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

TL;DR

This study shows how the hepatitis B virus organizes its RNA genome inside the capsid using a process called liquid-liquid phase separation, which helps in its replication.

Contribution

The study reveals a novel mechanism of HBV pgRNA structural organization via LLPS within the capsid.

Findings

HBV pgRNA forms a hollow shell-like condensate inside the capsid via LLPS.

LLPS promotes long-range RNA base-pairing and enhances polymerase mobility for reverse transcription.

RNA microphases exhibit nematically aligned hairpins and flexible linkers, balancing order and flexibility.

Abstract

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.

Viruses encapsulate their genome within a protein capsid. This study reveals that hepatitis B virus employs a liquid-liquid phase separation mechanism to organize its pregenomic RNA inside the capsid, thereby facilitating reverse transcription.

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