The hepatitis B virus (HBV) infection is a major global health problem, with chronic infection leading to liver complications and high death toll. Current treatments, such as nucleos(t)ide analogs and interferon-α, effectively suppress viral replication but rarely cure the infection. To address this, new antivirals targeting different components of the HBV molecular machinery are being developed. Here we investigated the hepatitis B core protein (HBc) that forms the viral capsids and plays a vital role in the HBV life cycle. We explored two distinct binding pockets on the HBV capsid: the central hydrophobic pocket of HBc-dimers and the pocket at the tips of capsid spikes. We synthesized a geranyl dimer that binds to the central pocket with micromolar affinity, and dimeric peptides that bind the spike-tip pocket with sub-micromolar affinity. Cryo-electron microscopy further confirmed the binding of peptide dimers to the capsid spike tips and their capsid-aggregating properties. Finally, we show that the peptide dimers induce HBc aggregation in vitro and in living cells. Our findings highlight two tractable sites within the HBV capsid and provide an alternative strategy to affect HBV capsids.

The phase separation of intrinsically disordered proteins is emerging as an important mechanism for cellular organization. However, efforts to connect protein sequences to the physical properties of condensates, that is, the molecular grammar, are hampered by a lack of effective approaches for probing high-resolution structural details. Using a combination of multiscale simulations and fluorescence lifetime imaging microscopy experiments, we systematically explored a series of systems consisting of diblock elastin-like polypeptides (ELPs). The simulations succeeded in reproducing the variation of condensate stability upon amino acid substitution and revealed different microenvironments within a single condensate, which we verified with environmentally sensitive fluorophores. The interspersion of hydrophilic and hydrophobic residues and a lack of secondary structure formation result in an interfacial environment, which explains both the strong correlation between ELP condensate stability and interfacial hydrophobicity scales, as well as the prevalence of protein-water hydrogen bonds. Our study uncovers new mechanisms for condensate stability and organization that may be broadly applicable.