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TSRI Scientific Report 2003

Structure, Function, and Applications of Virus Particles

J.E. Johnson, B. Bothner, A. Chatterji, G. Cingolani, L. Gan, M.-J. Kim, K. Lee, P. Natarajan, W. Fernandez-Ochoa, M. Paine, J. Tang, L. Tang, D. Taylor, H. Walukiewicz

For humans, viruses are two-edged swords. Many of these microorganisms are dangerous pathogens (e.g., HIV and the virus that causes severe acute respiratory syndrome) and cause extraordinary suffering, mortality, and economic loss. However, viruses benign to humans provide the means for understanding numerous biological processes that, by analogy, lead to an understanding of cellular functions crucial for the development of therapies for disease.

We investigate model virus systems that continue to provide insights for understanding the entry, localization, and replication of nonenveloped viruses. More recently, some of the benign viruses were recognized as practical reagents for applications in nanotechnology, chemistry, and biology. Using viral capsids as reagents, we discovered novel and, in some instances, unique properties of macromolecular organizations and dynamics. We investigate viruses that infect bacteria, insects, yeast, plants, and, recently, the extreme thermophile Sulfolobus. These viruses have genomes of single-stranded RNA, double-stranded RNA, and double-stranded DNA. In many instances, we use viruslike particles that do not contain infectious genomes.

We use a variety of physical methods to investigate structure-function relationships, including single-crystal and static and time-resolved solution x-ray diffraction, electron cryomicroscopy and image reconstruction, mass spectrometry, structure-based computational analyses, and methods associated with thermodynamic characterization of virus particles and their transitions. Biological methods we use include genetic engineering of viral genes and their expression in Escherichia coli, mammalian cells, insect cells, and yeast and the characterization of these gene products by the physical methods mentioned previously. For cytologic studies of viral entry and infection, we use fluorescence and electron microscopy and particles assembled in heterologous expression systems. Our studies depend on extensive consultations and collaborations with others at TSRI, including the groups led by C.L. Brooks, D.A. Case, B. Carragher, M.G. Finn, M.R. Ghadiri, T. Lin, M. Manchester, D.P. Millar, R.A. Milligan, C. Potter, V. Reddy, A. Schneemann, G. Siuzdak, K.F. Sullivan, and M. Yeager, and a variety of groups outside Scripps.

Doubled-Stranded DNA Viruses

HK97 is a double-stranded DNA bacterial virus similar to phage l. It undergoes a remarkable morphogenesis in its assembly and maturation, and this process can be recapitulated in vitro. The most dramatic event is the expansion from prohead (~500 Å in size) to head (~650 Å) in which both particles have identical protein composition. Two years ago, we determined the atomic resolution structure of the mature particle and discovered the mechanism used to concatenate the subunits of the particle into a chain-mail fabric similar to that used in the armor of medieval knights. Last year, we created a model of the procapsid on the basis of a 12-Å electron cryomicroscopy structure and the subunits defined in the head. Currently, we are using time-resolved intrinsic fluorescence, circular dichroism, and solution scattering to investigate the transition.

The data correlate remarkably well and indicate that a highly pH-sensitive early event (<10 minutes) is followed by a slow (hours) pH-independent late event. We also determined the kinetics of the subunit autoligation and showed that cross-linking begins earlier than expected, at the start of the second stage, but that the final cross-links can not occur until the particle is fully mature.

Yellowstone virus was isolated from the extreme thermophile (90°C, pH 2) Sulfolobus, which grows in the sulfur springs of Yellowstone National Park. The virus was isolated and its double-stranded DNA genome sequenced by our collaborator M. Young, Montana State University, Bozeman, Montana. Figure 1 shows the electron cryomicroscopy reconstruction of the virus we determined. The pseudo T = 31 capsid, including the pentons, is 1000 Å in diameter. The trimeric nature of the hexons strongly indicates that this virus is related to the human adenovirus, the PRD1 bacteriophage, and a virus that infects algae. So far as we know, Yellowstone virus is the first example of viruses with related architectures that infect all domains of life.

Single-Stranded RNA Viruses

Nudaurelia capensis w virus and helicoverpa armigera stunt virus are single-stranded RNA viruses with T = 4 (240 subunits) capsids that infect lepidopterans. We expressed the genes for both capsid proteins in the baculovirus system and assembled large quantities of particles that exist in 2 pH-dependent morphologies. The diameter of the particle is 450 Å at pH 7 and 410 Å at pH 5. Electron cryomicroscopy reconstructions have been done on both forms and on 2 intermediate structures for nudaurelia capensis w virus. We found that mutations in the internal helical domain dramatically affect assembly and morphology. A pH-dependent helix-coil transition seems to be the driving force for the particle dynamics. The particles are nanomachines with potential use as sensors and as "engines" for other nanodevices.

Cowpea mosaic virus was developed as a reagent for chemistry and nanotechnology. In collaboration with T. Lin, Department of Molecular Biology, and M.G. Finn, Department of Chemistry, we generated and produced a large variety of viable mutations of the virus in gram quantities for nanopatterning, molecular electronic scaffolds, and platforms for novel chemistry.


Chatterji, A., Burns, L.L., Taylor, S.S., Lomonossoff, G.P., Johnson, J.E., Lin, T., Porta, C. Cowpea mosaic virus: from the presentation of antigenic peptides to the display of active biomaterials. Intervirology 45:362, 2002.

Cheung, C.L., Camarero, J.A., Woods, B.W., Lin, T., Johnson, J.E., De Yoreo, J.J. Fabrication of assembled virus nanostructures on templates of chemoselective linkers formed by scanning probe nanolithography. J. Am. Chem. Soc. 125:6848, 2003.

Cingolani, G., Moore, S.D., Previlege, P.E., Jr., Johnson, J.E. Preliminary crystallographic analysis of the bacteriophage P22 portal protein. J. Struct. Biol. 139:46, 2002.

Da Poian, A.T., Johnson, J.E., Silva, J.L. Protein-RNA interactions and virus stability as probed by the dynamics of tryptophan side chains. J. Biol. Chem. 277:47596, 2002.

Damodaran, K.V., Reddy, V.S., Johnson, J.E., Brooks, C.L. III. A general method to quantify quasi-equivalence in icosahedral viruses. J. Mol. Biol. 324:723, 2002.

Fourme, R., Ascone, I., Kahn, R., Girard, E., Mezoua, M., Lin, T., Johnson, J. New trends in macromolecular crystallography at high hydrostatic pressure. In: International Conference on High Pressure Bioscience and Biotechnology. Winter, R. (Ed.). Springer Verlag, New York, in press.

Fourme, R., Ascone, I., Kahn, R., Mezouar, M., Bouvier, P., Girard, E., Lin, T., Johnson, J.E. Opening the high-pressure domain beyond 2 kbar to protein and virus crystallography. Structure (Camb.) 10:1409, 2002.

Johnson, J.E. Virus assembly and maturation. In: Folding and Self-Assembly of Macromolecules: Proceedings of the Deuxièmes Entretiens de Bures, Bures-sur-Yvette, France, 27 November-1 December 2001. Westhof, E., Hardy, N. (Eds.). World Scientific, River Edge, NJ, in press.

Johnson, J.E. Virus particle dynamics. Adv. Protein Chem., in press.

Liu, H., Qu, C., Johnson, J.E., Case, D.A. Pseudo-atomic models of swollen CCMV from cryo-electron microscopy data. J. Struct. Biol. 142:356, 2003.

Naitow, H., Tang, J., Canady, M., Wickner, R.B., Johnson, J.E. L-A virus at 3.4 Å resolution reveals particle architecture and mRNA decapping mechanism. Nat. Struct. Biol. 9:725, 2002.

Reddy, V., Schneemann, A., Johnson, J.E. Nodavirus endopeptidase. In: Handbook of Proteolytic Enzymes, 2nd ed. Barrett, A.J., Rawlings, N.D., Woessner, J.F. (Eds.). Academic Press, San Diego, in press.

Smith, J.C., Lee, K.-B., Wang, Q., Finn, M.G., Johnson, J.E., Mrksich, M., Mirkin, C.A. Nanopatterning the chemospecific immobilization of cowpea mosaic virus capsid. Nano Lett. 3:883, 2003.

Tang, L., Johnson, J.E. Structural biology of viruses by the combination of electron cryomicroscopy and x-ray crystallography. Biochemistry 41:11517, 2002.

Taylor, D.J., Krishna, N.K., Canady, M.A., Schneemann, A., Johnson, J.E. Large-scale, pH-dependent, quaternary structure changes in an RNA virus capsid are reversible in the absence of subunit autoproteolysis. J. Virol. 76:9972, 2002.

Wang, Q., Kaltgrad, E., Lin, T., Johnson, J.E., Finn, M.G. Natural supramolecular building blocks: wild-type cowpea mosaic virus. Chem. Biol. 9:805, 2002.

Wang, Q., Lin, T., Johnson, J.E., Finn, M.G. Natural supramolecular building blocks: cysteine-added mutants of cowpea mosaic virus. Chem. Biol. 9:813, 2002.

Willits, D., Zhao, X., Olson, N., Baker, T.S., Zlotnick, A., Johnson, J.E., Douglas, T., Young, M.J. Effects of the cowpea chlorotic mottle bromovirus ß-hexamer structure on virion assembly. Virology 306:280, 2003.



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