Guanidine Hydrochloride-Induced Hepatitis B Virus Capsid Disassembly Hysteresis.

Hepatitis B virus (HBV) displays remarkable self-assembly capabilities that interest the scientific community and biotechnological industries as HBV is leading to an annual mortality of up to 1 million people worldwide (especially in Africa and Southeast Asia). When the ionic strength is increased, hepatitis B virus-like particles (VLPs) can assemble from dimers of the first 149 residues of the HBV capsid protein core assembly domain (Cp149). Using solution small-angle X-ray scattering, we investigated the disassembly of the VLPs by titrating guanidine hydrochloride (GuHCl). Measurements were performed with and without 1 M NaCl, added either before or after titrating GuHCl. Fitting the scattering curves to a linear combination of atomic models of Cp149 dimer (the subunit) and T = 3 and T = 4 icosahedral capsids revealed the mass fraction of the dimer in each structure in all the titration points. Based on the mass fractions, the variation in the dimer-dimer association standard free energy was calculated as a function of added GuHCl, showing a linear relation between the interaction strength and GuHCl concentration. Using the data, we estimated the energy barriers for assembly and disassembly and the critical nucleus size for all of the assembly reactions. Extrapolating the standard free energy to [GuHCl] = 0 showed an evident hysteresis in the assembly process, manifested by differences in the dimer-dimer association standard free energy obtained for the disassembly reactions compared with the equivalent assembly reactions. Similar hysteresis was observed in the energy barriers for assembly and disassembly and the critical nucleus size. The results suggest that above 1.5 M, GuHCl disassembled the capsids by attaching to the protein and adding steric repulsion, thereby weakening the hydrophobic attraction.

Structure of the Hepatitis B virus capsid quasi-6-fold with a trapped C-terminal domain reveals capsid movements associated with domain exit.

Many viruses undergo transient conformational change to surveil their environments for receptors and host factors. In Hepatitis B virus (HBV) infection, after the virus enters the cell, it is transported to the nucleus by interaction of the HBV capsid with an importin α/ß complex. The interaction between virus and importins is mediated by nuclear localization signals on the capsid protein's C-terminal domain (CTD). However, CTDs are located inside the capsid. In this study, we asked where does a CTD exit the capsid, are all quasi-equivalent CTDs created equal, and does the capsid structure deform to facilitate CTD egress from the capsid? Here, we used Impß as a tool to trap transiently exposed CTDs and examined this complex by cryo-electron microscopy. We examined an asymmetric reconstruction of a T = 4 icosahedral capsid and a focused reconstruction of a quasi-6-fold vertex (3.8 and 4.0 Å resolution, respectively). Both approaches showed that a subset of CTDs extended through a pore in the center of the quasi-6-fold complex. CTD egress was accompanied by enlargement of the pore and subtle changes in quaternary and tertiary structure of the quasi-6-fold. When compared to molecular dynamics simulations, structural changes were within the normal range of capsid flexibility. Although pore diameter was enlarged in the Impß-bound reconstruction, simulations indicate that CTD egress does not exclusively depend on enlarged pores. In summary, we find that HBV surveillance of its environment by transient exposure of its CTD requires only modest conformational change of the capsid.

Engineering Metastability into a Virus-like Particle to Enable Triggered Dissociation.

For a virus-like particle (VLP) to serve as a delivery platform, the VLP must be able to release its cargo in response to a trigger. Here, we use a chemical biology approach to destabilize a self-assembling capsid for a subsequent triggered disassembly. We redesigned the dimeric hepatitis B virus (HBV) capsid protein (Cp) with two differentially addressable cysteines, C150 for reversibly crosslinking the capsid and C124 to react with a destabilizing moiety. The resulting construct, Cp150-V124C, assembles into icosahedral, 120-dimer VLPs that spontaneously crosslink via the C-terminal C150, leaving C124 buried at a dimer-dimer interface. The VLP is driven into a metastable state when C124 is reacted with the bulky fluorophore, maleimidyl BoDIPY-FL. The resulting VLP is stable until exposed to modest, physiologically relevant concentrations of reducing agent. We observe dissociation with FRET relaxation of polarization, size exclusion chromatography, and resistive-pulse sensing. Dissociation is slow, minutes to hours, with a characteristic lag phase. Mathematical modeling based on the presence of a nucleation step predicts disassembly dynamics that are consistent with experimental observations. VLPs transfected into hepatoma cells show similar dissociation behavior. These results suggest a generalizable strategy for designing a VLP that can release its contents in an environmentally responsive reaction.

Core Protein-Directed Antivirals and Importin ß Can Synergistically Disrupt Hepatitis B Virus 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 life cycle; 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., Impß) via Cp's 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 modulator (CpAM), a class of antivirals that inappropriately stimulates and misdirects HBV assembly and deforms capsids. CpAM impact on other aspects of the HBV life cycle is poorly understood. We investigate how HAP12 influences the interactions between empty or RNA-filled capsids with Impß and trypsin in vitro. We show that HAP12 can modulate CTD accessibility and capsid stability, depending on the saturation of HAP12-binding sites. We demonstrate that Impß synergistically contributes to capsid disruption at high levels of HAP12 saturation, using electron microscopy to visualize the disruption and rearrangement of Cp dimers into aberrant complexes. However, RNA-filled capsids resist the destabilizing effects of HAP12 and Impß. 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 life cycle and also cause the virus to become vulnerable to host innate immune responses. IMPORTANCE The 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 the potential to disrupt the virus life cycle and activate the innate immune system.

Hysteresis in Hepatitis B Virus (HBV) Requires Assembly of Near-Perfect Capsids.

The hepatitis B virus (HBV) must release its contents to initiate infection, making capsid disassembly critical to the viral life cycle. Capsid assembly proceeds through a cascade of weak interactions between copies of capsid protein (Cp) to yield uniform particles. However, there is a hysteresis to capsid dissociation that allows capsids to persist under conditions where they could not assemble. In this study, we have sought to define the basis of hysteresis by examining urea-induced dissociation of in vitro-assembled HBV capsids. In general, capsid samples show a mixture of two pools, differentiated by stability. Labile capsid dissociation corresponds to an ∼5 µM pseudocritical concentration of assembly (pcc), the same as that observed in assembly reactions. Dissociation of the stable pool corresponds to a subfemtomolar pcc, indicative of hysteresis. The fraction of stable capsids in an assembly reaction increases with the integrity of the Cp preparation and when association is performed at a higher ionic strength, which modifies the Cp conformation. Labile complexes are more prevalent when assembly conditions yield many kinetically trapped (incomplete and overgrown) products. Cp isolated from stable capsids reassembles into a mixture of stable and labile capsids. These results suggest that hysteresis arises from an ideal capsid lattice, even when some of the substituents in that lattice have defects. Consistent with structural studies that show a subtle difference between Cp dimers and Cp in capsid, we propose that hysteresis arises when HBV capsids undergo a lattice-dependent structural transition.

Disassembly of Single Virus Capsids Monitored in Real Time with Multicycle Resistive-Pulse Sensing.

Virus assembly and disassembly are critical steps in the virus lifecycle; however, virus disassembly is much less well understood than assembly. For hepatitis B virus (HBV) capsids, disassembly of the virus capsid in the presence of guanidine hydrochloride (GuHCl) exhibits strong hysteresis that requires additional chemical energy to initiate disassembly and disrupt the capsid structure. To study disassembly of HBV capsids, we mixed T = 4 HBV capsids with 1.0-3.0 M GuHCl, monitored the reaction over time by randomly selecting particles, and measured their size with resistive-pulse sensing. Particles were cycled forward and backward multiple times to increase the observation time and likelihood of observing a disassembly event. The four-pore device used for resistive-pulse sensing produces four current pulses for each particle during translocation that improves tracking and identification of single particles and increases the precision of particle-size measurements when pulses are averaged. We studied disassembly at GuHCl concentrations below and above denaturing conditions of the dimer, the fundamental unit of HBV capsid assembly. As expected, capsids showed little disassembly at low GuHCl concentrations (e.g., 1.0 M GuHCl), whereas at higher GuHCl concentrations (≥1.5 M), capsids exhibited disassembly, sometimes as a complex series of events. In all cases, disassembly was an accelerating process, where capsids catastrophically disassembled within a few 100 ms of reaching critical stability; disassembly rates reached tens of dimers per second just before capsids fell apart. Some disassembly events exhibited metastable intermediates that appeared to lose one or more trimers of dimers in a stepwise fashion.

Rapidly Forming Early Intermediate Structures Dictate the Pathway of Capsid Assembly.

There are ∼1030 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 initial assembly conditions. With the mildest conditions tested, the reaction appeared to be two-state from dimer to 120-dimer capsid with some dimers-of-dimers and trimers-of-dimers. In slightly more aggressive conditions, we observed transient accumulation of a decamer-of-dimers and the appearance of 90-dimer capsids. In conditions where there is measurable kinetic trapping, we found that 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.

Higher Resolution Charge Detection Mass Spectrometry.

Charge detection mass spectrometry is a single particle technique where the masses of individual ions are determined from simultaneous measurements of each ion's m/z ratio and charge. The ions pass through a conducting cylinder, and the charge induced on the cylinder is detected. The cylinder is usually placed inside an electrostatic linear ion trap so that the ions oscillate back and forth through the cylinder. The resulting time domain signal is analyzed by fast Fourier transformation; the oscillation frequency yields the m/z, and the charge is determined from the magnitudes. The mass resolving power depends on the uncertainties in both quantities. In previous work, the mass resolving power was modest, around 30-40. In this work we report around an order of magnitude improvement. The improvement was achieved by coupling high-accuracy charge measurements (obtained with dynamic calibration) with higher resolution m/z measurements. The performance was benchmarked by monitoring the assembly of the hepatitis B virus (HBV) capsid. The HBV capsid assembly reaction can result in a heterogeneous mixture of intermediates extending from the capsid protein dimer to the icosahedral T = 4 capsid with 120 dimers. Intermediates of all possible sizes were resolved, as well as some overgrown species. Despite the improved mass resolving power, the measured peak widths are still dominated by instrumental resolution. Heterogeneity makes only a small contribution. Resonances were observed in some of the m/z spectra. They result from ions with different masses and charges having similar m/z values. Analogous resonances are expected whenever the sample is a heterogeneous mixture assembled from a common building block.

Structural Differences between the Woodchuck Hepatitis Virus Core Protein in the Dimer and Capsid States Are Consistent with 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) has 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 assemblies to investigate the basis of assembly regulation. We determined the structures of the WHV capsid to 4.5-Å resolution by cryo-electron microscopy (cryo-EM) and of the WHV Cp dimer to 2.9-Å resolution by crystallography and examined the biophysical properties of the dimer. We found, in dimer, that the subdomain that makes protein-protein interactions is partially disordered and rotated 21° from its position in capsid. This subdomain is susceptible to proteolysis, consistent with local disorder. WHV assembly shows similar susceptibility to HBV antiviral molecules, suggesting that HBV assembly follows similar transitions. These data show that 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 a disorder-to-order transition and structural shifts. We suggest that a cascade of structural changes may be a common mechanism for regulating high-fidelity capsid assembly in HBV and other viruses.IMPORTANCE Virus capsids assemble spontaneously with surprisingly high fidelity. This requires strict geometry and a narrow range of association energies for these protein-protein interactions. It was hypothesized that requiring subunits to undergo a conformational change to become assembly active could regulate assembly by creating an energetic barrier and attenuating association. We found that woodchuck hepatitis virus capsid protein undergoes structural transitions between its dimeric and its 120-dimer capsid states. It is likely that the closely related hepatitis B virus capsid protein undergoes similar structural changes, which has implications for drug design. Regulation of assembly by structural transition may be a common mechanism for many viruses.

Competition between Normative and Drug-Induced Virus Self-Assembly Observed with Single-Particle Methods.

Disruption of virus capsid assembly has compelling antiviral potential that has been applied to hepatitis B virus (HBV). HBV core protein assembly can be modulated by heteroaryldihydropyrimidines (HAPs), and such molecules are collectively termed core protein allosteric modulators (CpAMs). Although the antiviral effects of CpAMs are acknowledged, the mechanism of action remains an open question. Challenging aspects of characterizing misdirected assembly are the large size and nonuniform nature of the final particles. In this study of HBV assembly, we observed a competition between normative and CpAM-induced aberrant assembly with electron microscopy and resistive-pulse sensing on nanofluidic devices. This competition was a function of the strength of the association energy between individual core proteins, which is proportional to ionic strength. At strong association energy, assembly reactions primarily yielded morphologically normal HBV capsids, despite the presence of HAP-TAMRA. At weak association energy, HAP-TAMRA led to increased assembly product size and disrupted morphology. The smallest particles were T = 4 icosahedra, whereas the larger particles were defective spheres, ellipsoids, and bacilliform cylinders, with regions of T = 4 geometry interspersed with flat regions. Deviation from spherical geometry progressively increased with particle size, which is consistent with the interpretation of a competition between two alternative assembly pathways.

Asymmetric Modification of Hepatitis B Virus (HBV) Genomes by an Endogenous Cytidine Deaminase inside HBV Cores Informs a Model of Reverse Transcription.

Cytidine deaminases inhibit replication of a broad range of DNA viruses by deaminating cytidines on single-stranded DNA (ssDNA) to generate uracil. While several lines of evidence have revealed hepatitis B virus (HBV) genome editing by deamination, it is still unclear which nucleic acid intermediate of HBV is modified. Hepatitis B virus has a relaxed circular double-stranded DNA (rcDNA) genome that is reverse transcribed within virus cores from a RNA template. The HBV genome also persists as covalently closed circular DNA (cccDNA) in the nucleus of an infected cell. In the present study, we found that in HBV-producing HepAD38 and HepG2.2.15 cell lines, endogenous cytidine deaminases edited 10 to 25% of HBV rcDNA genomes, asymmetrically with almost all mutations on the 5' half of the minus strand. This region corresponds to the last half of the minus strand to be protected by plus-strand synthesis. Within this half of the genome, the number of mutations peaks in the middle. Overexpressed APOBEC3A and APOBEC3G could be packaged in HBV capsids but did not change the amount or distribution of mutations. We found no deamination on pregenomic RNA (pgRNA), indicating that an intact genome is encapsidated and deaminated during or after reverse transcription. The deamination pattern suggests a model of rcDNA synthesis in which pgRNA and then newly synthesized minus-sense single-stranded DNA are protected from deaminase by interaction with the virus capsid; during plus-strand synthesis, when enough dsDNA has been synthesized to displace the remaining minus strand from the capsid surface, the single-stranded DNA becomes deaminase sensitive.IMPORTANCE Host-induced mutation of the HBV genome by APOBEC proteins may be a path to clearing the virus. We examined cytidine-to-thymidine mutations in the genomes of HBV particles grown in the presence or absence of overexpressed APOBEC proteins. We found that genomes were subjected to deamination activity during reverse transcription, which takes place within the virus capsid. These observations provide a direct insight into the mechanics of reverse transcription, suggesting that newly synthesized dsDNA displaces ssDNA from the capsid walls, making the ssDNA accessible to deaminase activity.

Assembly Properties of Hepatitis B Virus Core Protein Mutants Correlate with Their Resistance to Assembly-Directed Antivirals.

The hepatitis B virus (HBV) capsid or core protein (Cp) can self-assemble to form an icosahedral capsid. It is now being pursued as a target for small-molecule antivirals that enhance the rate and extent of its assembly to yield empty and/or aberrant capsids. These small molecules are thus called core protein allosteric modulators (CpAMs). We sought to understand the physical basis of CpAM-resistant mutants and how CpAMs might overcome them. We examined the effects of two closely related CpAMs, HAP12 and HAP13, which differ by a single atom but have drastically different antiviral activities, on the assembly of wild-type Cp and three T109 mutants (T109M, T109I, and T109S) that display a range of resistances. The T109 side chain forms part of the mouth of the CpAM binding pocket. A T109 mutant that has substantial resistance even to a highly active CpAM strongly promotes normal assembly. Conversely, a mutant that weakens assembly is more susceptible to CpAMs. In crystal and cryo-electron microscopy (cryo-EM) structures of T=4 capsids with bound CpAMs, the CpAMs preferentially fit into two of four quasi-equivalent sites. In these static representations of capsid structures, T109 does not interact with the neighboring subunit. However, all-atom molecular dynamics simulations of an intact capsid show that T109 of one of the four classes of CpAM site has a hydrophobic contact with the neighboring subunit at least 40% of the time, providing a physical explanation for the mutation's ability to affect capsid stability, assembly, and sensitivity to CpAMs.IMPORTANCE The HBV core protein and its assembly into capsids have become important targets for development of core protein allosteric modulators (CpAMs) as antivirals. Naturally occurring T109 mutants have been shown to be resistant to some of these CpAMs. We found that mutation of T109 led to changes in capsid stability and recapitulated resistance to a weak CpAM, but much less so than to a strong CpAM. Examination of HBV capsid structures, determined by cryo-EM and crystallography, could not explain how T109 mutations change capsid stability and resistance. However, by mining data from a microsecond-long all-atom molecular dynamics simulation, we found that the capsid was extraordinarily flexible and that T109 can impede entry to the CpAM binding site. In short, HBV capsids are incredibly dynamic and molecular mobility must be considered in discussions of antiviral mechanisms.

Effect of dsDNA on the Assembly Pathway and Mechanical Strength of SV40 VP1 Virus-like Particles.

Simian virus 40 (SV40) is a possible vehicle for targeted drug delivery systems because of its low immunogenicity, high infectivity, and high transfection efficiency. To use SV40 for biotechnology applications, more information is needed on its assembly process to efficiently incorporate foreign materials and to tune the mechanical properties of the structure. We use atomic force microscopy to determine the effect of double-stranded DNA packaging, buffer conditions, and incubation time on the morphology and strength of virus-like particles (VLPs) composed of SV40 VP1 pentamers. DNA-induced assembly results in a homogeneous population of native-like, ∼45 nm VLPs. In contrast, under high-ionic-strength conditions, the VP1 pentamers do not seem to interact consistently, resulting in a heterogeneous population of empty VLPs. The stiffness of both in-vitro-assembled empty and DNA-filled VLPs is comparable. Yet, the DNA increases the VLPs' resistance to large deformation forces by acting as a scaffold, holding the VP1 pentamers together. Both disulfide bridges and Ca2+, important in-vitro-assembly factors, affect the mechanical stability of the VLPs: the reducing agent DTT makes the VLPs less resistant to mechanical stress and prone to damage, whereas Ca2+-chelating EDTA induces a marked softening of the VLP. These results show that negatively charged polymers such as DNA can be used to generate homogeneous particles, thereby optimizing VLPs as vessels for drug delivery. Moreover, the storage buffer should be chosen such that VP1 interpentamer interactions are preserved.

Use of a Fluorescent Analogue of a HBV Core Protein-Directed Drug To Interrogate an Antiviral Mechanism.

Heteroaryldihydropyrimidines (HAPs) are antiviral small molecules that enhance assembly of HBV core protein (Cp), lead to assembly of empty and defective particles, and suppress viral replication. These core protein allosteric modulators (CpAMs) bind to the pocket at the interface between two Cp dimers and strengthen interdimer interactions. To investigate the CpAM mechanism, we wanted to examine the cellular distributions of Cp and the CpAM itself. For this reason, we developed a fluorescently labeled CpAM, HAP-ALEX. In vitro, HAP-ALEX modulated assembly of purified Cp and at saturating concentrations induced formation of large structures. HAP-ALEX bound capsids and not dimers, making it a capsid-specific molecular tag. HAP-ALEX labeled HBV in transfected cells, with no detectable background with a HAP-insensitive Cp mutant. HAP-ALEX caused redistribution of Cp in a dose-dependent manner consistent with its 0.7 µM EC50, leading to formation of large puncta and an exclusively cytoplasmic distribution. HAP-ALEX colocalized with the redistributed Cp, but large puncta accumulated long before they appeared saturated with the fluorescent CpAM. CpAMs affect HBV assembly and localization; with a fluorescent CpAM both drug and target can be identified.

Multiple Pathways in Capsid Assembly.

For a three-dimensional structure to spontaneously self-assemble from many identical components, the steps on the pathway must be kinetically accessible. Many virus capsids are icosahedral and assembled from hundreds of identical proteins, but how they navigate the assembly process is poorly understood. Capsid assembly is thought to involve stepwise addition of subunits to a growing capsid fragment. Coarse-grained models suggest that the reaction occurs on a downhill energy landscape, so intermediates are expected to be fleeting. In this work, charge detection mass spectrometry (CDMS) has been used to track assembly of the hepatitis B virus (HBV) capsid in real time. The icosahedral T = 4 capsid of HBV is assembled from 120 capsid protein dimers. Our results indicate that there are multiple pathways for assembly. Under conditions that favor a modest association energy there is no accumulation of large intermediates, which indicates that available pathways include ones on a downhill energy surface. Under higher salt conditions, where subunit interactions are strengthened, around half of the products of the initial assembly reaction have masses close to the T = 4 capsid and the other half are stalled intermediates which emerge abruptly at around 90 dimers, indicating a bifurcation in the ensemble of assembly paths. When incubated at room temperature, the 90-dimer intermediates accumulate dimers and gradually shift to higher mass and merge with the capsid peak. Though free subunits are present in solution, the stalled intermediates indicate the presence of a local minima on the energy landscape. Some intermediates may result from hole closure, where the growing capsid distorts to close the hole due to the missing capsid proteins or from a species where subsequent additions are particularly labile.

Characterization of Virus Capsids and Their Assembly Intermediates by Multicycle Resistive-Pulse Sensing with Four Pores in Series.

Virus self-assembly is a critical step in the virus lifecycle. Understanding how viruses assemble and disassemble provides needed insight into developing antiviral pharmaceuticals. Few tools offer sufficient resolution to study assembly intermediates that differ in size by a few dimers. Our goal is to improve resistive-pulse sensing on nanofluidic devices to offer better particle-size and temporal resolution to study intermediates and capsids generated along the assembly pathway. To increase the particle-size resolution of the resistive-pulse technique, we measured the same, single virus particles up to a thousand times, cycling them back and forth across a series of nanopores by switching the polarity of the applied potential, i.e., virus ping-pong. Multiple pores in series provide a unique multipulse signature during each cycle that improves particle tracking and, therefore, identification of a single particle and reduces the number of cycles needed to make the requisite number of measurements. With T = 3 and T = 4 hepatitis B virus (HBV) capsids, we showed the standard deviation of the particle-size distribution decreased with the square root of the number of measurements and approached discriminating particles differing in size by single dimers. We then studied in vitro assembly of HBV capsids and observed that the ensemble of intermediates shift to larger sizes over 2 days of annealing. On the contrary, assembly reactions diluted to lower dimer concentrations an hour after initiation had fewer intermediates that persisted after the 2 day incubation and had a higher ratio of T = 4 to T = 3 capsids. These reactions indicate that labile T = 4 intermediates are formed rapidly, and dependent on conditions, intermediates may be trapped as metastable species or progress to yield complete capsids.

Importin ß Can Bind Hepatitis B Virus Core Protein and Empty Core-Like Particles and Induce Structural Changes.

Hepatitis B virus (HBV) capsids are found in many forms: immature single-stranded RNA-filled cores, single-stranded DNA-filled replication intermediates, mature cores with relaxed circular double-stranded DNA, and empty capsids. A capsid, the protein shell of the core, is a complex of 240 copies of core protein. Mature cores are transported to the nucleus by a complex that includes both importin α and importin ß (Impα and Impß), which bind to the core protein's C-terminal domains (CTDs). Here we have investigated the interactions of HBV core protein with importins in vitro. Strikingly, empty capsids and free core protein can bind Impß without Impα. Cryo-EM image reconstructions show that the CTDs, which are located inside the capsid, can extrude through the capsid to be bound by Impß. Impß density localized on the capsid exterior near the quasi-sixfold vertices, suggested a maximum of 30 Impß per capsid. However, examination of complexes using single molecule charge-detection mass spectrometry indicate that some complexes include over 90 Impß molecules. Cryo-EM of capsids incubated with excess Impß shows a population of damaged particles and a population of "dark" particles with internal density, suggesting that Impß is effectively swallowed by the capsids, which implies that the capsids transiently open and close and can be destabilized by Impß. Though the in vitro complexes with great excess of Impß are not biological, these results have implications for trafficking of empty capsids and free core protein; activities that affect the basis of chronic HBV infection.

Molecular jenga: the percolation phase transition (collapse) in virus capsids.

Virus capsids are polymeric protein shells that protect the viral cargo. About half of known virus families have icosahedral capsids that self-assemble from tens to thousands of subunits. Capsid disassembly is critical to the lifecycles of many viruses yet is poorly understood. Here, we apply a graph and percolation theory to examine the effect of removing capsid subunits on capsid stability and fragmentation. Based on the structure of the icosahedral capsid of hepatitis B virus (HBV), we constructed a graph of rhombic subunits arranged with icosahedral symmetry. Though our approach neglects dependence on energetics, time, and molecular detail, it quantitatively predicts a percolation phase transition consistent with recent in vitro studies of HBV capsid dissociation. While the stability of the capsid graph followed a gradual quadratic decay, the rhombic tiling abruptly fragmented when we removed more than 25% of the 120 subunits, near the percolation threshold observed experimentally. This threshold may also affect results of capsid assembly, which also experimentally produces a preponderance of 90 mer intermediates, as the intermediate steps in these reactions are reversible and can thus resemble dissociation. Application of percolation theory to understanding capsid association and dissociation may prove a general approach to relating virus biology to the underlying biophysics of the virus particle.

Hepatitis B Virus Capsid Completion Occurs through Error Correction.

Understanding capsid assembly is important because of its role in virus lifecycles and in applications to drug discovery and nanomaterial development. Many virus capsids are icosahedral, and assembly is thought to occur by the sequential addition of capsid protein subunits to a nucleus, with the final step completing the icosahedron. Almost nothing is known about the final (completion) step because the techniques usually used to study capsid assembly lack the resolution. In this work, charge detection mass spectrometry (CDMS) has been used to track the assembly of the T = 4 hepatitis B virus (HBV) capsid in real time. The initial assembly reaction occurs rapidly, on the time scale expected from low resolution measurements. However, CDMS shows that many of the particles generated in this process are defective and overgrown, containing more than the 120 capsid protein dimers needed to form a perfect T = 4 icosahedron. The defective and overgrown capsids self-correct over time to the mass expected for a perfect T = 4 capsid. Thus, completion is a distinct phase in the assembly reaction. Capsid completion does not necessarily occur by inserting the last building block into an incomplete, but otherwise perfect icosahedron. The initial assembly reaction can be predominently imperfect, and completion involves the slow correction of the accumulated errors.