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

ABSTRACT 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 Mg 2 + 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.