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Ultrafiltration using polyethersulfone-membranes was evaluated as an efficient and preferred method for purifying Cowpea Chlorotic Mottle Virus (CCMV). Cesium chloride (CsCl) ultracentrifugation and ultrafiltration protocols are described, and comparative UV-spectroscopic and electron micrograph results are presented. CCMV purified by ultrafiltration are shown to be equivalent to CCMV purified by ultracentrifugation, while reducing purification time by two days and avoiding the need for expensive capital overheads such as ultracentrifuges, rotors and toxic CsCl chemical waste.

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Bacterial viral capsids in aqueous solution can be opened in vitro by addition of their specific receptor proteins, with consequent full ejection of their genomes. We demonstrate that it is possible to control the extent of this ejection by varying the external osmotic pressure. In the particular case of bacteriophage λ, the ejection is 50% inhibited by osmotic pressures (of polyethylene glycol) comparable to those operative in the cytoplasm of host bacteria; it is completely suppressed by a pressure of 20 atmospheres. Furthermore, our experiments monitor directly a dramatic decrease of the stress inside the unopened phage capsid upon addition of polyvalent cations to the host solution, in agreement with many recent theories of DNA interactions.

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We report a combined theoretical and experimental study of the structural failure of viral shells under mechanical stress. We find that discontinuities in the force-indentation curve associated with failure should appear when the so-called Foppl-von Karman (FvK) number exceeds a critical value. A nanoindentation study of a viral shell subject to a soft-mode instability, where the stiffness of the shell decreases with increasing pH, confirms the predicted onset of failure as a function of the FvK number.

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The elastic properties of capsids of the cowpea chlorotic mottle virus have been examined at pH 4.8 by nanoindentation measurements with an atomic force microscope. Studies have been carried out on W7 capsids, both empty and containing the RNA genome, and on full capsids of a salt-stable mutant and empty capsids of the subE mutant. Full capsids resisted indentation more than empty capsids, but all of the capsids were highly elastic. There was an initial reversible linear regime that persisted up to indentations varying between 20% and 30% of the diameter and applied forces of 0.6-1.0 nN; it was followed by a steep drop in force that is associated with irreversible deformation. A single point mutation in the capsid protein increased the capsid stiffness. The experiments are compared with calculations by finite element analysis of the deformation of a homogeneous elastic thick shell. These calculations capture the features of the reversible indentation region and allow Young's moduli and relative strengths to be estimated for the empty capsids.

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Bacterial viral capsids in aqueous solution can be opened in vitro by addition of their specific receptor proteins, with consequent full ejection of their genomes. We demonstrate that it is possible to control the extent of this ejection by varying the external osmotic pressure. In the particular case of bacteriophage lambda, the ejection is 50% inhibited by osmotic pressures (of polyethylene glycol) comparable to those operative in the cytoplasm of host bacteria, it is completely suppressed by a pressure of 20 atmospheres. Furthermore, our experiments monitor directly a dramatic decrease of the stress inside the unopened phage capsid upon addition of polyvalent cations to the host solution, in agreement with many recent theories of DNA interactions.

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The protein shells, or capsids, of nearly all spherelike viruses adopt icosahedral symmetry. In the present Letter, we propose a statistical thermodynamic model for viral self-assembly. We find that icosahedral symmetry is not expected for viral capsids constructed from structurally identical protein subunits and that this symmetry requires (at least) two internal "switching" configurations of the protein. Our results indicate that icosahedral symmetry is not a generic consequence of free energy minimization but requires optimization of internal structural parameters of the capsid proteins.

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Osmotic shock is a familiar means for rupturing viral capsids and exposing their genomes intact. The necessary conditions for providing this shock involve incubation in high-concentration salt solutions, and lower permeability of the capsids to salt ions than to water molecules. We discuss here how values of the capsid strength can be inferred from calculations of the osmotic pressure differences associated with measured values of the critical concentration of incubation solution.

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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 similar to1/3 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.

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Not just the repository of our genetic information, DNA is also a fascinating, shape-shifting molecule whose behavior in solution counters our intuition and challenges our physical understanding.

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We report a strategy for encapsulating and condensing DNA. When T5 phage binds to its membrane protein receptor, FhuA, its double stranded DNA (120,000 bp) is progressively released base pair after base pair in the surrounding medium. Using cryoelectron microscopy, we have visualized the structures formed after T5 phage DNA is released into neutral unilamellar proteoliposomes reconstituted with the receptor FhuA. In the presence of spermine, toroidal condensates of circumferentially wrapped DNA were formed. Most significantly, the sizes of these toroids were shown to vary, from 90 to 200 nm in their outer diameters, depending on the number of DNA stands transferred. We have also analyzed T5 DNA release in bulk solution containing the detergent-solubilized FhuA receptor. After DNA release in a spermine containing solution, huge DNA condensates with a diameter of about 300 nm were formed containing the DNAs from as many as 10-20 capsids. At alkaline pH, the condensates appeared as large hollow cylinders with a diameter of 200 nm and a height of 100-200 nm. Overall, the striking feature of our experiments is that, because of the progressive release of DNA from the phage capsid, the mechanism of toroid formation is fundamentally different from that in the classical studies in which highly dilute, "naked" DNA is condensed by direct addition of polyvalent rations, as a consequence, our method leads to toroids of arbitrary size.

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