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.

The assembly of hundreds of identical proteins into an icosahedral virus capsid is a remarkable feat of molecular engineering. How this occurs is poorly understood. Key intermediates have been anticipated at the end of the assembly reaction, but it has not been possible to detect them. In this work we have used charge detection mass spectrometry to identify trapped intermediates from late in the assembly of the hepatitis B virus T = 4 capsid, a complex of 120 protein dimers. Prominent intermediates are found with 104/105, 110/111, and 117/118 dimers. Cryo-EM observations indicate the intermediates are incomplete capsids and, hence, on the assembly pathway. On the basis of their stability and kinetic accessibility we have proposed plausible structures. The prominent trapped intermediate with 104 dimers is attributed to an icosahedron missing two neighboring facets, the 111-dimer species is assigned to an icosahedron missing a single facet, and the intermediate with 117 dimers is assigned to a capsid missing a ring of three dimers in the center of a facet.

Crystal structure determination has long provided insight into structure and bonding of small molecules. When those same small molecules are designed to come together in multimolecular assemblies, such as in coordination cages, supramolecular architectures and organic-based frameworks, their crystallographic characteristics closely resemble biological macromolecules. This resemblance suggests that biomacromolecular refinement approaches be used for structure determination of abiological molecular complexes that arise in an aggregate state. Following this suggestion we investigated the crystal structure of a pentagonal macrocycle, cyanostar, by means of biological structure analysis methods and compared results to traditional small molecule methods. Cyanostar presents difficulties seen in supramolecular crystallography including whole molecule disorder and highly flexible solvent molecules sitting in macrocyclic and intermolecule void spaces. We used the force-field assisted refinement method, molecular dynamics flexible fitting algorithm for X-ray crystallography (xMDFF), along with tools from the macromolecular structure determination suite PHENIX. We found that a standard implementation of PHENIX, namely one without xMDFF, either fails to produce a solution by molecular replacement alone or produces an inaccurate structure when using generic geometry restraints, even at a very high diffraction data resolution of 0.84 Å. The problems disappear when taking advantage of xMDFF, which applies an optimized force field to realign molecular models during phasing by providing accurate restraints. The structure determination for this model system shows excellent agreement with the small-molecule methods. Therefore, the joint xMDFF-PHENIX refinement protocol provides a new strategy that uses macromolecule methods for structure determination of small molecules and their assemblies.

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.Keywords: capsid; enveloped viruses; Ross River virus; self-assembly;

During the hepatitis B virus (HBV) life cycle, capsid assembly and disassembly must ensure correct packaging and release of the viral genome. Here we show that changes in the dynamics of the core protein play an important role in regulating these processes. The HBV capsid assembles from 120 copies of the core protein homodimer. Each monomer contains a conserved cysteine at position 61 that can form an intradimer disulfide that we use as a marker for dimer conformational states. We show that dimers in the context of capsids form intradimer disulfides relatively rapidly. Surprisingly, compared to reduced dimers, fully oxidized dimers assembled slower and into capsids that were morphologically similar but less stable. We hypothesize that oxidized protein adopts a geometry (or constellation of geometries) that is unfavorable for capsid assembly, resulting in weaker dimer–dimer interactions as well as slower assembly kinetics. Our results suggest that structural flexibility at the core protein intradimer interface is essential for regulating capsid assembly and stability. We further suggest that capsid destabilization by the C61–C61 disulfide has a regulatory function to support capsid disassembly and release of the viral genome.

Assembly of a hepatitis B virus (HBV) virion begins with the formation of an RNA-filled core composed of a symmetrical capsid (built of core protein), viral pregenomic RNA, and viral reverse transcriptase. To generate the circular dsDNA genome of HBV, reverse transcription requires multiple template switches within the confines of the capsid. To date, most anti-HBV therapeutics target this reverse transcription process. The detailed molecular mechanisms of this crucial process are poorly understood because of the lack of structural information. We hypothesized that capsid, RNA, and viral reverse transcriptase would need a precise geometric organization to accomplish reverse transcription. Here we present the asymmetric structure of authentic RNA-filled cores, determined to 14.5-Å resolution from cryo-EM data. Capsid and RNA are concentric. On the interior of the RNA, we see a distinct donut-like density, assigned to viral reverse transcriptase, which pins the viral pregenomic RNA to the capsid inner surface. The observation of a unique ordered structure inside the core suggests that assembly and the first steps of reverse transcription follow a single, determinate pathway and strongly suggests that all subsequent steps in DNA synthesis do as well.

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.

Remarkably, uniform virus-like particles self-assemble in a process that appears to follow a rapid kinetic mechanism. The mechanisms by which spherical viruses assemble from hundreds of capsid proteins around nucleic acid, however, are yet unresolved. Using time-resolved small-angle X-ray scattering (TR-SAXS), we have been able to directly visualize SV40 VP1 pentamers encapsidating short RNA molecules (500mers). This assembly process yields T = 1 icosahedral particles comprised of 12 pentamers and one RNA molecule. The reaction is nearly one-third complete within 35 ms, following a two-state kinetic process with no detectable intermediates. Theoretical analysis of kinetics, using a master equation, shows that the assembly process nucleates at the RNA and continues by a cascade of elongation reactions in which one VP1 pentamer is added at a time, with a rate of approximately 109 M–1 s–1. The reaction is highly robust and faster than the predicted diffusion limit. The emerging molecular mechanism, which appears to be general to viruses that assemble around nucleic acids, implicates long-ranged electrostatic interactions. The model proposes that the growing nucleo-protein complex acts as an electrostatic antenna that attracts other capsid subunits for the encapsidation process.

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.

Controlling self-assembly is critical to the advancement of nanotechnology. A rugged or crenated assembly energy surface can redirect assembly off path. By using a defined starting point and an energy surface made rough by a strong association energy, we can impose entirely new assembly paths and products. Normally, the coat protein (CP) of the Cowpea Chlorotic Mottle Virus (CCMV) assembles into virus-like 28 nm diameter icosahedral particles. Here we have started with the coat protein trapped in a rod-like structure in complex with DNA. When these 17 nm diameter rods are placed under the same condition, low pH, that normally leads to assembly of 28 nm diameter particles, we instead obtain 17 nm capsids. The extrusion of all-pentamer capsids from the hexagonal lattice of the rod demonstrates the importance of the starting state for controlled assembly.

Understanding self-assembly of icosahedral virus capsids is critical to developing assembly directed antiviral approaches and will also contribute to the development of self-assembling nanostructures. One approach to controlling assembly would be through the use of assembly inhibitors. Here we use Cp149, the assembly domain of the hepatitis B virus capsid protein, together with an assembly defective mutant, Cp149-Y132A, to examine the limits of the efficacy of assembly inhibitors. By itself, Cp149-Y132A will not form capsids. However, Cp-Y132A will coassemble with the wild-type protein on the basis of light scattering and size exclusion chromatography. The resulting capsids appear to be indistinguishable from normal capsids. However, coassembled capsids are more fragile, with disassembly observed by chromatography under mildly destabilizing conditions. The relative persistence of capsids assembled under conditions where association energy is weak compared to the fragility of those where association is strong suggests a mechanism of “thermodynamic editing” that allows replacement of defective proteins in a weakly associated complex. There is fine line between weak assembly, where assembly defective protein is edited from a growing capsid, and relatively strong assembly, where assembly defective subunits may dramatically compromise virus stability. Thus, attempts to control virus self-assembly (with small molecules or defective proteins) must take into account the competing process of thermodynamic editing.

Assembly of virus capsids and surface proteins must be regulated to ensure that the resulting complex is an infectious virion. In this review, we examine assembly of virus capsids, focusing on hepatitis B virus and bacteriophage MS2, and formation of glycoproteins in the alphaviruses. These systems are structurally and biochemically well-characterized and are simplest-case paradigms of self-assembly. Published data suggest that capsid and glycoprotein assembly is subject to allosteric regulation, that is regulation at the level of conformational change. The hypothesis that allostery is a common theme in viruses suggests that deregulation of capsid and glycoprotein assembly by small molecule effectors will be an attractive antiviral strategy, as has been demonstrated with hepatitis B virus.

Assembly of viruses that have hundreds of subunits or folding of proteins that have hundreds of amino acids—complex biological reactions—are often spontaneous and rapid. Here, we examine the complete set of intermediates available for the assembly of a hypothetical viruslike particle and the connectivity between these intermediates in a graph-theory-inspired study. Using a build-up procedure, assuming ideal geometry, we enumerated the complete set of 2,423,313 species for formation of an icosahedron from 30 dimeric subunits. Stability of each n-subunit intermediate was defined by the number of contacts between subunits. The probability of forming an intermediate was based on the number of paths to it from its precedecessors. When defining population subsets predicted to have the greatest impact on assembly, both stability- and probability-based criteria select a small group of compact and degenerate species; ergo, only a few hundred intermediates make a measurable contribution to assembly. Though the number of possible intermediates grows combinatorially with the number of subunits in the capsid, the number of intermediates that make a significant contribution to the reaction grows by a much smaller function, a result that may contribute to our understanding of assembly and folding reactions.

Hepatitis B virus core gene products can adopt different conformations to perform their functional roles. In this issue of Structure, DiMattia and colleagues show the crystal structure of immuno-modulating HBeAg and thereby reveal the similarities and differences between it and HBcAg, the variant found in virions.

•Phenylpropenamides induce tertiary and quaternary structural changes in HBV capsids•AT-130 binds to a promiscuous pocket at the dimer-dimer interface•AT-130 favors a unique quasiequivalent binding site in the capsid•Small molecules that do not disrupt capsid structure are still effective antiviralsHepatitis B virus (HBV) is a major cause of liver disease. Assembly of the HBV capsid is a critical step in virus production and an attractive target for new antiviral therapies. We determined the structure of HBV capsid in complex with AT-130, a member of the phenylpropenamide family of assembly effectors. AT-130 causes tertiary and quaternary structural changes but does not disrupt capsid structure. AT-130 binds a hydrophobic pocket that also accommodates the previously characterized heteroaryldihydropyrimidine compounds but favors a unique quasiequivalent location on the capsid surface. Thus, this pocket is a promiscuous drug-binding site and a likely target for different assembly effectors with a broad range of mechanisms of activity. That AT-130 successfully decreases virus production by increasing capsid assembly rate without disrupting capsid structure delineates a paradigm in antiviral design, that disrupting reaction timing is a viable strategy for assembly effectors of HBV and other viruses.Download high-res image (453KB)Download full-size image

The capsid of SV40 virion is comprised of 72 pentamers of the major capsid protein, VP1. We examined the synergism between pentamer–pentamer interaction and pentamer–DNA interaction using a minimal system of purified VP1 and a linear dsDNA 600-mer, comparing electrophoresis with electron microscopy and size exclusion chromatography. At low VP1/DNA ratios, large tubes were observed that apparently did not survive native agarose gel electrophoresis. As the VP1 concentration increased, electrophoretic migration was slower and tubes were replaced by 200 Å diameter particles and excess free pentamer. At high VP1/DNA ratios, a progressively larger fraction of particles was similar to 450 Å diameter virions. VP1 association with DNA is very strong compared to the concentrations in these experiments yet, paradoxically, stable complexes appear only at high ratios of VP1 to DNA. These data suggest a DNA saturation-dependent nucleation event based on non-specific pentamer–DNA interaction that controls assembly and the ultimate capsid geometry.

UV spectra of viruses are complicated by overlapping protein and RNA absorbance and light scattering. We describe and validate methodology for estimating RNA and protein concentration from such spectra. Importantly, we found that encapsidation did not substantially affect RNA absorbance. Combining absorbance data with a known T number, we confirmed that brome mosaic virus packages about 3100 nucleotides/capsid, consistent with its genome. E. coli-expressed hepatitis B virus (HBV) packages host RNA based on capsid charge and volume. We examined HBV capsid protein (Cp183, + 15 charge) and a less basic mutant (Cp183-EEE, + 12 charge) that mimics a phosphorylated state. Cp183-EEE packaged ~ 3450 nucleotides per T = 4 capsid and Cp183 packaged ~ 4800 nucleotides, correlating to the size of HBV's RNA pre-genome and mature DNA genome, respectively. The RNA:protein charge ratio (about 1.4 phosphates per positive charge) was consistent with that of other ssRNA viruses. This spectroscopic method is generalizable to any virus-like particle.

In vitro assembly of bacteriophage P22 procapsids requires coat protein and sub-stoichiometric concentrations of the internal scaffolding protein. If there is no scaffolding protein, coat protein assembles aberrantly, but only at higher concentrations. Too much scaffolding protein results in partial procapsids. By treating the procapsid as a lattice that can bind and be stabilized by scaffolding protein we dissect procapsid assembly as a function of protein concentration and scaffolding/coat protein ratio. We observe that (i) the coat–coat association is weaker for procapsids than for aberrant polymer formation, (ii) scaffolding protein makes a small but sufficient contribution to stability to favor the procapsid form, and (iii) there are multiple classes of scaffolding protein binding sites. This approach should be applicable to other heterogeneous virus assembly reactions and will facilitate our ability to manipulate such in vitro reactions to probe assembly, and for development of nanoparticles.

Many viruses package their genomes concomitant with assembly. Here, we show that this reaction can be described by three coefficients: association of capsid protein (CP) to nucleic acid (NA), KNA; CP-CP interaction, ω; and α, proportional to the work required to package NA. The value of α can vary as NA is packaged. A phase diagram of average lnα versus lnω identifies conditions where assembly is likely to fail or succeed. NA morphology can favor (lnα > 0) or impede (lnα < 0) assembly. As lnω becomes larger, capsids become more stable and assembly becomes more cooperative. Where (lnα + lnω) < 0, the CP is unable to contain the NA, so that assembly results in aberrant particles. This phase diagram is consistent with quantitative studies of cowpea chlorotic mottle virus, hepatitis B virus, and simian virus 40 assembling on ssRNA and dsDNA substrates. Thus, the formalism we develop is suitable for describing and predicting behavior of experimental studies of CP assembly on NA.

Hepatitis B Virus (HBV) cores assemble on viral RNA, which is reverse transcribed within the core to the partially dsDNA genome of mature HBV. However, constraining dsDNA, a stiff polymer, within a core necessarily requires far greater capsid stability than constraining ssRNA. We hypothesized that, unlike ssRNA, dsDNA would be a poor substrate for assembly. We examined titrations of ssDNA and dsDNA with purified HBV core protein, Cp183, by EMSA, EM, DLS, and etheno-DNA fluorescence. Cp183 bound ssDNA with high affinity to form virus-like capsids. However, Cp183 bound dsDNA poorly, forming a mixture of irregular complexes. Nonetheless, we observed some normal cores in dsDNA assembly reactions, indicating that the energy required to bend DNA could be similar to the protein–protein association energy. This similarity of energies suggests that dsDNA stresses mature HBV cores, in agreement with calculation, which may be the basis for the virus maturation signal and DNA release.

The assembly of hundreds of identical proteins into an icosahedral virus capsid is a remarkable feat of molecular engineering. How this occurs is poorly understood. Key intermediates have been anticipated at the end of the assembly reaction, but it has not been possible to detect them. In this work we have used charge detection mass spectrometry to identify trapped intermediates from late in the assembly of the hepatitis B virus T = 4 capsid, a complex of 120 protein dimers. Prominent intermediates are found with 104/105, 110/111, and 117/118 dimers. Cryo-EM observations indicate the intermediates are incomplete capsids and, hence, on the assembly pathway. On the basis of their stability and kinetic accessibility we have proposed plausible structures. The prominent trapped intermediate with 104 dimers is attributed to an icosahedron missing two neighboring facets, the 111-dimer species is assigned to an icosahedron missing a single facet, and the intermediate with 117 dimers is assigned to a capsid missing a ring of three dimers in the center of a facet.