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Scientific Report 2004

Molecular Biology

Structure, Function, and Applications of Virus Particles

J.E. Johnson, B. Bothner, A. Chatterji, W. Fernandez-Ochoa, L. Gan, I. Gertsman, R. Khayat, M.-J. Kim, G. Lander, J. Lanman, K. Lee, P. Natarajan, J. Speir, J. Tang, L. Tang, V. Volpetti, H. Walukiewicz, E. Wu

For humans, viruses are two-edged swords. Many of these microorganisms are dangerous pathogens (e.g., HIV and the coronavirus associated with severe acute respiratory syndrome) that 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, fish, 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 Scripps Research, 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.

Double-stranded DNA Viruses

HK97 is a double-stranded DNA bacterial virus similar to phage λ. 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. Four 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 seen in the armor of medieval knights. Three years ago, we created a model of the procapsid on the basis of a 12-Å electron cryomicroscopy structure and the subunits defined in the head. We investigated the transition by using time-resolved intrinsic fluorescence, circular dichroism, and solution scattering.

The data indicated 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 the structure of a mutant that is not cross-linked. The structure and the biochemical data indicate that the native quaternary structure depends on the covalent cross-link for formation. DNA-packaging studies of bacteriophage have progressed, with crystallographic and electron cryomicroscopy studies of the portal complex from bacteriophage P22 (Fig. 1).

Fig. 1. The structure of the portal complex from bacteriophage P22 at 8-Å resolution by electron cryomicroscopy. Double-stranded DNA bacteriophages and herpesviruses use similar mechanisms for DNA packaging. Central to this packaging is the portal complex located at a unique vertex of the virus, where it serves as a channel through which DNA enters. The P22 portal complex is the largest one reported so far, a dodecameric ring with a mass of approximately 1 million daltons.

Sulfolobus turreted icosahedral 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 was sequenced by our collaborator M. Young, Montana State University, Bozeman, Montana. Using electron cryomicroscopy reconstruction of the virus, we found that the capsid has pseudo T = 31 quasi-symmetry and is 1000 Å in diameter, including the gold pentons. 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. Recently, we obtained crystals of the isolated subunit and a structure determination is under way.

Single-Stranded RNA Viruses

Nudaurelia capensis ω virus and helicoverpa armigera stunt virus are single-stranded RNA viruses with T = 4 (240 subunits) capsids that infect Lepidoptera. 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 480 Å at pH 7 and 410 Å at pH 5. Electron cryomicroscopy reconstructions were done on both forms and on 2 intermediate structures for nudaurelia capensis ω virus. We found that mutations in the internal helical domain dramatically affect assembly and morphology. A pH-dependent helix-coil transition may be the driving force for the particle dynamics. The particles are nanomachines with potential as sensors and as “engines” for driving other nanodevices.

Cowpea mosaic virus is a pH-driven 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.


Brumfield, S., Willits, D., Tang, L., Johnson, J.E., Douglas, T., Young, M.J. Heterologous expression of the modified coat protein of cowpea chlorotic mottle bromovirus results in the assembly of protein cages with altered architectures and function. J. Gen. Virol. 85(Pt. 4):1049, 2004.

Chatterji, A., Ochoa, W., Paine, M., Ratna, B., Johnson, J.E., Lin, T. New addresses on an addressable virus nanoblock: uniquely reactive Lys residues on cowpea mosaic virus. Chem. Biol., in press.

Chatterji, A., Ochoa, W., Shamieh, L., Salakian, S.P., Wong, S.M., Clinton, G., Ghosh, P., Lin, T., Johnson, J.E. Chemical conjugation of heterologous proteins on the surface of cowpea mosaic virus. Bioconjug. Chem. 15:807, 2004.

Fourme, R., Ascone, I., Kahn, R., Girard, E., Mezouar, M., Lin, T., Johnson, J.E. New trends in macromolecular crystallography at high hydrostatic pressure. In: Advances in High Pressure Bioscience and Biotechnology II: Proceedings of the 2nd International Conference on High Pressure Bioscience and Biotechnology, Dortmund, September 16-19, 2002. Winter, R. (Ed.). Springer Verlag, New York, 2003, p. 161.

Fourme, R., Girard, E., Kahn, R., Ascone, I., Mezouar, M., Dhaussy, A.C., Lin, T., Johnson, J.E. Using a quasi-parallel x-ray beam of ultrashort wavelength for high pressure virus crystallography: implications for standard macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 59(Pt.10):1767, 2003.

Fourme, R., Girard, E., Kahn, R., Ascone, I., Mezouar, M., Lin, T., Johnson, J.E. State of the art and prospects of macromolecular crystallography at high hydrostatic pressure. In: High Pressure Crystallography. Katrusiak, A., McMillan, P. (Eds.). Kluwer Academic, Dordrecht, the Netherlands, 2004, p. 527. NATO Science Series, II: Mathematics, Physics, and Chemistry, Vol. 140.

Gan, L., Conway, J.F., Firek, B.A., Cheng, N., Hendrix, R.W., Steven, A.C., Johnson, J.E., Duda, R.L. Control of crosslinking by quaternary structure changes during bacteriophage HK97 maturation. Mol. Cell 14:559, 2004.

Helgstrand, C., Munshi, S., Johnson, J.E., Liljas, L. The refined structure of nudaurelia capensis ω virus reveals control elements for a T = 4 capsid maturation. Virology 318:192, 2004.

Helgstrand, C., Wikoff, W.R., Duda, R.L., Hendrix, R.W., Johnson, J.E., Liljas, L. The refined structure of a protein catenane: the HK97 bacteriophage capsid at 3.44 Å resolution. J. Mol. Biol. 334:885, 2003.

Johnson, J.E. An atomic model of a plant reovirus: rice dwarf virus. Structure (Camb.) 11:1193, 2003.

Johnson, J.E. Virus assembly and maturation. In: Folding and Self-Assembly of Biological Macromolecules: Proceedings of the Deuxièmes Entretiens de Bures, Bures-sur-Yvette, France, 27 November-1 December 2001. Westhoff, E., Hardy, N. (Eds.). World Scientific, Hackensack, NJ, 2003, p. 349.

Johnson, J.E. Virus particle dynamics. Adv. Protein Chem. 64:197, 2003.

Johnson, J.E. Virus structure analysis with synchrotron radiation: methods and results. J. Synchrotron Radiat. 11(Pt. 1):89, 2004.

Lee, K.K., Gan, L., Tsuruta, H., Hendrix, R.W., Duda, R.L., Johnson, J.E. Evidence that a local refolding event triggers maturation of HK97 bacteriophage capsid. J. Mol. Biol. 340:419, 2004.

Lee, K.K., Johnson, J.E. Complementary approaches to structure determination of icosahedral viruses. Curr. Opin. Struct. Biol. 13:558, 2003.

Lee, K.K., Tang, J., Taylor, D., Bothner, B., Johnson, J.E. Small compounds targeted to subunit interfaces arrest maturation in a nonenveloped, icosahedral animal virus. J. Virol. 78:7208, 2004.

Lin, T., Cavarelli, J., Johnson, J.E. Evidence for assembly-dependent folding of protein and RNA in an icosahedral virus. Virology 314:26, 2003.

Lin, T., Johnson, J.E. Structures of picorna-like plant viruses: implications and applications. Adv. Virus Res. 62:167, 2003.

Raja, K.S., Wang, Q., Gonzalez, M.J., Manchester, M., Johnson, J.E., Finn, M.G. Hybrid virus-polymer materials, 1: synthesis and properties of PEG-decorated cowpea mosaic virus. Biomacromolecules 4:472, 2003.

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

Rice, G., Tang, L., Stedman, K., Roberto, F., Spuhler, J., Gillitzer, E., Johnson, J.E., Douglas, T., Young, M.J. The structure of a thermophilic archeal virus shows a doubled-stranded DNA viral capsid type that spans all domains of life. Proc. Natl. Acad. Sci. U. S. A. 101:7716, 2004.

Taylor, D.J., Wang, Q., Bothner, B., Natarajan, P., Finn, M.G., Johnson, J.E. Correlation of chemical reactivity of nudaurelia capensis ω virus with a pH-induced conformational change. Chem. Commun. (Camb.) 2770, 2003, Issue 22.

Tihova, M., Dryden, K.A., Le, T.V., Harvey, S.C., Johnson, J.E., Yeager, M., Schneemann, A. Nodavirus coat protein imposes dodecahedral RNA structure independent of nucleotide sequence and length. J. Virol. 78:2897, 2004.

Wikoff, W.R., Che, Z., Duda, R.L., Hendrix, R.W., Johnson, J.E. Crystallization and preliminary analysis of a dsDNA bacteriophage capsid intermediate: prohead II of HK97. Acta Crystallogr. D Biol. Crystallogr. 59(Pt. 12):2060, 2003.

Yin, Z., Zheng, Y., Doerschuk, P.C., Natarajan, P., Johnson, J.E. A statistical approach to computer processing of cryo-electron microscope images: virion classification and 3-D reconstruction. J. Struct. Biol. 144:24, 2003.


John E. Johnson, Ph.D.

Johnson Web Site