An ATP-Driven N Protein-DDX21 Molecular Switch Dynamically Controls SARS-CoV-2 RNA G-Quadruplex Heterogeneity.

The SARS-CoV-2 RNA genome functions as a highly structured regulatory scaffold. Although bioinformatic analyses predict widespread RNA G-quadruplexes (G4s) across the viral genome, their structural diversity and regulatory mechanisms remain poorly understood. Here, we report a diverse landscape of viral G4s encompassing parallel and non-canonical topologies with remarkable thermostability. Unlike typical eukaryotic G4s, these two-tetrad viral G4s exhibit a hierarchical ion-dependent mechanism, in which K+ establishes the core fold, and Mg2 + acts as a secondary regulator promoting conformational compaction. Single-molecule FRET analysis further distinguishes rigid, long-lived G4 folds from highly dynamic, metastable species, defining a continuum of conformational states along the viral genome. Functionally, we identify a synergistic yet competitive interplay between the viral nucleocapsid (N) protein and host helicase DDX21. While the N protein acts as a molecular chaperone to promote G4 folding, DDX21 selectively resolves these structures in an ATP-dependent manner. Strikingly, N and DDX21 jointly constitute a finely tuned, ATP-driven molecular switch, where ATP availability dictates the equilibrium between G4-stabilized and resolved states. Our findings establish a mechanistic framework for the active regulation of SARS-CoV-2 RNA architecture and reveal a multilayered host-virus regulatory axis that modulates viral genome heterogeneity.