John E. Johnson

Structures of the native and swollen forms of cowpea chlorotic mottle virus determined by X-ray crystallography and cryo-electron microscopy.

BACKGROUND RNA-protein interactions stabilize many viruses and also the nucleoprotein cores of enveloped animal viruses (e.g. retroviruses). The nucleoprotein particles are frequently pleomorphic and generally unstable due to the lack of strong protein-protein interactions in their capsids. Principles governing their structures are unknown because crystals of such nucleoprotein particles that diffract to high resolution have not previously been produced. Cowpea chlorotic mottle virions (CCMV) are typical of particles stabilized by RNA-protein interactions and it has been found that crystals that diffract beyond 4.5 A resolution are difficult to grow. However, we report here the purification of CCMV with an exceptionally mild procedure and the growth of crystals that diffract X-rays to 3.2 A resolution. RESULTS The 3.2 A X-ray structure of native CCMV, an icosahedral (T = 3) RNA plant virus, shows novel quaternary structure interactions based on interwoven carboxyterminal polypeptides that extend from canonical capsid beta-barrel subunits. Additional particle stability is provided by intercapsomere contacts between metal ion mediated carboxyl cages and by protein interactions with regions of ordered RNA. The structure of a metal-free, swollen form of the virus was determined by cryo-electron microscopy and image reconstruction. Modeling of this structure with the X-ray coordinates of the native subunits shows that the 29 A radial expansion is due to electrostatic repulsion at the carboxyl cages and is stopped short of complete disassembly by preservation of interwoven carboxyl termini and protein-RNA contacts. CONCLUSIONS The CCMV capsid displays quaternary structural interactions that are unique compared with previously determined RNA virus structures. The loosely coupled hexamer and pentamer morphological units readily explain their versatile reassembly properties and the pH and metal ion dependent polymorphism observed in the virions. Association of capsomeres through inter-penetrating carboxy-terminal portions of the subunit polypeptides has been previously described only for the DNA tumor viruses, SV40 and polyoma.

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

VIPERdb2: an enhanced and web API enabled relational database for structural virology.

VIPERdb (http://viperdb.scripps.edu) is a relational database and a web portal for icosahedral virus capsid structures. Our aim is to provide a comprehensive resource specific to the needs of the virology community, with an emphasis on the description and comparison of derived data from structural and computational analyses of the virus capsids. In the current release, VIPERdb2, we implemented a useful and novel method to represent capsid protein residues in the icosahedral asymmetric unit (IAU) using azimuthal polar orthographic projections, otherwise known as Φ–Ψ (Phi–Psi) diagrams. In conjunction with a new Application Programming Interface (API), these diagrams can be used as a dynamic interface to the database to map residues (categorized as surface, interface and core residues) and identify family wide conserved residues including hotspots at the interfaces. Additionally, we enhanced the interactivity with the database by interfacing with web-based tools. In particular, the applications Jmol and STRAP were implemented to visualize and interact with the virus molecular structures and provide sequence–structure alignment capabilities. Together with extended curation practices that maintain data uniformity, a relational database implementation based on a schema for macromolecular structures and the APIs provided will greatly enhance the ability to do structural bioinformatics analysis of virus capsids.

The Structure of an Infectious p22 Virion Shows the Signal for Headful DNA Packaging

Bacteriophages, herpesviruses, and other large double-stranded DNA (dsDNA) viruses contain molecular machines that pump DNA into preassembled procapsids, generating internal capsid pressures exceeding, by 10-fold, that of bottled champagne. A 17 angstrom resolution asymmetric reconstruction of the infectious P22 virion reveals that tightly spooled DNA about the portal dodecamer forces a conformation that is significantly different from that observed in isolated portals assembled from ectopically expressed protein. We propose that the tight dsDNA spooling activates the switch that signals the headful chromosome packing density to the particle exterior.

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.

Evidence of viral capsid dynamics using limited proteolysis and mass spectrometry.

Virus particles are stable yet exhibit highly dynamic character given the events that shape their life cycle. Isolated from their hosts, the nucleoprotein particles are macromolecules that can be crystallized and studied by x-ray diffraction. During assembly, maturation and entry, however, they are highly dynamic and display remarkable plasticity. These dynamic properties can only be inferred from the x-ray structure and must be studied by methods that are sensitive to mobility. We have used matrix-assisted laser desorption/ionization mass spectrometry combined with time resolved, limited proteolysis (Cohen, S. L., Ferre-D’Amare, A. R., Burley, S. K., and Chait, B. T. (1995) Protein Sci. 4, 1088–1099; Kriwacki, R. W., Wu, J., Tennant, T., Wright, P. E., and Siuzdak, G. (1997) J. Chromatogr. 777, 23–30; Kriwacki, R. W., Wu, J., Siuzdak, G., and Wright, P. E. (1996)J. Am. Chem. Soc. 118, 5320–5321) to examine the viral capsid of flock house virus. Employing less than 10 μg of virus, time course digestion products were assigned to polypeptides of the subunit. Although surface regions in the three-dimensional structure were susceptible to cleavage on extended exposure to the protease, the first digestion products were invariably from parts of the subunit that are internal to the x-ray structure. Regions in the N- and C-terminal portions of the subunit, located within the shell in the x-ray structure, but implicated in RNA neutralization and RNA release and delivery, respectively, were the most susceptible to cleavage demonstrating transient exposure of these polypeptides to the viral surface.

Hybrid Virus−Polymer Materials. 1. Synthesis and Properties of PEG-Decorated Cowpea Mosaic Virus

Cowpea mosaic virus was derivatized with poly(ethylene glycol) to give well-controlled loadings of polymer on the outer surface of the coat protein assembly. The resulting conjugates displayed altered densities and immunogenicities, consistent with the known chemical and biological properties of PEG. These studies make CPMV potentially useful as a tailored vehicle for drug delivery.

Virus Particle Explorer (VIPER), a Website for Virus Capsid Structures and Their Computational Analyses

The number of icosahedral-capsid structures determined at a near-atomic level of resolution is growing rapidly as advances in synchrotron radiation sources, fast-readout detectors, and computer hardware and software are made. Hence, there is an increasing need to organize these mega-assemblies into a uniform and easy-to-use database. The coordinates of the icosahedral-capsid structures deposited in the Protein Data Bank (PDB) (2) follow a variety of conventions in which the icosahedral symmetry axes are oriented differently in the orthogonal coordinate system. While trying to analyze the various capsid structures en masse, we became aware of the need for a database in which all capsid structures (coordinates) are stored in a standard icosahedral orientation. Such a structural database of viral capsids would indeed facilitate the development of tools for high-throughput analyses of the virus structures. We report here the creation of a web-base (website and database) of virus structures, the Virus Particle Explorer (VIPER), which can be accessed through the World Wide Web (WWW) at the uniform resource locator (URL) http://mmtsb.scripps.edu /viper/. The organization of the VIPER database is shown in Fig. ​Fig.1.1. FIG. 1 Flow chart showing the organization of the contents of the VIPER site. The VIPER database contains the structures of viral capsids determined at a nearly atomic-level resolution. Coordinates of the capsid structures are stored in the z(2)-3-5-x(2) convention. ...

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.