Tianwei Lin

Icosahedral Virus Particles as Addressable Nanoscale Building Blocks

Nanochemistry is the synthesis and study of well-defined structures with dimensions of 1 ± 100 nanometers (nm), and thus spans the size range between molecules and materials.[1] While supramolecular chemistry (making small molecules bigger) and microfabrication techniques (making big structures smaller) attack from the flanks, biology employs many constructs of this size. Examples include the photosynthetic reaction center, the ribosome, and membrane-bound receptor-signaling complexes, all notable because of their sophisticated yet modular function. The burgeoning field of nanotechnology[2] seeks to mimic the information-handling, materials-building, and responsive sensing capabilities of biological systems at the nanometer scale. The special requirements of this enterprise would be well served by building blocks of the proper size with predictable and programmable chemistry. Cowpea mosaic virus (CPMV) particles are 30 nm-diameter icosahedra, formed by 60 copies of two different types of protein subunits (Figure 1a).[3] The physical, biological, and genetic properties of CPMV have been well characterized.[4] Approximately one gram of virus is easily and routinely obtained from a kilogram of infected leaves of the black-eye pea plant. The structure of CPMV has been characterized at 2.8 ä resolution by X-ray crystallography and an atomic model of the particle has been constructed.[5] The virion displays icosahedral symmetry to the resolution of the crystal structure and an infectious clone of the virus allows sitedirected and insertional mutagenesis to be performed in a straightforward and rapid manner.[6] The particles are remarkably stable; they maintain their integrity at 60 C (pH 7) for at least one hour and at pH values from 3.5 to 9 indefinitely at room temperature. Different crystal forms of the virus can be readily produced under well-defined conditions (Figure 1d).[7, 8] Here we report on the selective Experimental Section

Natural Supramolecular Building Blocks: Cysteine-Added Mutants of Cowpea Mosaic Virus

Wild-type Cowpea mosaic virus (CPMV) displays no cysteine side chains on the exterior capsid surface and is therefore relatively unreactive with thiol-selective reagents. Four CPMV mutants bearing cysteine residues in one of two exterior positions of the asymmetric unit were created. The mutants were shown to aggregate by virtue of disulfide bond formation in the absence of added reducing agent, bind to metallic gold, and undergo selective reactions at the introduced thiol residues. Controlled aggregation by virtue of biotin-avidin interactions was demonstrated, as was the independent derivatization of reactive lysine and cysteine positions. The ability to introduce such reactivity into a system that can be readily prepared and isolated in gram quantities should open new doors to applications in biochemistry, materials science, and catalysis.

Natural Supramolecular Building Blocks: Wild-Type Cowpea Mosaic Virus

Cowpea mosaic virus (CPMV) can be isolated in gram quantities, possesses a structure that is known to atomic resolution, and is quite stable. It is therefore of potential use as a molecular entity in synthesis, particularly as a building block on the nanochemical scale. CPMV was found to possess a lysine residue with enhanced reactivity in each asymmetric unit, and thus 60 such lysines per virus particle. The identity of this residue was established by a combination of acylation, protein digestion, and mass spectrometry. Under forcing conditions, up to four lysine residues per asymmetric unit can be addressed. In combination with engineered cysteine reactivity described in the accompanying paper, this provides a powerful platform for the alteration of the chemical and physical properties of CPMV particles.

The Structure of Pariacoto Virus Reveals a Dodecahedral Cage of Duplex RNA

The 3.0 Å resolution crystal structure of Pariacoto virus (PaV) reveals extensive interactions between portions of the viral RNA genome and the icosahedral capsid. Under the protein shell of the T = 3 quasi equivalent capsid lies a dodecahedral cage composed of RNA duplex that accounts for ∼35% of the single-stranded RNA genome. The highly basic N-terminal regions (residues 7–54) of the subunits, forming pentamers (A subunits) are clearly visible in the density map and make numerous interactions with the RNA cage. The C-terminal segments (residues 394–401) of the A subunits lie in channels near the quasi three-fold axes. Electron cryo-microscopy and image reconstruction of PaV particles clearly show the dodecahedral RNA cage.

Orphan nuclear receptor TR3 acts in autophagic cell death via mitochondrial signaling pathway.

Autophagy is linked to cell death, yet the associated mechanisms are largely undercharacterized. We discovered that melanoma, which is generally resistant to drug-induced apoptosis, can undergo autophagic cell death with the participation of orphan nuclear receptor TR3. A sequence of molecular events leading to cellular demise is launched by a specific chemical compound, 1-(3,4,5-trihydroxyphenyl)nonan-1-one, newly acquired from screening a library of TR3-targeting compounds. The autophagic cascade comprises TR3 translocation to mitochondria through interaction with the mitochondrial outer membrane protein Nix, crossing into the mitochondrial inner membrane through Tom40 and Tom70 channel proteins, dissipation of mitochondrial membrane potential by the permeability transition pore complex ANT1-VDAC1 and induction of autophagy. This process leads to excessive mitochondria clearance and irreversible cell death. It implicates a new approach to melanoma therapy through activation of a mitochondrial signaling pathway that integrates a nuclear receptor with autophagy for cell death.

Structure-Based Engineering of an Icosahedral Virus for Nanomedicine and Nanotechnology

A quintessential tenet of nanotechnology is the self-assembly of nanometer-sized components into devices. Biological macromolecular systems such as viral particles were found to be suitable building blocks for nanotechnology for several reasons: viral capsids are extremely robust and can be produced in large quantities with ease, the particles self-assemble into monodisperse particles with a high degree of symmetry and polyvalency, they have the propensity to form arrays, and they offer programmability through genetic and chemical engineering. Here, we review the recent advances in engineering the icosahedral plant virus Cowpea mosaic virus (CPMV) for applications in nano-medicine and -technology. In the first part, we will discuss how the combined knowledge of the structure of CPMV at atomic resolution and the use of chimeric virus technology led to the generation of CPMV particles with short antigenic peptides for potential use as vaccine candidates. The second part focuses on the chemical addressability of CPMV. Strategies to chemically attach functional molecules at designed positions on the exterior surface of the viral particle are described. Biochemical conjugation methods led to the fabrication of electronically conducting CPMV particles and networks. In addition, functional proteins for targeted delivery to mammalian cells were successfully attached to CPMV. In the third part, we focus on the utilization of CPMV as a building block for the generation of 2D and 3D arrays. Overall, the potential applications of viral nanobuilding blocks are manifold and range from nanoelectronics to biomedical applications.

Structure-based design of peptide presentation on a viral surface: the crystal structure of a plant/animal virus chimera at 2.8 å resolution

BACKGROUND We employed a genetically engineered icosahedral plant virus, cowpea mosaic virus (CPMV), as an expression and presentation system to display a 14 amino acid linear antigenic epitope found in a capsid protein of human rhinovirus 14 (HRV14). RESULT Gram quantities of the CPMV/HRV 14 chimera were made in plants and purified particles were crystallized in a form isomorphous with wild-type CPMV. The 2.8 A resolution structure of the chimera shows that the inserted loop is well ordered and that if the loop were intact, a phenylalanine residue of CPMV would be placed in a hydrophilic environment. The resultant strain may make the loop an attractive substrate for endogenous plant proteases, as roughly 80% of the inserted polypeptides are cleaved, allowing the phenylalanine to be partially buried. Altering the phenylalanine to an arginine could relieve the stress, reducing the propensity for cleavage and increasing the likelihood that the peptide will assume a structure closely similar to its structure in HRV14. CONCLUSIONS Successful crystallization of other CPMV chimeras in forms isomorphous with the native virus suggests that this is a viable system for structure-based design of peptide presentation.

Cowpea mosaic virus: from the presentation of antigenic peptides to the display of active biomaterials.

The potential of cowpea mosaic virus (CPMV), a plant icosahedral virus, for the presentation of foreign peptides and proteins is reported. The most prominent feature at the virus surface is a region of the smaller of the two coat proteins (S) which has been extensively used for the insertion of foreign peptides. Given the availability of the three-dimensional structure of the native virus and the amenability of foreign peptide-expressing CPMV chimeras to crystallisation, immunological data can be correlated with the conformational state of the foreign insert. The latter is influenced by proteolysis which occurs within the foreign inserts. In an effort to offer an alternative context for peptide expression, extensive exploration of a second region of the S protein is reported with respect to tolerance to small insertions. Moreover, to make CPMV suitable for a wider spectrum of presentation, a technique was developed to allow surface coupling of a peptide which can serve as the anchoring point for a range of proteins. This new approach is also widely applicable for the direct chemical cross-linking of peptides and full-length protein domains to the viral capsid.

3D Domain Swapping Modulates the Stability of Members of an Icosahedral Virus Group

BACKGROUND Rice yellow mottle virus (RYMV) is a major pathogen that dramatically reduces rice production in many African countries. RYMV belongs to the genus sobemovirus, one group of plant viruses with icosahedral capsids and single-stranded, positive-sense RNA genomes. RESULTS The structure of RYMV was determined and refined to 2.8 A resolution by X-ray crystallography. The capsid contains 180 copies of the coat protein subunit arranged with T = 3 icosahedral symmetry. Each subunit adopts a jelly-roll beta sandwich fold. The RYMV capsid structure is similar to those of other sobemoviruses. When compared with these viruses, however, the betaA arm of the RYMV C subunit, which is a molecular switch that regulates quasi-equivalent subunit interactions, is swapped with the 2-fold-related betaA arm to a similar, noncovalent bonding environment. This exchange of identical structural elements across a symmetry axis is categorized as 3D domain swapping and produces long-range interactions throughout the icosahedral surface lattice. Biochemical analysis supports the notion that 3D domain swapping increases the stability of RYMV. CONCLUSIONS The quasi-equivalent interactions between the RYMV proteins are regulated by the N-terminal ordered residues of the betaA arm, which functions as a molecular switch. Comparative analysis suggests that this molecular switch can also modulate the stability of the viral capsids.

Influence of three-dimensional structure on the immunogenicity of a peptide expressed on the surface of a plant virus.

The influence of peptide structure on immunogenicity has been investigated by constructing a series of cowpea mosaic virus (CPMV) chimaeras expressing the 14 amino acid NIm‐1A epitope from human rhinovirus 14 (HRV‐14) at different positions on the capsid surface. Biochemical and crystallographic analysis of a CPMV/HRV chimaera expressing the NIm‐1A epitope inserted into the βC′–βC″ loop of the S protein revealed that, although the inserted peptide was free at its C‐terminus, it adopted a conformation distinct from that previously found when a similarly cleaved peptide was expressed in the βB–βC loop of the S protein. Adjustment of the site of insertion within the βB–βC loop resulted in the isolation of a chimaera in which cleavage at the C‐terminus of the epitope was much reduced. Crystallographic analysis confirmed that in this case the epitope was presented as a closed loop. Polyclonal antisera raised against the CPMV/HRV chimaera presenting the NIm‐1A epitope as a closed loop had a significantly enhanced ability to bind to intact HRV‐14 particles compared with antisera raised against chimaeras presenting the same sequence as peptides with free C‐termini. These results demonstrate that the mode of presentation of an epitope on a heterologous carrier can dramatically affect its immunological properties. Copyright