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Crystallographic Analyses of Viruses and Macromolecular Assemblies

J. Johnson, A. Schneemann, V. Reddy, T. Lin, W. Wikoff, A. Kumar, P. Natarajan, J. Tate, N. Krishna, F. Dong, M. Canady, C. Qu, Z. Che, H. Giesing, B. Sheehan, H. Langedijk,* L. Liljas,** G. Lomonossoff***

* Institute for Animal Science and Health, Lelystad, the Netherlands
** Uppsala University, Uppsala, Sweden
*** John Innes Centre, Cambridge, England

Our group investigates the structure and function of viruses to elucidate molecular mechanisms of infection. On the basis of this information, we determine potential targets for antiviral agents and use viruses as reagents for understanding and exploiting the biology of the cell. We use a variety of virus families for our investigations; each family has novel properties appropriate for specific lines of investigation.

For research on assembly, we use virus groups readily studied in vitro and in vivo. An area of specific interest is the formation of quasi-equivalent viral capsids in which the same gene product participates in polymorphic interactions to generate both hexamers and pentamers in the formation of icosahedral shells. These structures are the biological equivalent of Buckminster Fuller's geodesic domes. They provide particles large enough to package and protect the genome and have targeting properties to deliver the genome to host cells for viral replication.

Knowledge of the structure of cowpea chlorotic mottle virus has enabled us to determine the parts of the subunit that control different aspects of assembly. In vitro assembly with structure-based mutations has been studied in collaboration with M. Young at Montana State University, who developed an Escherichia coli expression system for the cowpea chlorotic mottle virus subunit and can purify, refold, and assemble the protein into viruslike particles. These studies confirm the postulated functional roles, based on crystallographic studies, of different regions of the subunit.

To further understand assembly polymorphism, we examined the different patterns of assembly of the coat protein of alfalfa mosaic virus. In Nature, this multipartite RNA virus exists as 4 bacilliform particles in which capsid protein forms a particle proportional to the size of the RNA genome segment packaged. Purified viral protein can assemble in vitro into an icosahedral particle in the absence of RNA. We determined the structure of this assembly product at 4.0-Å resolution. Using the coordinates of the subunit structure, we created a model of the bacilliform particles that agrees in detail with the hexagonal lattice observed in electron microscopy studies.

We are exploring structure-function relationships in the nodavirus and tetravirus families of animal viruses. Our studies indicate that capsid polymorphism is controlled by a protein switch and/or RNA, that the maturation cleavage required for infection is autocatalytic and depends on assembly and the binding of calcium ions, that the release of RNA probably depends on a specific protein-RNA interaction that occurs only once in the context of the symmetric capsid, and that membrane translocation of RNA is probably mediated by a pentameric helical bundle that is rendered covalently independent from the subunit by the maturation cleavage.

Although the quaternary structures and subunits of nodaviruses and tetraviruses differ dramatically in size, our studies have revealed an evolutionary relationship between the 2 families. Using heterologous protein expression systems in which the capsid protein spontaneously assembles to form particles, we crystallized and solved the structures of particles specifically mutated to reveal the atomic details of the phenomena described earlier in this report. Recently, we used computational chemistry methods to explore assembly trajectories and the dependence of the trajectories on intersubunit stabilities.

We are investigating the use of plant viral particles as carriers for genetically inserted, heterologous polypeptides up to 40 amino acids long. These polypeptides have been used to generate neutralizing antibodies against HIV by presenting an epitope from the gp41 protein and to present peptide antagonists that trigger cell-surface phenomena. Determinations of the structures of these chimeric viruses have enabled us to design presentations that increase the efficacy of inserted peptides and to better understand factors that affect peptide folding and viral assembly.

We are continuing crystallographic studies of the capsid of HK97, a -like double-stranded DNA bacteriophage. The T = 7, 620-Å diameter particles were assembled by expressing the gene for the capsid protein gene in E coli. These particles were crystallized, and x-ray diffraction patterns beyond 3.5-Å resolution were obtained. A data set was collected at the Cornell High Energy Synchrotron Source, processed to 7-Å resolution, and merged with ultralow-resolution data (200- to 16-Å resolution) collected at the Stanford Synchrotron Radiation Laboratory. Phases between 200- and 50-Å resolution were computed with the particle model determined by cryo-electron microscopy and image reconstruction at 35-Å resolution. Phases for the higher resolution data were determined by using extension procedures and the 60-fold noncrystallographic symmetry. The structure shows a classical T = 7 lattice with the subunits formed predominantly of -helices. On the basis the x-ray structure and a cryo-electron microscopy reconstruction of the 450-Å diameter procapsid, we have proposed a detailed mechanism for particle maturation.

PUBLICATIONS

Bailey, M., Schulten, K., Johnson, J. The use of solid physical models for the study of macromolecular assembly. Curr. Opin. Struct. Biol. 8:202, 1998.

Bothner, B., Dong, X., Bibbs, L., Johnson, J., Siuzdak, G. Evidence of viral capsid dynamics using limited proteolysis and mass spectrometry. J. Biol. Chem. 273:673, 1998.

Chandrasekar, V., Johnson, J. The structure of tobacco ringspot virus: A link in the evolution of icosahedral capsids in the picornavirus superfamily. Structure 6:157, 1998.

Chapman, M., Blanc, E., Johnson, J., McKenna, R., Munshi, S., Rossmann, M., Tsao, J. Use of non-crystallographic symmetry for ab initio phasing of virus structures. In: Direct Methods for Solving Macromolecular Structures. Kluwer, Boston, 1998, p. 433.

Dong, X., Natarajan, P., Tihova, M., Johnson, J., Schneemann, A. Particle polymorphism caused by deletion of a peptide molecular switch in a quasi-equivalent icosahedral virus. J. Virol., in press.

Fox, J., Wang, G., Speir, J., Olson, N., Johnson, J., Baker, T., Young, M. Comparison of the native CCMV virion with in vitro assembled CCMV virions by cryo-electron microscopy and image reconstruction. Virology 244:212, 1998.

Gaspar, L., Johnson, J., Silva, J., Dapoian, A. Partially folded states of the capsid protein of cowpea severe mosaic virus in the disassembly pathway. J. Mol. Biol. 273:456, 1997.

Johnson, J., Reddy, V. Structural studies of noda and tetraviruses. In: The Insect Viruses. Miller, L., Ball, L. (Eds.). Plenum, New York, 1998, p. 171.

Munshi, S., Liljas, L., Johnson, J. The structure determination of Nudaurelia capensis omega virus. Acta Crystallogr. D, in press.

Natarajan, P., Johnson, J. Molecular packing in virus crystals: Geometry, chemistry and biology. J. Struct. Biol., in press.

Reddy, V., Giesing, H., Morton, R., Kumar, A., Post, C., Brooks, C., Johnson, J. Energetics of quasi-equivalence: Computational analysis of protein-protein interactions in icosahedral viruses. Biophys. J. 74:546, 1998.

Schneemann, A., Reddy, V., Johnson, J. The structure and function of nodavirus particles: A paradigm for understanding chemical biology. Adv. Virus Res. 50:381, 1998.

Spall, V., Porta, C., Taylor, K., Lin, T., Johnson, J., Lomonossoff, G. Antigen expression on the surface of a plant virus for vaccine production. In: Engineering Crops for Industrial End Uses. Shewry, P.R. (Ed.). Portland Press, London, in press.

Stewart, M., Johnson. J. Structural cell biology: Functional integration in macromolecular assemblages [Editorial Overview]. Curr. Opin. Struct. Biol. 8:139, 1998.

Wikoff, W., Duda, R., Hendrix, R., Johnson, J. Crystallization and preliminary x-ray analysis of the dsDNA bacteriophage HK97 mature empty capsid. Virology 243:113, 1998.

Zlotnick, A., Natarajan, P., Munshi, S., Johnson, J. Resolution of space group ambiguity and the structure determination of nodamura virus to 3.3-Å resolution from pseudo R32 (monoclinic) crsytals. Acta Crystallogr. D 53:738, 1997.

 

 







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