Elucidating the Thermodynamic Driving Forces of Polyanion-Templated Virus-like Particle Assembly.

A virus in its most simple form is comprised of a protein capsid that surrounds and protects the viral genome. The self-assembly of such structures, however, is a highly complex, multiprotein, multiinteraction process and has been a topic of study for a number of years. This self-assembly process is driven by the (mainly electrostatic) interaction between the capsid proteins (CPs) and the genome as well as by the protein-protein interactions, which primarily rely on hydrophobic interactions. Insight in the thermodynamics that is involved in virus and virus-like particle (VLP) formation is crucial in the detailed understanding of this complex assembly process. Therefore, we studied the assembly of CPs of the cowpea chlorotic mottle virus (CCMV) templated by polyanionic species (cargo), that is, single-stranded DNA (ssDNA), and polystyrene sulfonate (PSS) using isothermal titration calorimetry. By separating the electrostatic CP-cargo interaction from the full assembly interaction, we conclude that CP-CP interactions cause an enthalpy change of -3 to -4 kcal mol-1 CP. Furthermore, we quantify that upon reducing the CP-CP interaction, in the case of CCMV by increasing the pH to 7, the CP-cargo starts to dominate VLP formation. This is highlighted by the three times higher affinity between CP and PSS compared to CP and ssDNA, resulting in the disassembly of CCMV at neutral pH in the presence of PSS to yield PSS-filled VLPs.

Experimental and Theoretical Determination of the pH inside the Confinement of a Virus-Like Particle.

In biology, a variety of highly ordered nanometer-size protein cages is found. Such structures find increasing application in, for example, vaccination, drug delivery, and catalysis. Understanding the physiochemical properties, particularly inside the confinement of a protein cage, helps to predict the behavior and properties of new materials based on such particles. Here, the relation between the bulk solution pH and the local pH inside a model protein cage, based on virus-like particles (VLPs) built from the coat proteins of the cowpea chlorotic mottle virus, is investigated. The pH is a crucial parameter in a variety of processes and is potentially significantly influenced by the high concentration of charges residing on the interior of the VLPs. The data show a systematic more acidic pH of 0.5 unit inside the VLP compared to that of the bulk solution for pH values above pH 6, which is explained using a theoretical model based on a Donnan equilibrium. The model agrees with the experimental data over almost two orders of magnitude, while below pH 6 the experimental data point to a buffering capacity of the VLP. These results are a first step in a better understanding of the physiochemical conditions inside a protein cage.

Nanoreactors via Encapsulation of Catalytic Gold Nanoparticles within Cowpea Chlorotic Mottle Virus Protein Cages.

Viral protein cage-based nanoreactors can be generated by encapsulation of catalytic metal nanoparticles within the capsid structure. In this method, coat proteins of the cowpea chlorotic mottle virus (CCMV) are used to sequester gold nanoparticles (Au NPs) in buffered solutions at neutral pH to form CCMV-Au hybrid nanoparticles. This chapter describes detailed methods for the encapsulation of Au NPs into CCMV protein cages. Protocols for the reduction of nitroarenes by using CCMV-Au NPs as catalyst are described as an example for the catalytic activity of Au NPs in the protein cages.

Oligonucleotide Length-Dependent Formation of Virus-Like Particles.

Understanding the assembly pathway of viruses can contribute to creating monodisperse virus-based materials. In this study, the cowpea chlorotic mottle virus (CCMV) is used to determine the interactions between the capsid proteins of viruses and their cargo. The assembly of the capsid proteins in the presence of different lengths of short, single-stranded (ss) DNA is studied at neutral pH, at which the protein-protein interactions are weak. Chromatography, electrophoresis, microscopy, and light scattering data show that the assembly efficiency and speed of the particles increase with increasing length of oligonucleotides. The minimal length required for assembly under the conditions used herein is 14 nucleotides. Assembly of particles containing such short strands of ssDNA can take almost a month. This slow assembly process enabled the study of intermediate states, which confirmed a low cooperative assembly for CCMV and allowed for further expansion of current assembly theories.

Effect of Variations in Micropatterns and Surface Modulus on Marine Fouling of Engineering Polymers.

We report on the marine fouling and fouling release effects caused by variations of surface mechanical properties and microtopography of engineering polymers. Polymeric materials were covered with hierarchical micromolded topographical patterns inspired by the shell of the marine decapod crab Myomenippe hardwickii. These micropatterned surfaces were deployed in field static immersion tests. PDMS, polyurethane, and PMMA surfaces with higher elastic modulus and hardness were found to accumulate more fouling and exhibited poor fouling release properties. The results indicate interplay between surface mechanical properties and microtopography on antifouling performance.

Protein Cages as Containers for Gold Nanoparticles.

Abundant and highly diverse, viruses offer new scaffolds in nanotechnology for the encapsulation, organization, or even synthesis of novel materials. In this work the coat protein of the cowpea chlorotic mottle virus (CCMV) is used to encapsulate gold nanoparticles with different sizes and stabilizing ligands yielding stable particles in buffered solutions at neutral pH. The sizes of the virus-like particles correspond to T = 1, 2, and 3 Caspar-Klug icosahedral triangulation numbers. We developed a simple one-step process enabling the encapsulation of commercially available gold nanoparticles without prior modification with up to 97% efficiency. The encapsulation efficiency is further increased using bis-p-(sufonatophenyl)phenyl phosphine surfactants up to 99%. Our work provides a simplified procedure for the preparation of metallic particles stabilized in CCMV protein cages. The presented results are expected to enable the preparation of a variety of similar virus-based colloids for current focus areas.

Combining Protein Cages and Polymers: from Understanding Self-Assembly to Functional Materials.

Protein cages, such as viruses, are well-defined biological nanostructures which are highly symmetrical and monodisperse. They are found in various shapes and sizes and can encapsulate or template non-native materials. Furthermore, the proteins can be chemically or genetically modified giving them new properties. For these reasons, these protein structures have received increasing attention in the field of polymer-protein hybrid materials over the past years, however, advances are still to be made. This Viewpoint highlights the different ways polymers and protein cages or their subunits have been combined to understand self-assembly and create functional materials.