Rapidly Forming Early Intermediate Structures Dictate the Pathway of Capsid Assembly

Abstract

There are ∼ 10 30 possible intermediates on the assembly path from hepatitis B capsid protein dimers to the 120-dimer capsid. If every intermediate was tested, assembly would often get stuck in an entropic trap and essentially every capsid would follow a unique assembly path. Yet, capsids assemble rapidly with minimal trapped intermediates, a realization of the Levinthal paradox. To understand the fundamental mechanisms of capsid assembly it is critical to resolve the early stages of the reaction. We have used Time-Resolved Small Angle X-ray Scattering, which is sensitive to solute size and shape and has millisecond temporal resolution. Scattering curves were fit to a thermodynamically curated library of assembly intermediates, using the principle of maximum entropy. Maximum entropy also provides a physical rationale for the selection of species. We found that the capsid assembly pathway was exquisitely sensitive to 1 initial assembly conditions. With the mildest conditions tested, the reaction appeared two-state from dimer to 120-dimer capsid with some dimers-of-dimers and trimers-ofdimers. In slightly more aggressive conditions, we observed transient accumulation of a decamer-of-dimers and appearance of 90-dimer capsids. In conditions where there is measurable kinetic trapping, we found that a highly diverse early intermediates accumulated within a fraction of a second and propagated into long-lived kinetically trapped states (≥ 90-mer). In all cases, intermediates between 35 and 90 subunits did not accumulate. These results are consistent with the presence of low barrier paths that connect early and late intermediates and direct the ultimate assembly path to late intermediates where assembly can be paused.

Virus self-assembly proceeds through contact-rich energy minima

“…Our results complement the recent study by Asor et al ( 34 , 44 ), which examined the assembly of empty HBV capsids for assembly domain dimers using time-resolved small-angle x-ray scattering (SAXS). The SAXS experiments used much higher protein concentrations than HS-AFM to obtain an interpretable signal at a resolution that allowed distinguishing intermediates.…”

“…Our results indicate that the diamond-shape pentameric arrangement of Cps is a critical participant in HBV assembly, as a minimal stable structure of Cps able to condense single-stranded nucleic acids. Previous studies have reported the accumulation of small HBV assembly intermediates for truncated versions of Cp (without the CTD) and in the absence of nucleic acids ( 34 , 40 , 42 – 44 ). The assembly model proposed here ( Fig.…”

Discovery of New Small Molecule Hits as Hepatitis B Virus Capsid Assembly Modulators: Structure and Pharmacophore-Based Approaches

“…The HBV capsid is composed of 120 core protein (Cp) dimers that primarily assemble to form capsids with T = 4 icosahedral symmetry (~95%), with a small number of T = 3 capsids having been observed in HBV-infected human liver samples and in vitro-assembled capsids from recombinant HBV Cp [ 7 , 8 , 9 , 10 ]. HBV genome synthesis from pgRNA via reverse transcription is highly dependent on properly assembled viral capsids [ 11 ].…”

Asymmetrizing an icosahedral virus capsid by hierarchical assembly of subunits with designed asymmetry

“…Although capsid assembly has nucleation and elongation phases, there are no discrete stopping points for manipulation and modification of specific intermediates 27 – 29 . Here we used Hepatitis B Virus (HBV) capsid assembly as a model system and demonstrated an effective approach to break the spontaneous assembly into addressable steps 28 , 30 , 31 . HBV capsid dimeric subunits can assemble two species of capsids, T = 3 capsid with 90 dimers and T = 4 capsid with 120 dimers.…”