Assembly Reactions of Hepatitis B Capsid Protein into Capsid Nanoparticles Follow a Narrow Path through a Complex Reaction Landscape

For many viruses, capsids (biological nanoparticles) assemble to protect genetic material and dissociate to release their cargo. To understand these contradictory properties, we analyzed capsid assembly for hepatitis B virus; an endemic pathogen with an icosahedral, 120-homodimer capsid. We used solution X-ray scattering to examine trapped and equilibrated assembly reactions. To fit experimental results, we generated a library of distinct intermediates, selected by umbrella sampling of Monte Carlo simulations. The number of possible capsid intermediates is immense, ∼10³⁰, yet assembly reactions are rapid and completed with high fidelity. If the huge number of possible intermediates were actually present, maximum entropy analysis shows that assembly reactions would be blocked by an entropic barrier, resulting in incomplete nanoparticles. When an energetic term was applied to select the stable species that dominated the reaction mixture, we found only a few hundred intermediates, mapping out a narrow path through the immense reaction landscape. This is a solution to a viral application of the Levinthal paradox. With the correct energetic term, the match between predicted intermediates and scattering data was striking. The grand canonical free energy landscape for assembly, calibrated by our experimental results, supports a detailed analysis of this complex reaction. There is a narrow range of energies that supports on-path assembly. If association energy is too weak or too strong, progressively more intermediates will be entropically blocked, spilling into paths leading to dissociation or trapped incomplete nanoparticles, respectively. These results are relevant to many viruses and provide a basis for simplifying assembly models and identifying new targets for antiviral intervention. They provide a basis for understanding and designing biological and abiological self-assembly reactions.