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Virus Structure and Function
J. Johnson, A. Schneemann, V. Reddy, T. Lin, W. Wikoff, P. Natarajan, J. Tate, F. Dong, M. Canady, C. Qu, Z. Che, L. Tang, H. Naitow, H. Giesing, H. Langedijk,* L. Liljas,** G. Lomonossoff***
* Institute for Animal Science and Health, Lelystad, the Netherlands
** Uppsala University, Uppsala, Sweden
*** John Innes Centre, Cambridge, England
Viruses are highly accessible subjects for investigating the properties of macromolecular assemblies. These microorganisms allow an understanding of structural biology greater than that usually achieved by the reductionist study of individual proteins, and they provide insights as to why "the whole is more than the sum of its parts." We investigate RNA viruses that infect plants and animals and double-stranded DNA viruses that infect bacteria (bacteriophages).
Many of the systems that we study can be isolated in different polymorphic forms that reflect the dynamic nature of the maturation from noninfectious provirions to infectious mature particles. Static methods, such as crystallography and electron cryo-microscopy, were used to image these different forms at resolutions of 20--2.5 Å. Dynamic methods, such as solution x-ray scattering, limited proteolysis combined with mass spectrometry, and computational simulations, were used to investigate dynamic features of the particles.
Recent studies done by us and in collaboration with groups at TSRI led by C. Brooks, D. Case, A. Schneemann, G. Siuzdak, and M. Yeager in the Department of Molecular Biology and R. Ghadiri in the Department of Chemistry revealed that even a simple virus is a complex organism. We determined the structural basis for a variety of functions encoded within a single gene product that are expressed through assembly of viral particles with 60, 120, 180, 240, or 420 copies of the product. All of the viruses have been investigated as authentic, infectious virions or as viruslike particles that assemble spontaneously in recombinant protein expression systems. The investigations described in the following sections allow a better understanding of viral evolution, an integrated view of the diverse mechanisms for gene packaging and delivery, and insights for the development of antiviral agents that may obstruct one or more steps in infection. Although the methods described have been applied to viral nucleoprotein particles, they have application to a broad range of macromolecular complexes. A long-term goal of our work is to develop the means of producing atomic-resolution models for cellular megastructures that may reveal features of function that are not obvious from the structures of the components of the megastructures.
RNA PLANT VIRUSES
Different polymorphic forms of bromoviridae were studied with electron cryo-microscopy, crystallography, and computational chemistry to develop atomic-resolution and pseudo--atomic-resolution models of icosahedral and bacilliform particles. A variety of chimeric viruses in which foreign polypeptides were genetically inserted in surface loops of cowpea mosaic virus were prepared, analyzed for biological activity, and studied by using crystallography. A link in the evolution of icosahedral capsids of plant and animal viruses in the picornavirus superfamily was established with the determination of the structure of tobacco ringspot virus. The connectivity of the nascent capsid polyprotein in picornaviruses was conclusively established by this study.
RNA ANIMAL VIRUSES
Studies on RNA animal viruses have focused on viruses that infect insectsbecause of the structural, genetic, and biological simplicity of these viruses and because they readily assemble as viruslike particles when the capsid protein is expressed in a baculovirus system. Despite this simplicity, physical and biological studies revealed that subtle complexity and high precision govern all levels of protein and RNA association. Although capsids with T = 3 symmetry readily form as viruslike particles with foreign RNA packaged, they are about 10 times more susceptible to protease digestion than are authentic particles. Surprisingly, these particles are indistinguishable from authentic virions by crystallography at 3.0-Å resolution.
Studies of T = 4 viruslike particles of nudaurelia capensis o virus revealed the first example of large-scale polymorphism in the maturation of a simple RNA virus. Expression in the baculovirus system allowed a normally transient assembly intermediate to be trapped, studied by electron cryo-microscopy, and modeled with subunits determined by crystallographic analysis of the mature particle. The 2.4-Å structure and genetic organization of a picornavirus-like insect virus that we recently studied indicated that RNA viruses can exchange functional genetic modules through recombination.
DOUBLE-STRANDED DNA VIRUSES
The structure of the capsid of a l-like phage called HK97 was determined at 3.6-Å resolution. The particle has T = 7l quasi symmetry, is 650 Å in diameter, and is stabilized by chemical cross-links, formed autocatalytically, between lysine and asparagine side chains that physically chain-link hexamers and pentamers in a form similar to the chain mail worn by medieval knights. The fold of the 282 residues is unique compared with the folds of other protein structures and has a nearly 2-dimensional appearance, with a radial thickness of 20 Å or less. The empty particle has the appearance of a molecular balloon. The chain mail results from a simple, but unexpected, mode of hexamer-pentamer interlinking across 2-fold symmetry axes. The structure should lead to mechanisms for the autocatalytic chemical cross-link, the extraordinary expansion that occurs during DNA packaging, and the refolding of the subunit that occurs during the maturation. Recently, crystals of the 450-Å diameter procapsid of the particle were produced; these diffract to a resolution of 6 Å.
Bothner, B., Schneemann, A., Marshall, D., Reddy, V., Johnson, J., Siuzdak, G. Crystallographically identical virus capsids display different properties in solution. Nat. Struct. Biol. 6:114, 1999.
Brennan, F., Jones, T., Gilleland, L., Bellaby, T., Xu, F., North, P., Thompson, A., Staczek, J., Lin, T., Johnson, J., Hamilton, W., Gilleland, H. Pseudomonas aeruginosa outer membrane protein F epitopes are highly immunogenic when expressed on a plant virus. Microbiology 145:211, 1999.
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. 72:6024, 1998.
Janshoff, A., Bong, D., Steinem, C., Johnson, J., Ghadiri, M. An animal derived peptide switches membrane morphology: Possible relevance to nodaviral transfection process. Biochemistry, in press.
Johnson, J., Reddy, V. Biggest virus molecular structure yet. Nat. Struct. Biol. 5:849, 1998.
Johnson, J., Schneemann, A. Nodavirus endopeptidase. In: Handbook of Proteolytic Enzymes. Barret, A., Rawlings, N., Woessner, J. (Eds.). Academic Press, San Diego, 1998, p. 964.
Johnson, J., Speir, J. (Eds.). Principles of Virus St ructure. Academic Press, San Diego, 1999.
Johnson, J., Wikoff, W. Macromolecular assembly: Chainmail stabilization of a viral capsid. Curr. Biol. 8:914, 1998.
Munshi, S., Liljas, L., Johnson, J. Structure determination of Nudaurelia capensis omega virus. Acta Crystallogr. D 54:1295, 1998.
Rapaport, D., Johnson, J., Skolnick, J. Supramolecular self-assembly: Molecular dynamics modeling of polyhedral shell formation. Comput. Phys. Commun., in press.
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., Napier, J., Davis, P. (Eds.). Portland Press, London, 1998, p. 35.
Tate, J., Liljas, L., Scotti, P., Christian, P., Lin, T., Johnson, J. The crystal structure of cricket paralysis virus provides the first view of a new virus family. Nat. Struct. Biol., in press.
Taylor, K., Porta, C., Lin, T., Johnson, J., Barker, P., Lomonossoff, G. Position-dependent processing of peptides presented on the surface of cowpea mosaic virus. Biol. Chem. 380:387, 1999.
Tsuruta, H., Reddy, V., Wikoff, W., Johnson, J. Imaging RNA and dynamic protein segments with low resolution virus crystallography: Experimental design, data processing and implications of electron density maps. J. Mol. Biol. 284:1439, 1998.
Wikoff, W., Duda, R., Hendrix, R., Johnson, J. Crystallographic analysis of the dsDNA bacteriophage HK97 mature empty capsid. Acta Crystallogr. D 55:763, 1999.
Wikoff, W., Johnson, J. Virus assembly: Imaging a molecular machine. Curr. Biol. 9:296, 1999.
Zheng, Y., Doerschuk, P., Johnson, J. Symmetry-constrained interpolation of viral x-ray crystallography data. IEEE Trans. Signal Process., in press.