Jodi A. Hadden-Perilla

Human papillomavirus (HPV) is a significant health burden and leading cause of virus-induced cancers. The mechanisms of HPV receptor binding and host entry are not completely understood, although it is known that heparan sulfate proteoglycans (HSPGs) mediate entry. HPV16 quasivirus, composed of L1 and L2 capsid proteins with a packaged cottontail rabbit papillomavirus genome was incubated with heparin. The complex was vitrified, and data were collected for cryoEM single particle analysis. Subparticles were extracted and hexavalent and pentavalent capsomers were refined separately. Here we present the resulting 1.9 Å resolution structure, with heparin visualized around the capsomer at the icosahedral fivefold vertex. A model of the asymmetric unit was built unambiguously into the atomic resolution cryoEM map. Hydrogen bonds are predicted between L1 N-terminal regions, which are supported by molecular dynamics. The heparin binding site was identified, along with local L1 conformational changes and global flexibility changes. These changes induced by heparin binding likely reflect the structure of HPV during early stages of entry and provide a framework for future HPV biochemical, genetic, and biophysical studies.

Protein function often depends on ligand binding pockets that fluctuate among conformational states, altering their size, shape, topology, and accessibility, yet quantitative comparison of these dynamic cavities remains challenging because their boundaries are often inherently ambiguous. The measure volinterior algorithm uses fuzzy-boundary detection to characterize enclosed molecular spaces; here, the hepatitis B virus (HBV) capsid assembly modulator (CAM) binding site is used as a model system to develop and validate a practical workflow for applying the method to dynamic protein binding pockets. The resulting methodology provides practical guidance for parameter selection and evaluation, establishes a standardized protocol for quantitative characterization of the HBV CAM pocket, and demonstrates robust, reproducible performance across conformational ensembles derived from molecular dynamics (MD) simulations. More broadly, this work provides a reproducible strategy for adapting measure volinterior to other dynamic binding pockets, enabling consistent comparison of pocket geometry among independent structural studies. Graphical

There is an unmet need to develop vehicles for delivering protein cargo and ribonucleoprotein complexes to cells. Virus capsids, or virus-like particles, have numerous advantages as self-assembling protein platforms, but there remains a need to develop robust methods for the specific internal packaging of cargo. In this work, we use the Hepatitis B Virus (HBV) capsid as our delivery vehicle. To specifically associate protein cargo with the capsid, we covalently attached a derivative of an HBV sulfamoylbenzamide (SBA) antiviral to green fluorescent protein (GFP). We show that SBA-tagged GFP can associate with HBV capsids, but efficient internal packaging requires engineering the assembly of capsids around a cargo. Under standard co-assembly conditions, SBA-tagged GFP predominantly displayed on the capsid exterior, reaching ∼40 GFP cargo proteins per capsid. We tested two strategies to drive encapsidation of protein cargo. First, remodeling the capsid interior was nominally effective. Conversely, His-tag/Ni2+-mediated clustering converts the cargo into a multivalent capsid-binding scaffold that templates assembly, enabling internal packaging of up to ∼30 cargo proteins per capsid. Based on capsid geometry, we suggest the practical limit is approximately 30 cargo proteins per T = 4 HBV capsid, indicating that this strategy approaches maximal loading. Together, these results provide a highly effective method for packaging proteins inside VLPs.

Hepatitis B virus (HBV) infections cause chronic liver disease, resulting in about one million deaths per year, and there is currently no cure. Recent work has shown that a class of small molecules called capsid assembly modulators (CAMs) is promising for treating HBV. CAMs bind to HBV capsid protein subunits and alter their assembly, leading to non-functional and malformed structures rather than functional, closed shells. However, the mechanisms by which CAMs alter capsid assembly pathways remain unclear. Here, we extend a recently-developed kinetic Monte Carlo (KMC) model for HBV capsid assembly to simulate how CAMs affect assembly. In the model, CAMs alter assembly by preferentially binding to interfaces between certain quasi-equivalent subunit conformations. Simulations of the model reproduce experimental assembly product distributions. By analyzing assembly trajectories, we clarify the roles of thermodynamics and kinetics in determining assembly products, identify assembly mechanisms, and predict the key intermediates that lead to either capsids or malformed structures. Our findings enhance our fundamental understanding of capsid assembly, help advance the development of CAMs as a treatment for HBV and, more broadly, inform efforts to direct self-assembly pathways toward specific products.

In an infection, Hepatitis B Virus (HBV) core protein (HBc) normally assembles into icosahedral capsids. Capsid Assembly Modulators (CAMs) are direct acting antivirals that induce HBc mis-assembly and are the subject of active research and development. Two versions of HBc are used in structural studies of CAM-HBc complexes: Cp150 and Cp149-Y132A. Cp150 forms empty icosahedral capsids that are structurally indistinguishable from those found in virions. The Y132A mutation of Cp149 leads to an assembly defective soluble protein that crystalizes as flat hexagonal sheets, where the hexagons resemble icosahedral quasi-sixfold vertices. In this study, we compare structures of CAM-bound Cp150 to CAM-bound Cp149-Y132A. In capsids, the residues forming the CAM site shift to match the structure of bound CAMs, an induced fit. In Cp149-Y132A crystals, CAM sites show little structural adjustment in response to different CAMs binding. In turn, the array of residues that interact with CAMs varies from CAM to CAM in capsid structures but remains nearly constant in Cp149-Y132A crystals. These results illustrate important differences between CAM binding in Cp149-Y132A and Cp150 structures that will contribute to future CAM design.

The symmetric protein cage of the brome mosaic virus provides a relatively rigid, chemically addressable, deterministic molecular scaffold capable of being covalently labeled with organic dyes at high densities, with the overall scaffold structure remaining intact. Homogeneous short interchromophore distances endow the dye-decorated cage with network-like connectivity that supports efficient energy flow. Network connectivity varies in time in a way that is determined by the relative rates of reversible and irreversible phenomena, including Förster resonant energy transfer, prototropism, and photobleaching. Through a combined experimental and computational analysis, we disentangle these effects and advance a simplified model that reveals the role of the virus shell scaffold in shaping the fluorescence dynamics, which is distinct from those of other luminescent nanoparticles.

Capsid assembly modulators (CAMs) represent a promising antiviral strategy against hepatitis B virus (HBV), but their effects on pre-formed capsids remain incompletely understood. Here, all-atom molecular dynamics (MD) simulations of intact HBV capsids complexed with prototypical CAM-As (HAP1, HAP18) and CAM-Es (AT130), reveal how structural changes induced by small molecule binding in the interdimer interfaces propagate through the shell lattice to yield global morphological consequences. Each quasi-equivalent interface exhibits a unique response: A sites, located within the pentameric capsomers, are unfilled in these systems and altered marginally by the presence of CAMs in neighboring interfaces. B sites are the most open and “CAM-ready,” suggesting uptake requires minimal conformational perturbation on the local or global level. C sites emerge as hubs of allosteric control and the key drug target, as their occupancy creates local distortion that is broadcast to adjacent sites, driving capsid faceting and – in the case of CAM-As – the destabilization that precedes dissociation in favor of aberrant assembly. D sites, unfilled in these systems, act as structural sinks, absorbing distortions from adjacent interfaces within the hexameric capsomers. The extent of C site adjustment and the nature of D site counterbalance varies with CAM chemotype, highlighting the divergent effects of CAM-As versus CAM-Es. The tensegrity relationship between the four quasi-equivalent interfaces couples them into a global network for strain redistribution that is functionally allosteric, with CAM binding sites displaying signs of both positive and negative cooperativity. These new insights into HBV capsid dynamics clarify how CAMs alter them on the microsecond timescale and suggest that targeting strain redistribution in mature core particles could be leveraged therapeutically.

Superradiance (SR) arises when fluorophores radiate collectively rather than independently, producing ultrabright emission bursts. Recent experiments show that dye-labeled virus-like particles (VLPs) can exhibit SR at room temperature, where the VLP scaffold organizes dyes into a macrodipole of coherent emitters under pulsed excitation. To elucidate the biophysical basis of this phenomenon, microsecond-scale all-atom molecular dynamics (MD) simulations were performed on intact Brome mosaic virus (BMV) VLPs, comparing the native scaffold to a capsid decorated with Oregon Green 488 (OG). Results show that OG conjugation at K105 induces only subtle structural changes: a slight expansion of VLP volume and marginally higher inter-subunit flexibility, while overall shell morphology and pore architecture remain intact. However, dianionic OG alters VLP electrostatics, increasing water exchange across the shell and reducing chloride permeability. Dyes bound in pentamers are more closely packed and conformationally restricted than those in hexamers, and their transition dipoles more frequently adopt parallel orientations predicted to favor excitonic coupling and cooperative emission. By examining the role of scaffold architecture in dye interactions and dynamics, this work offers a mechanistic framework and design principles for engineering SR-VLPs as next-generation fluorescent probes and photonic materials.

Capsid assembly modulators (CAMs) represent a promising antiviral strategy against hepatitis B virus (HBV), but their effects on preformed capsids remain incompletely understood. Here, all-atom molecular dynamics (MD) simulations of intact HBV capsids complexed with prototypical CAM-As (HAP1, HAP18) and CAM-Es (AT130), reveal how structural changes induced by small molecule binding in the interdimer interfaces propagate through the shell lattice to yield global morphological consequences. Each quasi-equivalent interface exhibits a unique response: A sites, located within the pentameric capsomers, are unfilled in these systems and altered marginally by the presence of CAMs in neighboring interfaces. B sites are the most open and “CAM-ready,” suggesting uptake requires minimal conformational perturbation on the local or global level. C sites emerge as hubs of allosteric control and the key drug target, as their occupancy creates local distortion that is broadcast to adjacent sites, driving capsid faceting and – in the case of CAM-As – the destabilization that precedes dissociation in favor of aberrant assembly. D sites, unfilled in these systems, act as structural sinks, absorbing distortions from adjacent interfaces within the hexameric capsomers. The extent of C site adjustment and the nature of D site counterbalance varies with CAM chemotype, highlighting the divergent effects of CAM-As versus CAM-Es. The tensegrity relationship between the four quasi-equivalent interfaces couples them into a global network for strain redistribution that is functionally allosteric, with CAM binding sites displaying signs of both positive and negative cooperativity. These new insights into HBV capsid dynamics clarify how CAMs alter them on the microsecond timescale and suggest that targeting strain redistribution in mature core particles could be leveraged therapeutically. Author summary: Hepatitis B is a major cause of chronic liver disease worldwide. The virus relies on a protein shell, called the capsid, to protect and deliver its genetic material to the host cell during infection. Some experimental drug molecules attack this shell, either forcing it to assemble incorrectly or breaking it apart after it has formed. To understand how these molecules work, we used powerful computer simulations to model the capsid at the level of individual atoms. We discovered that when molecules bind the capsid at certain sites, they create strain that spreads across the shell, sometimes leading to large distortions and instability. These insights explain how small molecules can disrupt the virus and point the way toward designing better antiviral therapies.