Structural and functional insights into the SARS-CoV-2 SUD domain and its interaction with RNA G-Quadruplexes.

The SARS-CoV-2 pandemic has caused a global health crisis due to its high pathogenicity. The SARS-Unique Domain (SUD) in the non-structural protein Nsp3 of SARS-CoV-2 is hypothesized to play a critical role in viral replication and pathogenesis by interacting with host RNA G-quadruplex (G4) structures, but the molecular mechanisms remain unclear. In this study, we used a multidisciplinary approach, including fluorescence assays, CD, single-molecule FRET, SAXS, G4-unfolding experiments and MD simulations, to investigate the interaction between SUD and RNA G4 structures. We found that SUD exhibited a strong binding affinity for RNA three-layer G4 structures with 3'-tail preference but did not unfold G4; instead, it stabilized the G4 conformation, suggesting a role in modulating viral RNA stability and translation. SAXS revealed that G4 binds to a surface groove formed by the N- and C-termini of SUD, enhancing its conformational stability. MD simulations identified key interaction sites and confirmed the induced-fit binding mechanism. These findings provide critical insights into the role of SUD in modulating viral RNA stability and translation, and offers potential targets for antiviral strategies targeting SUD/G4 interactions.