Biology subjects

Zlotnick, A.

Publications and source records attributed to Zlotnick, A..

10 recordsLinked to original sources

bioRxiv2026

Toward Robust Characterization of Dynamic Binding Pockets: Lessons from the HBV Capsid Assembly Modulator Site

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 Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/743403v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@540692org.highwire.dtl.DTLVardef@7b94dborg.highwire.dtl.DTLVardef@169c478org.highwire.dtl.DTLVardef@e1de80_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Modeling Reveals How Direct-Acting Antivirals Redirect HBV Capsid Assembly Pathways to Noninfectious Products

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.

biophysics↗

bioRxiv2026

Assembly-active and -inactive forms of HBV capsid protein provide distinctly different binding sites for capsid assembly modulators

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.

biochemistry↗

bioRxiv2026

DNA synthesis inside the hepatitis B virus creates a high-energy spool

The Hepatitis B Virus (HBV) capsid assembles around an RNA pregenome which is reverse-transcribed into double-stranded DNA. It remains unclear how this DNA, which is stiff, bulky, and has significant negative charge, is accommodated within the capsid. The organization of the genome will answer this and lend insight into the viral reverse transcription reaction. We determined the structures of mature HBV and Duck HBV, finding that the DNA forms a spool that is coaxial with the capsids fivefold symmetry and interacts with charges on the capsid interior. We calculate that the energy of DNA bending would create a metastable capsid that just awaits a trigger for DNA release. Thus, we see how nature has created a fragile energy minimum that enables genome uncoating.

microbiology↗

bioRxiv2024

A narrow ratio of nucleic acid to SARS-CoV-2 N-protein enables phase separation

SARS-CoV-2 Nucleocapsid protein (N) is a viral structural protein that packages the 30kb genomic RNA inside virions and forms condensates within infected cells through liquid-liquid phase separation (LLPS). N, in both soluble and condensed forms, has accessory roles in the viral life cycle including genome replication and immunosuppression. The ability to perform these tasks depends on phase separation and its reversibility. The conditions that stabilize and destabilize N condensates and the role of N-N interactions are poorly understood. We have investigated LLPS formation and dissolution in a minimalist system comprised of N protein and an ssDNA oligomer just long enough to support assembly. The short oligo allows us to focus on the role of N-N interaction. We have developed a sensitive FRET assay to interrogate LLPS assembly reactions from the perspective of the oligonucleotide. We find that N alone can form oligomers but that oligonucleotide enables their assembly into a three-dimensional phase. At a [~]1:1 ratio of N to oligonucleotide LLPS formation is maximal. We find that a modest excess of N or of nucleic acid causes the LLPS to break down catastrophically. Under the conditions examined here assembly has a critical concentration of about 1 {micro}M. The responsiveness of N condensates to their environment may have biological consequences. A better understanding of how nucleic acid modulates N-N association will shed light on condensate activity and could inform antiviral strategies targeting LLPS.

biochemistry↗

bioRxiv2022

Multiscale Modeling of Hepatitis B Virus Capsid Assembly and its Dimorphism

Hepatitis B Virus (HBV) is an endemic, chronic virus that leads to 800,000 deaths per year. Central to the HBV lifecycle, the viral core has a protein capsid assembled from many copies of a single protein. The capsid protein adopts different (quasi-equivalent) conformations to form icosahedral capsids containing 180 or 240 proteins, T=3 or T=4 respectively in Caspar-Klug nomenclature. HBV capsid assembly has become an important target for new antivirals; nonetheless the assembly pathways and mechanisms that control HBV dimorphism remain unclear. We describe computer simulations of HBV assembly, using a coarse-grained model that has parameters learned from all-atom molecular dynamics simulations of a complete HBV capsid, and yet is computationally tractable. Dynamical simulations with the resulting model reproduce experimental observations of HBV assembly pathways and products. By constructing Markov state models and employing transition path theory, we identify pathways leading to T=3, T=4, and other experimentally observed capsid morphologies. The analysis identifies factors that control this polymorphism, in particular, the conformational free energy landscape of the capsid proteins and their interactions.

biophysics↗

bioRxiv2021

Core Protein-Directed Antivirals and Importinβ Can Synergistically Disrupt HBV Capsids

Viral structural proteins can have multiple activities. Antivirals that target structural proteins have potential to exhibit multiple antiviral mechanisms. Hepatitis B Virus (HBV) core protein (Cp) is involved in most stages of the viral lifecycle: it assembles into capsids, packages viral RNA, is a metabolic compartment for reverse transcription, interacts with nuclear trafficking machinery, and disassembles to release the viral genome into the nucleus. During nuclear localization, HBV capsids bind to host importins (e.g. Imp{beta}) via Cps C-terminal domain (CTD); the CTD is localized to the interior of the capsid and is transiently exposed on the exterior. We used HAP12 as a representative Cp Allosteric Modulators (CpAMs), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV lifecycle is poorly understood. We investigated how HAP12 influenced the interactions between empty or RNA-filled capsids with Imp{beta} and trypsin in vitro. We showed that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrated that Imp{beta} synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resisted the destabilizing effects of HAP12 and Imp{beta}. In summary, we show host protein-induced catalysis of capsid disruption, an unexpected additional mechanism of action for CpAMs. Potentially, untimely capsid disassembly can hamper the HBV lifecycle and also cause the virus to become vulnerable to host innate immune responses. IMPORTANCEThe HBV core, an icosahedral complex of 120 copies of the homodimeric core (capsid) protein with or without packaged nucleic acid, is transported to the host nucleus by its interaction with host importin proteins. Importin-core interaction requires the core protein C-terminal domain, which is inside the capsid, to "flip" to the capsid exterior. Core-protein directed drugs that affect capsid assembly and stability have been developed recently. We show that these molecules can, synergistically with importins, disrupt capsids. This mechanism of action, synergism with host protein, has potential to disrupt the virus lifecycle and activate the innate immune system.

microbiology↗

bioRxiv2020

Local stabilization of subunit-subunit contacts causes global destabilization of Hepatitis B virus capsids

Development of antiviral molecules that bind virion is a strategy that remains in its infancy and the details of their mechanisms are poorly understood. Here we investigate the behavior of DBT1, a dibenzothiazapine, which specifically interacts with the capsid protein of Hepatitis B Virus (HBV). We found that DBT1 stabilizes protein-protein interaction, accelerates capsid assembly, and can induce formation of aberrant particles. Paradoxically, DBT1 can cause pre-formed capsids to dissociate. These activities may lead to (i) assembly of empty and defective capsids, inhibiting formation of new virus and (ii) disruption of mature viruses, which are metastable, to inhibit new infection. Using cryo-electron microscopy we observed that DBT1 led to asymmetric capsids where well-defined DBT1 density was bound at all inter-subunit contacts. These results suggest that DBT1 can support assembly by increasing buried surface area but induce disassembly of metastable capsids by favoring asymmetry to induce structural defects.

biochemistry↗

bioRxiv2019

Identification of Chikungunya virus nucleocapsid core assembly modulators

AbstractThe alphavirus Chikungunya virus is transmitted to humans via infected mosquitos. Most infected humans experience symptoms which can range from short-term fatigue and fever to debilitating arthritis that can last for months or years. Some patients relapse and experience symptoms months or years after the initial bout of disease. The capsid protein of Chikungunya virus forms a shell around the viral RNA genome; this structure is called the nucleocapsid core. The core protects the genome during virus transmission and with the correct environmental trigger, this proteinaceous shell dissociates and releases the viral genome to initiate infection. We hypothesized that targeting compounds to interfere with the nucleocapsid cores function would constrain virus spread either by inhibiting the release of viral genomes during entry or by reducing the number of infectious virus particles assembled. We implemented a high throughput, in vitro, FRET-based assay to monitor nucleic acid packaging by purified Chikungunya capsid protein as a proxy for nucleocapsid core assembly and disassembly. We screened 10,000 compounds and found 45 that substantially modulated the assembly of core-like particles. A subset of compounds was selected to study their effects in virus-infected vertebrate cells. Our results show that four compounds inhibit infectious virus production by at least 90% in a dose-dependent manner. The most promising inhibitor was tested and found to reduce the amount of nucleocapsid cores inside the cell during Chikungunya virus infection. These compounds could be the foundation for anti-viral therapeutics.\n\nHighlightsO_LIA FRET-based assay to detect nucleic acid packaging by Chikungunya virus capsid protein\nC_LIO_LIIdentification of small molecules that modulate core-like particle assembly\nC_LIO_LIA subset of compounds that interfere with in vitro assembly also inhibit Chikungunya virus production in cell culture\nC_LIO_LIIdentification of antiviral molecules that may not be identified by assays using reporter viruses\nC_LIO_LIPotential starting compounds for developing direct-acting antivirals\nC_LI

microbiology↗

bioRxiv2019

Structural Differences Between the Woodchuck Hepatitis Virus Core Protein in Dimer and Capsid States Indicate Entropic and Conformational Regulation of Assembly

Hepadnaviruses are hepatotropic enveloped DNA viruses with an icosahedral capsid. Hepatitis B virus (HBV) causes chronic infection in an estimated 240 million people; Woodchuck Hepatitis virus (WHV), an HBV homologue, has been an important model system for drug development. The dimeric capsid protein (Cp) plays multiple functions during the viral life cycle and thus has become an important target for a new generation of antivirals. Purified HBV and WHV Cp spontaneously assemble into 120-dimer capsids. Though they have 65% identity, WHV Cp has error-prone assembly with stronger protein-protein association. We have taken advantage of the differences in assembly to investigate the basis of assembly regulation. We have determined the structures of the WHV capsid to 4.5 [A] resolution by cryo-EM and the WHV Cp dimer to 2.9 [A] resolution by crystallography and examined the biophysical properties of the dimer. We found, in dimer, the subdomain that makes protein-protein interactions is partially disordered and rotated 21{degrees} from its position in capsid. This subdomain is susceptible to proteolysis, consistent with local disorder. These data show there is an entropic cost for assembly that is compensated for by the energetic gain of burying hydrophobic interprotein contacts. We propose a series of stages in assembly that incorporate disorder-to-order transition and structural shifts. WHV assembly shows similar susceptibility to HBV antiviral molecules, suggesting that HBV assembly follows similar transitions and indicating WHVs importance as a model system for drug development. We suggest that a cascade of structural changes may be a common mechanism for regulating high fidelity capsid assembly in HBV and other viruses.\n\nAuthor summaryCapsid assembly is a key step in the Hepatitis B virus (HBV) life cycle; it requires sophisticated regulation to ensure the production of capsids and viruses with high-fidelity. HBV capsids are constructed from 120 self-assembling capsid protein dimers (Cp). It was hypothesized that changes in Cp structure regulated the assembly reaction. Here we determined structures of dimer and capsid for Woodchuck Hepatitis virus (WHV), an HBV homologue. We observed that the component of the dimer involved in subunit-subunit interactions undergoes a disorder-order transition and changes structure concomitant with assembly. Meanwhile WHV Cp displays similar susceptibility to HBV antiviral. We propose that this structural transition entropically regulates assembly and may be a common theme in other viruses.

biophysics↗