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Results point out that weak protein-protein interactions, influenced by salt environment, propel capsid assembly as revealed by the assembly energetics of hepatitis B virus. Data indicate that salt induces conformational change in the dimer shifting it from an assembly-inactive to assembly-active form.

The assembly of hepatitis B virus capsids begins with a trimer of dimers and continues by adding dimeric subunits. It may be possible to design drugs that block this process.

A small portion of the C-terminus plays an important role in deciding whether hepatitis B virus (HBV) capsid protein will assemble and whether T = 3 or T = 4 capsids are formed. A study conducted on mutant constructs without the predominantly basic C-terminal amino acids indicates that constructs terminating at and beyond residue 140 make a mix of T = 3 and T = 4 particles. The smallest construct with 138 residues fails to assemble. Larger deletions at the C-terminus result in an increase in the T = 3 capsids. The assays use ultracentrifugation and fractionation techniques.

The various features of the assembly inhibitors formed by the combination of the assembly domain of the hepatitis B virus capsid protein and the defective mutant, Cp149-Y132A are discussed. These coassembled capsids are shown to be extremely fragile, as compared to the conventional ones.

Micromolar concentrations of Zn(super 2+) are sufficient to initiate assembly of capsid protein, whereas other mono- and divalent cations elicited assembly only at millimolar concentrations, similar to those required for NaCl-induced assembly. Zinc binding enhances the kinetics of assembly by promoting formation of an intermediate that is readily consumed in the reaction.

A study aims at assembling a structure that can be used as a platform for building more complex nanostructures. An application of virus capsid proteins in generating structures that are a highly organized scaffold from which more complex networks may be prepared where the tunable length distribution of the coat protein nanotubes contributes to this utility.

Chronic hepatitis B virus (HBV) poses a significant public health burden worldwide, encouraging the search for curative antivirals. One approach is capsid assembly modulators (CAMs), which are assembly agonists. CAMs lead to empty and defective capsids, inhibiting the formation of new viruses, and can also lead to defects in the release of the viral genome, inhibiting new infections. In this study, we employed hydrogen-deuterium exchange mass spectrometry (HDX-MS) to assess the impact of one such CAM, HAP18, on HBV dimers, capsids composed of 120 (or 90) capsid protein dimers, and cross-linked capsids (xl-capsids). HDX analysis revealed hydrogen bonding networks within and between the dimers. HAP18 disrupted the hydrogen bonding network of dimers, demonstrating a previously unappreciated impact on the dimer structure. Conversely, HAP18 stabilized both unmodified and cross-linked capsids. Intriguingly, cross-linking the capsid, which was accomplished by forming disulfides between an engineered C-terminal cysteine, increased the overall rate of HDX. Moreover, HAP18 binding induced conformational changes beyond the binding sites. Our findings provide evidence for allosteric communication within and between capsid protein dimers. These results show that CAMs are capable of harnessing this allosteric network to modulate the dimer and capsid dynamics.

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 dibenzothiazepine that 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 preformed 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 intersubunit 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.

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, ∼10 30 , 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.

The Hepatitis B Virus (HBV) core protein homodimers self-assemble to form an icosahedral capsid that packages the viral genome. Disassembly occurs in the nuclear basket to release the mature genome to the nucleus. Small molecules have been developed that bind to a pocket at the interdimer interface to accelerate assembly and strengthen interactions between subunits; these are under development as antiviral agents. Here, we explore the role of the dimer-dimer interface by mutating sites in the drug-binding pocket to cysteine and examining the effect of covalently linking small molecules to them. We find that ligands bound to the pocket may trigger capsid disassembly in a dose-dependent manner. This result indicates that, at least transiently, the pocket adopts a destabilizing conformation. We speculate that this pocket also plays a role in virus disassembly and genome release by binding ligands that are incompatible with virus stability, "unwanted guests." Investigating protein-protein interactions, especially large protein polymers, offers new and unique challenges. By using an engineered addressable thiol, we provide a means to examine the effects of modifying an interface without requiring drug-like properties for the ligand.

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.

Simian virus 40 capsid protein (VP1) is a unique system for studying substrate-dependent assembly of a nanoparticle. Here, we investigate a simplest case of this system where 12 VP1 pentamers and a single polyanion, e.g., RNA, form a T = 1 particle. To test the roles of polyanion substrate length and structure during assembly, we characterized the assembly products with size exclusion chromatography, transmission electron microscopy, and single-particle resistive-pulse sensing. We found that 500 and 600 nt RNAs had the optimal length and structure for assembly of uniform T = 1 particles. Longer 800 nt RNA, shorter 300 nt RNA, and a linear 600 unit poly(styrene sulfonate) (PSS) polyelectrolyte produced heterogeneous populations of products. This result was surprising as the 600mer PSS and 500-600 nt RNA have similar mass and charge. Like ssRNA, PSS also has a short 4 nm persistence length, but unlike RNA, PSS lacks a compact tertiary structure. These data indicate that even for flexible substrates, shape as well as size affect assembly and are consistent with the hypothesis that work, derived from protein-protein and protein-substrate interactions, is used to compact the substrate.

To improve the precision of resistive-pulse measurements, we have used a focused ion beam instrument to mill nanofluidic devices with 2, 4, and 8 pores in series and compared their performance. The in-plane design facilitates the fabrication of multiple pores in series which, in turn, permits averaging of the series of pulses generated from each translocation event. The standard deviations (σ) of the pulse amplitude distributions decrease by 2.7-fold when the average amplitudes of eight pulses are compared to the amplitudes of single pulses. Similarly, standard deviations of the pore-to-pore time distributions decrease by 3.2-fold when the averages of the seven measurements from 8-pore devices are contrasted to single measurements from 2-pore devices. With signal averaging, the inherent uncertainty in the measurements decreases; consequently, the resolution (mean/σ) improves by a factor equal to the square root of the number of measurements. We took advantage of the improved size resolution of the 8-pore devices to analyze in real time the assembly of Hepatitis B Virus (HBV) capsids below the pseudocritical concentration. We observe that abundances of assembly intermediates change over time. During the first hour of the reaction, the abundance of smaller intermediates decreased, whereas the abundance of larger intermediates with sizes closer to a T = 4 capsid remained constant.

Virus assembly is a coordinated process in which typically hundreds of subunits react to form complex, symmetric particles. We use resistive-pulse sensing to characterize the assembly of hepatitis B virus core protein dimers into T = 3 and T = 4 icosahedral capsids. This technique counts and sizes intermediates and capsids in real time, with single-particle sensitivity, and at biologically relevant concentrations. Other methods are not able to produce comparable real-time, single-particle observations of assembly reactions below, near, and above the pseudocritical dimer concentration, at which the dimer and capsid concentrations are approximately equal. Assembly reactions across a range of dimer concentrations reveal three distinct patterns. At dimer concentrations as low as 50 nM, well below the pseudocritical dimer concentration of 0.5 μM, we observe a switch in the ratio of T = 3 to T = 4 capsids, which increases with decreasing dimer concentration. Far above the pseudocritical dimer concentration, kinetically trapped, incomplete T = 4 particles assemble rapidly, then slowly anneal into T = 4 capsids. At all dimer concentrations tested, T = 3 capsids form more rapidly than T = 4 capsids, suggesting distinct pathways for the two forms.

Weak association energy can lead to uniform nanostructures: defects can anneal due to subunit lability. What happens when strong association energy leads to particles where defects are trapped? Alphaviruses are enveloped viruses whose icosahedral nucleocapsid core can assemble independently. We used a simplest case system to study Ross River virus (RRV) core-like particle (CLP) self-assembly using purified capsid protein and a short DNA oligomer. We find that capsid protein binds the oligomer with high affinity to form an assembly competent unit (U). Subsequently, U assembles with concentration dependence into CLPs. We determined that U-U pairwise interactions are very strong (ca. -6 kcal/mol) compared to other virus assembly systems. Assembled RRV CLPs appeared morphologically uniform and cryo-EM image reconstruction with imposed icosahedral symmetry yielded a T = 4 structure. However, 2D class averages of the CLPs show that virtually every class had disordered regions. These results suggested that irregular cores may be present in RRV virions. To test this hypothesis, we determined 2D class averages of RRV virions using authentic virions or only the core from intact virions isolated by computational masking. Virion-based class averages were symmetrical, geometric, and corresponded well to projections of image reconstructions. In core-based class averages, cores and envelope proteins in many classes were disordered. These results suggest that partly disordered components are common even in ostensibly well-ordered viruses, a biological realization of a patchy particle. Biological advantages of partly disordered complexes may arise from their ease of dissociation and asymmetry.

Controlling the geometry of self-assembly will enable a greater diversity of nanoparticles than now available. Viral capsid proteins, one starting point for investigating self-assembly, have evolved to form regular particles. The polyomavirus SV40 assembles from pentameric subunits and can encapsidate anionic cargos. On short ssRNA (≤814 nt), SV40 pentamers form 22 nm diameter capsids. On RNA too long to fit a T = 1 particle, pentamers forms strings of 22 nm particles and heterogeneous particles of 29-40 nm diameter. However, on dsDNA SV40 forms 50 nm particles composed of 72 pentamers. A 7.2-Å resolution cryo-EM image reconstruction of 22 nm particles shows that they are built of 12 pentamers arranged with T = 1 icosahedral symmetry. At 3-fold vertices, pentamers each contribute to a three-helix triangle. This geometry of interaction is not seen in crystal structures of T = 7 viruses and provides a structural basis for the smaller capsids. We propose that the heterogeneous particles are actually mosaics formed by combining different geometries of interaction from T = 1 capsids and virions. Assembly can be trapped in novel conformations because SV40 interpentamer contacts are relatively strong. The implication is that by virtue of their large catalog of interactions, SV40 pentamers have the ability to self-assemble on and conform to a broad range of shapes.

Protein cages providing a controlled environment to encapsulated cargo are a ubiquitous presence in any biological system. Well-known examples are capsids, the regular protein shells of viruses, which protect and deliver the viral genome. Since some virus capsids can be loaded with nongenomic cargoes, they are interesting for a variety of applications ranging from biomedical delivery to energy harvesting. A question of vital importance for such applications is how does capsid stability depend on the size of the cargo? A nanoparticle-templated assembly approach was employed here to determine how different polymorphs of the Hepatitis B virus icosahedral capsid respond to a gradual change in the encapsulated cargo size. It was found that assembly into complete virus-like particles occurs cooperatively around a variety of core diameters, albeit the degree of cooperativity varies. Among these virus-like particles, it was found that those of an outer diameter corresponding to an icosahedral array of 240 proteins (T = 4) are able to accommodate the widest range of cargo sizes.

We report characterization of hepatitis B virus (HBV) capsids by resistive-pulse sensing through single track-etched conical nanopores formed in poly(ethylene terephthalate) membranes. The pores were ∼40 nm in diameter at the tip, and the pore surface was covalently modified with triethylene glycol to reduce surface charge density, minimize adsorption of the virus capsids, and suppress electroosmotic flow in the pore. The HBV capsids were assembled in vitro from Cp149, the assembly domain of HBV capsid protein. Assembled T = 3 (90 Cp149 dimer) and T = 4 (120 dimer) capsids are 31 and 36 nm in diameter, respectively, and were easily discriminated by monitoring the change in current as capsids passed through an electrically biased pore. The ratio of the number of T = 3 to T = 4 capsids transiting a pore did not reflect actual concentrations, but favored transport of smaller T = 3 capsids. These results combined with longer transit times for the T = 4 capsids indicated that the capsids must overcome an entropic barrier to enter a pore.

Understanding the biological self-assembly process of virus capsids is key to understanding the viral life cycle, as well as serving as a platform for the design of assembly-based antiviral drugs. Here we identify and characterize the phenylpropenamide family of small molecules, known to have antiviral activity in vivo, as assembly effectors of the hepatitis B virus (HBV) capsid. We have found two representative phenylpropenamides to be assembly accelerators, increasing the rate of assembly with only modest increases in the stability of the HBV capsids; these data provide a physical-chemical basis for their antiviral activity. Unlike previously described HBV assembly effectors, the phenylpropenamides do not misdirect assembly; rather, the accelerated reactions proceed on-path to produce morphologically normal capsids. However, capsid assembly in the presence of phenylpropenamides is characterized by kinetic trapping of assembly intermediates. These traps resolve under conditions close to physiological, but we found that trapped intermediates persist under conditions that favor phenylpropenamide binding and strong core protein-protein interactions. The phenylpropenamides serve as chemical probes of the HBV capsid assembly pathway by trapping on-path assembly intermediates, illustrating the governing influence of reaction kinetics on capsid assembly.