CPMV (cowpea mosaic virus), a plant virus, is a naturally occurring sphere-like nanoparticle, and is used as a synthon and/or template in bionanoscience. The virions formed by CPMV can be regarded as programmable nanobuilding blocks with a diameter of ∼30 nm. A range of molecules have been attached to this viral nanoscaffold, yielding stable nanoparticles that display multiple copies of the desired molecule. It has been shown that, in addition to surface amine groups, surface carboxy groups are also addressable, and a procedure has been developed that enables introduction of reactive thiols at the virion surface that avoids virus aggregation. Furthermore, the virions can be functionalized to form electroactive nanoparticles. Methods for the construction of arrays and multilayers, using a layer-by-layer approach, have been established. As proof of concept, for example, CPMV particles have been immobilized on surfaces and arranged in defined layers. Engineered variants of CPMV can be used as templates for mineralization with, for example, silica to give monodisperse robust silica nanoparticles of ∼32 nm. SIRV2 (Sulfolobus islandicus rod-shaped virus 2), is a robust archaeal virus, resistant to high temperature and low pH. SIRV2 can act as a template for site-selective and spatially controlled chemical modification. Both the ends and the body of the virus, or the ends only, can be chemically addressed; SIRV2 can be regarded as a structurally unique nanobuilding block.
Since the turn of the century, there has been a growing interest in the exploitation of viruses, especially, but not exclusively, plant viruses, for the fabrication of new nanomaterials. VNPs (viral nanoparticles) are generally extremely robust and rigid, making them an excellent tool for bionanotechnological applications. In the present article, I summarize the progress we have made over the last 4 years working with the plant virus CPMV (cowpea mosaic virus), and more recent results demonstrating that an archaeal virus, SIRV2 (Sulfolobus islandicus rod-shaped virus 2), can be considered a novel nanobuilding block. By its very nature, this article will be egocentric, so I draw the reader's attention to some recent reviews that provide a broader picture [1–4].
CPMV is the type member of the Comovirus genus in the family Comoviridae. It has a narrow host range, normally infecting legumes, and, in Nature, it is transmitted by leaf-feeding beetles, thrips and grasshoppers. In systemic infected plants, CPMV causes mosaic or mottling symptoms. Plant viruses are non-infectious to other organisms and present no biological hazard. Inoculation and purification are simple and quick to perform; high expression levels can be obtained in a short time and gram-scale yields can be obtained from 1 kg of infected leaf material . CPMV virions exhibit an icosahedral symmetry, are of 28 nm diameter and are formed by 60 copies each of two different types of CP (coat protein): the small subunit and the large subunit [6–8]. The capsids of CPMV are robust and maintain their integrity at 60°C for at least 1 h and at pH values from 3.5 to 9.0 indefinitely at room temperature . Together, these properties make CPMV an ideal nanobuilding block, synthon and template for use in bionanoscience.
The exposed amino acid residues on the external surface of the CPMV virion are chemically addressable. This was first shown in 2002 for lysine residues . There are potentially 300 lysine residues on the surface presenting amines that can be modified by a range of different bioconjugation strategies, although only 240 of these are readily addressable. We have shown that surface carboxy groups can also be decorated with a carboxy-selective chemical dye, N-cyclohexyl-N′-[4-(dimethylamino)naphthyl]carbodi-imide ; UV–visible spectroscopy and native gel electrophoresis confirmed covalent chemical modification and denaturing gel electrophoresis showed that carboxy groups of both the small and the large subunit were addressed. We have also developed a strategy to chemically introduce thiol groups on to the surface of CPMV that avoids virus aggregation . Although mutants of CPMV with cysteine residues exposed on the surface can be prepared by genetic engineering [12,13], they have a penchant for inter-particle aggregation by the formation of disulfide bonds . Using the reagent N-succinimidyl-S-acetylthiopropionate, exposed lysine residues can be modified to give CPMV particles carrying thioacetate (SAc) groups. The major advantage of this is that the chemically introduced thiol groups in CPMV–SAcn are chemically protected and so the VNPs cannot aggregate by disulfide bond formation. When required, the protecting group can be removed easily with hydroxylamine hydrochloride. Conveniently, deprotected CPMV–SHn particles also do not aggregate over a period of at least 3 weeks in solution, owing to the position of the introduced thiol groups on the capsid surface.
Our studies, together with those of others, have established that CPMV particles can be considered useful building blocks in bionanoscience applications. They possess a range of different residues on their surface, including lysine residues, carboxy groups, tyrosine residues and thiols (introduced either by genetic or chemical engineering) that can be coupled to a wide variety of chemical and biological entities using a selection of conjugation strategies [1–4].
Layer and array formation
One objective of bionanoscience researchers is the ability to immobilize biomolecules on surfaces and to assemble biomolecules into defined arrays. In the present paper, I describe two approaches that we have used to construct arrays of CPMV on surfaces. The first one exploits the strong, complementary, non-covalent interaction between SAv (streptavidin) and biotin, and the second, the fabrication of multilayer structures by electrostatic interactions. CPMV particles can be used for the construction of mono-, bi- and multi-layers in a controlled manner.
The decoration of CPMV particles with both biotin and a fluorescent dye enabled the construction of arrays, from the bottom up by a layer-by-layer approach, and their differential detection (Figure 1) . CPMV bilayers composed of [CPMV–biotin–AF568] and [CPMV–biotin–AF488], where AF568 and AF488 are different Alexa Fluor® dyes, and vice versa were constructed on SAv-functionalized gold surfaces and analysed by fluorescence microscopy imaging. The VNPs were spread evenly and densely throughout each layer. The merged images confirmed that the virions sat one on top of the other; this is in contrast with a mixed-layer monolayer, where the images do not line up, as the particles are occupying the same layer and competing for binding sites. QCMD (quartz crystal microbalance with dissipation monitoring) studies of the alternating deposition of biotin-modified CPMV, with different biotin-spacer length, and SAv on a biotinylated solid-supported lipid bilayer showed that the level of biotin coverage and the length of the biotin spacer affect the degree of intra- and inter-layer cross-linking . A long spacer and high biotin coverage gave the most regular and densest arrays; the degree of layer coverage and of cross-linking can therefore be highly tuned. These properties become important when designing nanostructures. In some cases, it is important to maintain channels between particles (e.g. catalytic or enzymatic systems); in others, it is desirable to minimize the distance between the particles (e.g. electronically active arrays). The construction of triple-layered arrays has also been demonstrated; the first layer in this case being a CPMVCYS mutant bound directly to the gold surface .
Bilayers and a mixed monolayer of biotinylated and fluorescently labelled CPMV particles on gold slides imaged via fluorescence microscopy
The second approach involves layer-by-layer build-up of multilayers of cationic polyethyleneimine and anionic polyacrylic acid that form a polyelectrolyte molecular thin film. The negatively charged sphere-like CPMV particles are able to substitute for a polyanion layer, and the VNPs bind to the positively charged polymer and become partially embedded within it . Further fabrication leads to a self-assembled alternating structure of polyelectrolytes and VNPs. Interestingly, and in contrast, we have found that rod-shaped tobacco mosaic virus particles are excluded from the polyelectrolyte assembly and float, in an ordered arrangement, on top of the polyionic layered structure; apparently, not only size, but also “shape matters, too” .
Functionalized CPMV particles
Having established that CPMV particles can be used as building blocks for the fabrication of layers and arrays, it is necessary to demonstrate that they can be functionalized. For example, we have shown that electroactive CPMV particles can be readily prepared, isolated and characterized. Ferrocene carboxylate can be used for the facile covalent decoration of amine groups, arising from lysine residues, on the capsid surface by standard coupling protocols . Electrochemical studies then established the presence of redox-active nanoparticles and enabled the quantification of the number of ferrocenes per virus particle. The CPMV–ferrocene conjugate possesses an electrochemically reversible ferrocene–ferrocenium couple. The bound ferrocenyl molecules each behave as independent electronically isolated units similar to the situation found for metallodendrimers. By using the Randles–Sevcik equation, it was calculated that there were approx. 240 ferrocene molecules on each CPMV particle.
Using a similar approach, an organic redox-active Viologen derivative, methyl(aminopropyl)viologen, can be coupled to carboxy groups on the capsid surface . The CPMV–Viologen conjugate exhibited the expected two successive reversible one-electron processes of Methyl Viologen. Again, the attached groups behave as independent electronically isolated units. The Randles–Sevcik equation gave the number of Viologens per CPMV particle as ∼180. That is, three carboxy groups on each virus asymmetric unit are decorated, providing the first quantification of the number of addressable carboxy groups on the capsid surface.
These robust, monodisperse, electronically active VNPs may act as multielectron reservoirs that could be developed for use in nanodevices such as molecular batteries or capacitors, or be used as nanoscale electron-transfer mediators in biosensors, molecular recognition or redox catalysis.
In addition to surface modification and multilayer array assembly of CPMV VNPs, engineered versions of CPMV can also be used to template mineralization processes. CPMV particles exhibit the ideal characteristics of a nanotemplate, in both their size and regular symmetric structure. It has been established that short peptides can be inserted into the highly exposed surface βB–βC loop of the small protein of the capsid [18,19]. We have designed, constructed, propagated, isolated and purified a CPMV chimaera that templates the mineralization of silica on the virion surface to give silica nanoparticles of ∼32 nm diameter . The CPMVsilica chimaera was produced by engineering a specific peptide sequence (YSDQPTQSSQRP), selected previously for silica nucleation by phage display , into the CP loop by using established cloning procedures [22,23]. The chimaeric construct was then introduced into cowpea plants by agro-inoculation, and virus particles were produced after passaging the infection to further plants . Yields of the chimaera are comparable with those of wild-type.
Having obtained the CPMVsilica chimaera, silication was achieved via a sol–gel process, using tetraethoxysilane and aminopropyltriethoxysilane, at pH 7 in aqueous buffer and at temperatures no higher than 45°C. The purified dense silicated CPMVsilica nanoparticles can be easily visualized in unstained TEM (transmission electron microscopy) images (Figure 2). In contrast, CPMVsilica before mineralization was not visible in unstained TEM images. The diameter of particles from TEM and dynamic light scattering measurements is ∼32 nm, consistent with an average silica coating on each particle of 2 nm. Dynamic light scattering showed that the particles were monodisperse, and energy dispersive X-ray unambiguously confirmed the presence of silicon and oxygen on the silicated CPMVsilica particles. This is the first example of a cage-like virus being mineralized on its outer surface . We are now exploring how these and other mineralized chimaeras, prepared using this methodology under environmentally benign conditions, may be exploited in various bionanotechnological applications.
TEM images of CPMVsilica chimaera and silicated CPMVsilica particles
SIRV2, a novel nanobuilding block
The archaeal virus SIRV2 is from the family Rudiviridae that infects strains of the archaeal genus Sulfolobus, which is isolated from Icelandic solfataric hot acidic springs with a temperature of 88°C and pH 2.5 [24,25]. It is a hyperthermophilic and acidophilic virus. SIRV2 particles are rod-shaped with dimensions of 23 nm×900 nm, with a central channel of approx. 6 nm that encapsidates the DNA genome. At each terminus of the rod are unique structures: there is a plug of approx. 48 nm length and 6 nm diameter that fills the terminal portion of the cavity, together with three tail fibres of approx. 28 nm length. SIRV2 is composed of two structural proteins. The major CP of ∼20 kDa forms the virus body, with the subunits arranged in a helical confirmation of periodicity of 4.3 nm . The level of minor CP is less than 1% of the major CP. The minor CP of ∼100 kDa has been shown to be located in the tail fibres at the end of the virus particle .
SIRV2, by its nature, is an extremely stable VNP; it is resistant to high temperature and low pH. SIRV2 has also been shown to be resistant to non-natural harsh conditions . For example, SIRV2 remains intact and infectious in DMSO/water mixtures (1:1, v/v) for at least 6 days. These properties make SIRV2 an ideal candidate for chemical modification to produce novel nanomaterials for future technological and/or biomedical applications. We have demonstrated that SIRV2 offers sites for site-specific and spatially controlled attachment of functional ligands . On the surface of SIRV2 are amino acid residues that can be easily addressed, including lysine residues, providing modifiable amine groups, and aspartic and glutamic acid residues, providing modifiable carboxy groups. In addition, the surface of the virion is glycosylated, providing modifiable carbohydrate groups after mild oxidation. The addressability of these groups was probed with biotin, using three different selective bioconjugation chemistries: amines were labelled at pH 7 with succinimide-activated biotin to give a stable amide bond; carboxy groups were activated with 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide and reacted with biotin hydrazide; carbohydrates were oxidized and reacted with biotin hydrazide to give a stable hydrazone linkage. Biotin can be readily detected with either fluorescently labelled SAv or with immunogold staining by anti-biotin antibodies. We confirmed that SIRV2 particles provide multiple and various attachment sites. Biotin labels were installed at carboxy, carbohydrate and amine sites, but the labelling was different in each case (Figure 3). Covalent attachment of biotins to carboxy groups or carbohydrate gave particles decorated with biotin throughout the virion, both virus body and virus end. In contrast, labelling of amines produced particles where the terminal tail fibres on the end of the virion were labelled exclusively. This selective and spatial control is a property not possessed by other rigid rod-shaped viruses. SIRV2 can therefore be regarded as a new, extremely stable and structurally unique viral nanobuilding block.
Immunogold staining of biotinylated SIRV2 particles using gold-labelled anti-biotin antibodies and TEM
By working at the interface of biology, chemistry and materials science, in those areas of bionanoscience and synthetic biology that exploit biomaterials for the fabrication of new nanomaterials and nanodevices, we have demonstrated the versatility of the plant virus CPMV as a synthon, building block and template. In addition, we have identified and begun to explore the properties of a robust archaeal virus as a new nanobuilding block. Further research in this exciting area is expected, in the fullness of time, to find applications in, among others, nanoelectronics, magneto-storage devices, catalytic or enzymatic nanofactories, biosensors and biomedicine.
Bionanotechnology II: from Biomolecular Assembly to Applications: Biochemical Society Focused Meeting held at Robinson College, Cambridge, U.K., 7–9 January 2009. Organized and Edited by Tony Cass (Imperial College London, U.K.) and Dek Woolfson (Bristol, U.K.).
Members of my group, past and present, and my collaborators are thanked for their contributions to the work described here.
This work was supported by the Biotechnology and Biological Sciences Research Council [Core Strategic Grant to the John Innes Centre and grant number BB/E024939/1] and the EU Marie Curie Early Stage Training Scheme [grant number MEST-CT-2004-504273].