Structural organization of HBV pgRNA genome driven by phase separation in capsid confinement.

Viruses rely on the precise packaging of their genomes within a capsid to execute essential life-cycle events, yet the principles governing genome structural organization in this confined environment remain elusive. Here, we reveal that hepatitis B virus (HBV) pregenomic RNA (pgRNA) exploits liquid-liquid phase separation (LLPS) inside the capsid to sculpt its architecture. Multiscale molecular dynamics (MD) simulations, supplemented by biochemical assays, show that pgRNA coalesces into a hollow, shell-like condensate along the inner capsid surface, with coexisting low- and high-density regions. Electrostatic interactions between pgRNA and the disordered C-terminal domain of capsid protein primarily govern condensate formation. LLPS drives the establishment of microphases composed of nematically aligned RNA hairpin arrays interspersed by domains rich in flexible single-stranded RNA linkers, achieving an optimal balance between structural order and dynamic flexibility. Intriguingly, although the ensemble-averaged pgRNA density exhibits icosahedral symmetry, individual simulation snapshots display pronounced heterogeneity, indicating symmetry breaking at the single-particle level. In addition, LLPS-induced hollow-shell architecture of pgRNA genome promotes long-range RNA base-pairing and enhances polymerase mobility, which may facilitate the functional dynamics of polymerase during reverse transcription. Our findings uncover a capsid-confined LLPS mechanism that orchestrates viral genome structure and dynamics, offering new targets for antiviral intervention.