Cowpea Chlorotic Mottle Virus Particles Research Articles

Self-Assembly and Stabilization of Hybrid Cowpea Chlorotic Mottle Virus Particles under Nearly Physiological Conditions.

Capsids of the cowpea chlorotic mottle virus (CCMV) hold great promise for use as nanocarriers in vivo. A major drawback, however, is the lack of stability of the empty wild-type virus particles under physiological conditions. Herein, the assembly behavior and stability under nearly physiological conditions of protein-based block copolymers composed of the CCMV capsid protein and two hydrophobic elastin-like polypeptides are reported. UV/Vis spectroscopy studies, dynamic light-scattering analysis, and TEM measurements demonstrate that both hybrid variants form stable capsids at pH 7.5, physiological NaCl concentration, and 37 °C. The more hydrophobic variant also remains stable in a cell culture medium. These engineered, hybrid CCMV capsid particles can therefore be regarded as suitable candidates for in vivo applications.

Packaging DNA Origami into Viral Protein Cages.

The DNA origami technique is a widely used method to create customized, complex, spatially well-defined two-dimensional (2D) and three-dimensional (3D) DNA nanostructures. These structures have huge potential to serve as smart drug-delivery vehicles and molecular devices in various nanomedical and biotechnological applications. However, so far only little is known about the behavior of these novel structures in living organisms or in cell culture/tissue models. Moreover, enhancing pharmacokinetic bioavailability and transfection properties of such structures still remains a challenge. One intriguing approach to overcome these issues is to coat DNA origami nanostructures with proteins or lipid membranes. Here, we show how cowpea chlorotic mottle virus (CCMV) capsid proteins (CPs) can be used for coating DNA origami nanostructures. We present a method for disassembling native CCMV particles and isolating the pure CP dimers, which can further bind and encapsulate a rectangular DNA origami shape. Owing to the highly programmable nature of DNA origami, packaging of DNA nanostructures into viral protein cages could find imminent uses in enhanced targeting and cellular delivery of various active nano-objects, such as enzymes and drug molecules.

Self-assembly and modular functionalization of three-dimensional crystals from oppositely charged proteins.

Multicomponent crystals and nanoparticle superlattices are a powerful approach to integrate different materials into ordered nanostructures. Well-developed, especially DNA-based, methods for their preparation exist, yet most techniques concentrate on molecular and synthetic nanoparticle systems in non-biocompatible environment. Here we describe the self-assembly and characterization of binary solids that consist of crystalline arrays of native biomacromolecules. We electrostatically assembled cowpea chlorotic mottle virus particles and avidin proteins into heterogeneous crystals, where the virus particles adopt a non-close-packed body-centred cubic arrangement held together by avidin. Importantly, the whole preparation process takes place at room temperature in a mild aqueous medium allowing the processing of delicate biological building blocks into ordered structures with lattice constants in the nanometre range. Furthermore, the use of avidin–biotin interaction allows highly selective pre- or post-functionalization of the protein crystals in a modular way with different types of functional units, such as fluorescent dyes, enzymes and plasmonic nanoparticles.

Self-assembly and optically triggered disassembly of hierarchical dendron–virus complexes

Nature offers a vast array of biological building blocks that can be combined with synthetic materials to generate a variety of hierarchical architectures. Viruses are particularly interesting in this respect because of their structure and the possibility of them functioning as scaffolds for the preparation of new biohybrid materials. We report here that cowpea chlorotic mottle virus particles can be assembled into well-defined micrometre-sized objects and then reconverted into individual viruses by application of a short optical stimulus. Assembly is achieved using photosensitive dendrons that bind on the virus surface through multivalent interactions and then act as a molecular glue between the virus particles. Optical triggering induces the controlled decomposition and charge switching of dendrons, which results in the loss of multivalent interactions and the release of virus particles. We demonstrate that the method is not limited to the virus particles alone, but can also be applied to other functional protein cages such as magnetoferritin.

Rapid and efficient purification of Cowpea chlorotic mottle virus by sucrose cushion ultracentrifugation

A simple, economical and efficient method for isolation of Cowpea chlorotic mottle virus (CCMV) particles was developed. Abundant viral particles could be obtained with three steps of 10 min duration each of conventional differential centrifugations and one sucrose cushion ultracentrifugation step of 2 h. Characterization of purified virus assessed by transmission electron microscopy, polyacrylamide gel electrophoresis (PAGE) and Western blot revealed the typical isometric particles of CCMV without any visible contamination or degradation. RNA extracted from purified CCMV particles showed all four RNA components by agarose gel electrophoresis. Both purified virus particles and RNA were biologically active and initiated successful infection upon inoculation to cowpea.

Expression and self-assembly of cowpea chlorotic mottle virus-like particles in Pseudomonas fluorescens

Coat protein of the cowpea chlorotic mottle virus (CCMV), a plant bromovirus, has been expressed in a soluble form in a prokaryote, Pseudomonas fluorescens, and assembled into virus-like particles (VLPs) in vivo that were structurally similar to the native CCMV particles derived from plants. The CCMV VLPs were purified by PEG precipitation followed by separation on a sucrose density gradient and analyzed by size exclusion chromatography, UV spectrometry, and transmission electron microscopy. DNA microarray experiments revealed that the VLPs encapsulated very large numbers of different host RNAs in a non-specific manner. The development of a P. fluorescens expression system now enables production of CCMV VLPs by bacterial fermentation for use in pharmaceutical or nanotechnology applications.

Electrostatic properties of cowpea chlorotic mottle virus and cucumber mosaic virus capsids

Electrostatic properties of cowpea chlorotic mottle virus (CCMV) and cucumber mosaic virus (CMV) were investigated using numerical solutions to the Poisson-Boltzmann equation. Experimentally, it has been shown that CCMV particles swell in the absence of divalent cations when the pH is raised from 5 to 7. CMV, although structurally homologous, does not undergo this transition. An analysis of the calculated electrostatic potential confirms that a strong electrostatic repulsion at the calcium-binding sites in the CCMV capsid is most likely the driving force for the capsid swelling process during the release of calcium. The binding interaction between the encapsulated genome material (RNA) inside of the capsid and the inner capsid shell is weakened during the swelling transition. This probably aids in the RNA release process, but it is unlikely that the RNA is released through capsid openings due to unfavorable electrostatic interaction between the RNA and capsid inner shell residues at these openings. Calculations of the calcium binding energies show that Ca(2+) can bind both to the native and swollen forms of the CCMV virion. Favorable binding to the swollen form suggests that Ca(2+) ions can induce the capsid contraction and stabilize the native form.

Virus-ribosome complexes from cell-free translation systems supplemented with cowpea chlorotic mottle virus particles

When particles of cowpea chlorotic mottle virus (CCMV) were added to cell-free extracts from wheat germ, the encapsidated viral genome was translated into polypeptides similar to the translation products specified by unencapsidated viral RNA (as shown before by M. J. Brisco, R. Hull, and T. M. A. Wilson, 1986, Virology 148, 210–217). The rate of protein synthesis observed upon addition of virus particles was much slower than that of extracted RNA and the quantity of protein formed was only 10% of that of extracted RNA. Using sucrose and cesium-chloride gradient analysis, virus-ribosome complexes, containing up to four ribosomes per virus particle, were isolated from translation mixtures supplemented with CCMV particles. These complexes, with densities intermediate of those of virus (1.36 g cm −3) and ribosomes (1.58 g cm −3), were analyzed and quantified in the electron microscope. Less than 5% of the particles was found in association with ribosomes. To verify whether these complexes were involved in the process of cotranslational disassembly, tobacco mosaic virus was analyzed with the same techniques and methods. The results found for TMV were similar to those found for CCMV except that virus-ribosome complexes with up to 20 ribosomes per virus particle were observed. The implications of the process of virion-directed translation for the structure of the particle as well as the role of this process in vivo are discussed.

Interference in Infections of Tobacco Protoplasts with Two Bromoviruses

SUMMARY Freshly prepared tobacco mesophyll protoplasts behave as if they contain two classes, one of which is resistant to infection but the other is susceptible and can readily be doubly infected with brome mosaic virus (BMV) and cowpea chlorotic mottle virus (CCMV). BMV dominates in all double infections and although both types of virus particles are produced, only those of BMV are infectious. The defective CCMV particles contain normal amounts of RNA but only RNA 3 could be detected in them. No CCMV RNA 2 could be detected in doubly infected protoplasts, showing that there was only partial replication of the genome of CCMV. The proteins encoded by CCMV RNA 3 were produced in mixed infections. It is possible that BMV may exploit the gene products of CCMV while suppressing synthesis of infectious CCMV particles. There was no evidence that BMV RNA was encapsidated in CCMV coat protein.

A negative staining-carbon film technique for studying viruses in the electron microscope: III. The formation of two-dimensional and three-dimensional crystalline arrays of cowpea chlorotic mottle virus

The negative staining-carbon film technique was applied to purified and highly concentrated suspensions of cowpea chlorotic mottle virus (CCMV) wild type and a salt-stable mutant. Both two-dimensional and three-dimensional crystals were rapidly formed at the surface of mica using CCMV suspensions containing about 2.0 to 14.0 mg/ml of virus. When mixed with 3% ammonium molybdate at pH 5.2 the crystals contained CCMV particles predominantly packed in a hexagonal array at the mica surface. At pH 7.0, the crystals were observed to be predominantly in square arrays. Under the preparative conditions used it was possible to form crystalline sheets with sides of several micrometers and in some instances covering entire grid squares. The crystals were found to be ideal for producing low-angle electron diffraction patterns and optical diffraction patterns from the micrographs with spectra extending out to about 2.0 nm. Experiments carried out on CCMV wild type and the salt stable mutants also showed that rapid morphological degradation occurs following storage at 4°C. The viral components from the degraded virus were observed to influence both the formation of the crystals and resolution obtainable from the specimens.

A Direct Estimate of the Number of Cowpea Chlorotic Mottle Virus Particles Absorbed by Tobacco Protoplasts that become Infected

Summary At the most, 760 cowpea chlorotic mottle virus particles are sufficient to infect a tobacco protoplast.