYG11B. Mutagenesis studies identified key residues within ORF10 that are key players in its interaction with CUL2^ZYG11B both in ITC assay and in vivo cells. In addition, we prove that enhancement of CUL2^ZYG11B activity for IFT46 degradation by which ORF10-mediated correlates with the binding affinity between ORF10 and CUL2^ZYG11B. 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^ZYG11B 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^ZYG11B interaction, while pinpointing a promising target for the development of treatments for COVID-19.