Production and applications of engineered viral capsids

As biological agents, viruses come in an astounding range of sizes, with varied shapes and surface morphologies. The structures of viral capsids are generally assemblies of hundreds of copies of one or a few proteins which can be harnessed for use in a wide variety of applications in biotechnology, nanotechnology, and medicine. Despite their complexity, many capsid types form as homogenous populations of precise geometrical assemblies. This is important in both medicine, where well-defined therapeutics are critical for drug performance and federal approval, and nanotechnology, where precise placement affects the properties of the desired material. Here we review the production of viruses and virus-like particles with methods for selecting and manipulating the size, surface chemistry, assembly state, and interior cargo of capsid. We then discuss many of the applications used in research today and the potential commercial and therapeutic products from engineered viral capsids.     

Blueprints for viral capsids in the family of polyomaviridae

In a seminal paper, Caspar and Klug [1962. Physical principles in the construction of regular viruses. Cold Spring Harbor Symp. Quant. Biol. 27, 1-24] derived a family of surface lattices as blueprints for the structural organisation of the protein shells, called viral capsids, which encapsulate and hence protect the viral genome. These lattices schematically encode, and hence predict, the locations of the proteins in the viral capsids. Despite the huge success and numerous applications of this theory in virology, experimental results have provided evidence for the fact that it is too restrictive to describe all known viruses [Casjens, S., 1985. Virus Structure and Assembly. Jones and Bartlett, Boston, MA]. Especially, the family of Polyomaviridae, which contains cancer-causing viruses, falls out of the scope of this theory.In [Twarock, R., 2004. A tiling approach to virus capsid assembly explaining a structural puzzle in virology. J. Theor. Biol. 226, 477], we have shown that a member of the family of Polyomaviridae can be described via an icosahedrally symmetric tiling. We show here that all viruses in this family can be described by tilings with vertices corresponding to subsets of a quasi-lattice that is constructed based on an affine extended Coxeter group, and we use this methodology to derive their coordinates explicitly. Since the particles appear as different subsets of the same quasi-lattice, their relative sizes are predicted by this approach, and there hence exists only one scaling factor that relates the sizes of all particles collectively to their biological counterparts. It is the first mathematical result that provides a common organisational principle for different types of viral particles in the family of Polyomaviridae, and paves the way for modelling Polyomaviridae polymorphism.     

Crosslinking in viral capsids via tiling theory

A vital part of a virus is its protein shell, called the viral capsid, that encapsulates and hence protects the viral genome. It has been shown in Twarock [2004. A tiling approach to vius capsids assembly explaining a structural puzzle in virology. J. Theor. Biol. 226, 477-482] that the surface structures of viruses with icosahedrally symmetric capsids can be modelled in terms of tilings that encode the locations of the protein subunits. This theory is extended here to multi-level tilings in order to model crosslinking structures. The new framework is demonstrated for the case of bacteriophage HK97, and it is shown, how the theory can be used in general to decide if crosslinking, and what type of crosslinking, is compatible from a mathematical point of view with the geometrical surface structure of a virus.     

Master equation approach to the assembly of viral capsids

The distribution of inequivalent geometries occurring during self-assembly of the major capsid protein in thermodynamic equilibrium is determined based on a master equation approach. These results are implemented to characterize the assembly of SV40 virus and to obtain information on the putative pathways controlling the progressive build-up of the SV40 capsid. The experimental testability of the predictions is assessed and an analysis of the geometries of the assembly intermediates on the dominant pathways is used to identify targets for anti-viral drug design.     

Influence of nonuniform geometry on nanoindentation of viral capsids

A series of recent nanoindentation experiments on the protein shells (capsids) of viruses has established atomic force microscopy (AFM) as a useful framework for probing the mechanics of large protein assemblies. Specifically these experiments provide an opportunity to study the coupling of the global assembly response to local conformational changes. AFM experiments on cowpea chlorotic mottle virus, known to undergo a pH-controlled swelling conformational change, have revealed a pH-dependent mechanical response. Previous theoretical studies have shown that homogeneous changes in shell geometry can play a significant role in the mechanical response. This article develops a method for accurately capturing the heterogeneous geometry of a viral capsid and explores its effect on mechanical response with a nonlinear continuum elasticity model. Models of both native and swollen cowpea chlorotic mottle virus capsids are generated from x-ray crystal structures, and are used in finite element simulations of AFM indentation along two-, three-, and fivefold icosahedral symmetry orientations. The force response of the swollen capsid model is observed to be softer by roughly a factor of two, significantly more nonlinear, and more orientation-dependent than that of a native capsid with equivalent elastic moduli, demonstrating that capsid geometric heterogeneity can have significant effects on the global structural response.     

Pseudorabies virus Us9 directs axonal sorting of viral capsids

Pseudorabies virus (PRV) mutants lacking the Us9 gene cannot spread from presynaptic to postsynaptic neurons in the rat visual system, although retrograde spread remains unaffected. We sought to recapitulate these findings in vitro using the isolator chamber system developed in our lab for analysis of the transneuronal spread of infection. The wild-type PRV Becker strain spreads efficiently to postsynaptic neurons in vitro, whereas the Us9-null strain does not. As determined by indirect immunofluorescence, the axons of Us9-null infected neurons do not contain the glycoproteins gB and gE, suggesting that their axonal sorting is dependent on Us9. Importantly, we failed to detect viral capsids in the axons of Us9-null infected neurons. We confirmed this observation by using three different techniques: by direct fluorescence of green fluorescent protein-tagged capsids; by transmission electron microscopy; and by live-cell imaging in cultured, sympathetic neurons. This finding has broad impact on two competing models for how virus particles are trafficked inside axons during anterograde transport and redefines a role for Us9 in viral sorting and transport.     

Forces and pressures in DNA packaging and release from viral capsids

In a previous communication (Kindt et al., 2001) we reported preliminary results of Brownian dynamics simulation and analytical theory which address the packaging and ejection forces involving DNA in bacteriophage capsids. In the present work we provide a systematic formulation of the underlying theory, featuring the energetic and structural aspects of the strongly confined DNA. The free energy of the DNA chain is expressed as a sum of contributions from its encapsidated and released portions, each expressed as a sum of bending and interstrand energies but subjected to different boundary conditions. The equilibrium structure and energy of the capsid-confined and free chain portions are determined, for each ejected length, by variational minimization of the free energy with respect to their shape profiles and interaxial spacings. Numerical results are derived for a model system mimicking the lambda-phage. We find that the fully encapsidated genome is highly compressed and strongly bent, forming a spool-like condensate, storing enormous elastic energy. The elastic stress is rapidly released during the first stage of DNA injection, indicating the large force (tens of pico Newtons) needed to complete the (inverse) loading process. The second injection stage sets in when approximately 1/3 of the genome has been released, and the interaxial distance has nearly reached its equilibrium value (corresponding to that of a relaxed torus in solution); concomitantly the encapsidated genome begins a gradual morphological transformation from a spool to a torus. We also calculate the loading force, the average pressure on the capsid's walls, and the anisotropic pressure profile within the capsid. The results are interpreted in terms of the (competing) bending and interaction components of the packing energy, and are shown to be in good agreement with available experimental data.     

Hydrogen-deuterium exchange as a probe of folding and assembly in viral capsids

The dynamics of proteins within large cellular assemblies are important in the molecular transformations that are required for macromolecular synthesis, transport, and metabolism. The capsid expansion (maturation) accompanying DNA packaging in the dsDNA bacteriophage P22 represents an experimentally accessible case of such a transformation. A novel method, based on hydrogen-deuterium exchange was devised to investigate the dynamics of capsid expansion. Mass spectrometric detection of deuterium incorporation allows for a sensitive and quantitative determination of hydrogen-deuterium exchange dynamics irrespective of the size of the assembly. Partial digestion of the exchanged protein with pepsin allows for region-specific assignment of the exchange. Procapsids and mature capsids were probed under native and slightly denaturing conditions. These experiments revealed regions that exhibit different degrees of flexibility in the procapsid and in the mature capsid. In addition, exchange and deuterium trapping during the process of expansion itself was observed and allowed for the identification of segments of the protein subunit that become buried or stabilized as a result of expansion. This approach may help to identify residues participating in macromolecular transformations and uncover novel patterns and hierarchies of interactions that determine functional movements within molecular machines.     

Osmotic shock of fertilized mouse ova.

The effect of osmotic changes on fertilized mouse ova was studied by measuring their survival, defined as development into hatching blastocysts, after exposure to various concentrations of ethanediol (ethylene glycol). In addition, a Boyle-van't Hoff plot was derived from exposing ova to hypotonic and hypertonic solutions ranging from 0.1 to 2.8 osmol. Volume of ova was inversely proportional to osmolality over this range. Extrapolation of this relationship yielded a nonosmotic volume of the ova of 22.5%. Eighty-five per cent or more of the ova survived exposure to this wide range of concentrations and developed into blastocysts. The rate of development of ova exposed to anisotonic solutions was the same as that of controls. Ova underwent osmotic shock when abruptly diluted out of concentrated solutions of ethanediol with an isotonic solution. Their survival was highly dependent on the ethanediol concentration with which they had equilibrated before dilution, and the manner, rate and temperature of dilution. The longer the exposure to ethanediol the greater was the sensitivity of the ova to osmotic shock, reflecting permeation of ethanediol into the ova. Osmotic shock could be alleviated by dilution at a high temperature, and prevented by the use of sucrose as an osmotic buffer at 37 degrees C. Identification of the variables that influence osmotic shock of ova will be helpful in the systematic study of their cryopreservation.     

Modeling and simulation of the mechanical response from nanoindentation test of DNA-filled viral capsids

Viruses can be described as biological objects composed mainly of two parts: a stiff protein shell called a capsid, and a core inside the capsid containing the nucleic acid and liquid. In many double-stranded DNA bacterial viruses (aka phage), the volume ratio between the liquid and the encapsidated DNA is approximately 1:1. Due to the dominant DNA hydration force, water strongly mediates the interaction between the packaged DNA strands. Therefore, water that hydrates the DNA plays an important role in nanoindentation experiments of DNA-filled viral capsids. Nanoindentation measurements allow us to gain further insight into the nature of the hydration and electrostatic interactions between the DNA strands. With this motivation, a continuum-based numerical model for simulating the nanoindentation response of DNA-filled viral capsids is proposed here. The viral capsid is modeled as large- strain isotropic hyper-elastic material, whereas porous elasticity is adopted to capture the mechanical response of the filled viral capsid. The voids inside the viral capsid are assumed to be filled with liquid, which is modeled as a homogenous incompressible fluid. The motion of a fluid flowing through the porous medium upon capsid indentation is modeled using Darcy’s law, describing the flow of fluid through a porous medium. The nanoindentation response is simulated using three-dimensional finite element analysis and the simulations are performed using the finite element code Abaqus. Force-indentation curves for empty, partially and completely DNA-filled capsids are directly compared to the experimental data for bacteriophage λ. Material parameters such as Young’s modulus, shear modulus, and bulk modulus are determined by comparing computed force-indentation curves to the data from the atomic force microscopy (AFM) experiments. Predictions are made for pressure distribution inside the capsid, as well as the fluid volume ratio variation during the indentation test.     

Use of liposomes, viral capsids, and nanoparticles as DNA carriers

We tested a variety of liposomes for parameters such as DNA binding capacity and DNase I protection of incorporated and attached DNA to elucidate their use as vehicles for DNA transfer into cells and animals. The results were compared to other potential DNA vehicles, empty viral capsids, and nanoparticles. Maximal binding capacity was achieved for positively charged nanoparticles, DNase I protection was observed for most preparations with neosome preparations being least efficient. The uptake of radiolabeled DNA by cells in culture was determined for cationic and nonionic surfactant vesicles, viral capsids, and nanoparticles. Cellular DNA uptake was best for dioleoyl-derived positively charged liposomes (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride; DOTMA) and the DNA could be shown to be physiologically active. The recombination rate for DNA fragments transfected in polyoma capsids in live mice was higher than for liposome mediated transfection. Homologous recombination could be observed for both DOTMA and polyoma-mediated DNA transfer.     

Intra- and inter-subunit disulfide bond formation is nonessential in adeno-associated viral capsids

The capsid proteins of adeno-associated viruses (AAV) have five conserved cysteine residues. Structural analysis of AAV serotype 2 reveals that Cys289 and Cys361 are located adjacent to each other within each monomer, while Cys230 and Cys394 are located on opposite edges of each subunit and juxtaposed at the pentamer interface. The Cys482 residue is located at the base of a surface loop within the trimer region. Although plausible based on molecular dynamics simulations, intra- or inter-subunit disulfides have not been observed in structural studies. In the current study, we generated a panel of Cys-to-Ser mutants to interrogate the potential for disulfide bond formation in AAV capsids. The C289S, C361S and C482S mutants were similar to wild type AAV with regard to titer and transduction efficiency. However, AAV capsid protein subunits with C230S or C394S mutations were prone to proteasomal degradation within the host cells. Proteasomal inhibition partially blocked degradation of mutant capsid proteins, but failed to rescue infectious virions. While these results suggest that the Cys230/394 pair is critical, a C394V mutant was found viable, but not the corresponding C230V mutant. Although the exact nature of the structural contribution(s) of Cys230 and Cys394 residues to AAV capsid formation remains to be determined, these results support the notion that disulfide bond formation within the Cys289/361 or Cys230/394 pair appears to be nonessential. These studies represent an important step towards understanding the role of inter-subunit interactions that drive AAV capsid assembly.     

Recombinant viral capsids as an efficient vehicle of oligonucleotide delivery into cells

Delivery of oligonucleotides (ON) into cells is a technical challenge. In this study, we utilized the capsid of the hepatitis B virus (HBV) to meet this goal. A single and short open reading frame of the virus programs efficient capsid production in bacteria. We show that these capsids can encapsulate ON in vitro and then mediate their delivery into cells with extreme efficiency. This process is cell type non-specific, rendering the recombinant HBV capsid a potentially valuable vehicle for ON delivery into a wide range of cultured cells.     

Ejection dynamics of polymeric chains from viral capsids: effect of solvent quality

We present simulations investigating the effects of solvent quality on the dynamics of flexible (RNA-like) and semiflexible (DNA-like) polymers ejecting from spherical viral capsids. We find that the mean ejection time increases and the ejection time distributions are broadened as the solvent quality decreases. Our results thus suggest that DNA ejection may be very efficiently controlled by tuning the salt concentration in the environment, in agreement with recent experimental findings. We also observe random pauses in the ejection. These become extremely long for semiflexible polymers at lower solvent quality, and we interpret this as a signature of a low driving force for ejection. We find that, for most polymers, ejection is an all-or-nothing process at the solvent conditions we investigated: polymers normally completely eject once the process is initiated.     

Packaging double-helical DNA into viral capsids: structures, forces, and energetics

Small, icosahedral double-stranded DNA bacteriophage pack their genomes tightly into preformed protein capsids using an ATP-driven motor. Coarse-grain molecular-mechanics models provide a detailed picture of DNA packaging in bacteriophage, revealing how conformation depends on capsid size and shape, and the presence or absence of a protein core. The forces that oppose packaging have large contributions from both electrostatic repulsions and the entropic penalty of confining the DNA into the capsid, whereas elastic deformations make only a modest contribution. The elastic deformation energy is very sensitive to the final conformation, whereas the electrostatic and entropic penalties are not, so the packaged DNA favors conformations that minimize the bending energy.     

渗透压冲击法快速高效提取青霉素酰化酶

青霉素在临床上的大量使用造成了细菌的耐药性增强,青霉素本身又具有不宜口服、过敏性强等缺点,人们正致力于研究有诸多优良特性的半合成青霉素。大肠杆菌青霉素酰化酶用于裂解青霉素生产6-氨基青霉烷酸(即6-ＡＰＡ,半合成青霉素的重要中间体),该酶的提取、纯化和固定化研究在半合成青霉素工业有重要的意义[1]。大肠杆菌青霉素酰化酶属胞内酶,文献报道多采用超声波法提取,该法得到的粗酶液比活低,一般要经过四、五步纯化才能得到较高比活[2,3,4]的酶液。采用渗透压冲击法提取青霉素酰化酶,得到的粗酶液比活高,只需经过硫酸铵沉淀一步纯化就…     

Osmotic shock induces structural damage on equine spermatozoa plasmalemma and mitochondria

The present study aimed to elucidate the effects that osmotic shock exerts on equine spermatozoa. To achieve this goal, a retrospective study of the cellular volume of 40 equine ejaculates subjected to osmolarities ranging from 75 to 900 mOsm in Biggers-Whitten-Whittingham (BWW) media was performed using a Multisizer³ Coulter Counter®. The 300 mOsm BWW solution was used as control. The sperm volume ranged between 37.93 ± 0.6 (mean ± Standard Error of the Mean (SEM)) in 75 mOsm BWW to 21.61 ± 0.27 (mean ± SEM) for 900 mOsm BWW. Thus the spermatozoa behaved as linear osmometers when adjusted to the Boyle Van't Hoff equation (R² = 0.9808). After the different osmotic challenges, spermatozoa were returned to 300 mOsm BWW and the cellular volume was measured again. The results showed that the spermatozoa were able to retrieve the isosmolar volume (20.81 ± 0.34; mean ± SEM). Also, an ultrastructural study of spermatozoa membrane and mitochondria was accomplished using Transmission Electron Microscopy (TEM) after the osmotic challenges in 2 ejaculates. As observed by TEM, sperm plasmalemma swelled and detached from the sperm head in hypotonic conditions (75 mOsm), with blebbing on return to isosmolarity. When subjected to 900 mOsm, the sperm plasmalemma shrank, with disarrangement and blebbing when returned to isosmolarity. Mitochondria were also found to change their volume; the main pathologic change was irreversible vacuolization and changes in their arrangement for all the osmotic challenges tested. The present work leads to a better understanding of how osmotic shock adversely affects equine spermatozoa structure.