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Scientific Report 2006

Molecular Biology

Structure, Function, and Applications of Virus Particles

J.E. Johnson, M. Banerjee, A. Chatterji, Z. Chen, I. Gertsman, R. Huang, R. Khayat, G. Lander, J. Lanman, K.K. Lee, T. Matsui, P. Natarajan, A. Odegard, J. Speir

We investigate model virus systems that provide insights for understanding assembly, maturation, entry, localization, and replication. We have also developed viruses as reagents for applications in nanomedicine, chemistry, and biology. We investigate viruses that infect bacteria, insects, plants, and the extreme thermophile Sulfolobus. These viruses have genomes of single-stranded RNA and double-stranded DNA.

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 physical methods. 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 groups led by C.L. Brooks, D.A. Case, B. Carragher, M.G. Finn, M. Manchester, D.R. Millar, R.A. Milligan, C. Potter, V. Reddy, A. Schneemann, G. Siuzdak, J.R. Williamson, and M.J. Yeager, and a variety of groups outside of Scripps.

Double-Stranded DNA Viruses

HK97 is a double-stranded DNA virus similar to phage λ. It undergoes a remarkable morphogenesis in its assembly and maturation, and this process can be recapitulated in vitro. We determined the atomic resolution structure of the 650-Å mature, head II particle and discovered the mechanism used to concatenate the subunits of the particle into a chain-mail fabric similar to that seen in armor of medieval knights. In the past year, we focused on the dynamics of maturation.

Prohead II is a 500-Å metastable intermediate at pH 7 that can be induced to begin maturation by lowering the pH to 4. Solution x-ray scattering and single-molecule fluorescence showed that the initial transition to a particle of about 560 Å occurs as a highly cooperative, stochastic event with no detectable intermediates that takes place in less than 1 second for an individual particle. A quorum of cross-links must form in this particle to generate the second expansion intermediate (about 650 Å), which also forms cooperatively with no detectable intermediates. At pH 4, formation of cross-links continues, with 360 formed per particle. Limited pentamer dynamics (established from crystallography and electron cryomicroscopy) prevents the last 60 cross-links from forming, but pentamer trajectories extend at pH 7, allowing these cross-links to form, completing maturation.

Bacteriophage P22 is the prototype of the Podoviridae, which are characterized by a T = 7 capsid with a short tail structure incorporated into a unique 5-fold vertex. We determined an asymmetric reconstruction of this particle that revealed spooled DNA, the dodecameric portal, and the location of the 9 gene products known to be in the particle.

Sulfolobus turreted icosahedral virus is an archaeal virus isolated from Sulfolobus, which grows in the acidic hot sulfur springs (pH 2–4, 72°C–92°C) in Yellowstone National Park. An electron cryomicroscopy reconstruction of the virus showed that the capsid has pseudo T = 31 quasi symmetry and is 1000 Å in diameter, including the pentons. We solved the x-ray structure of the major capsid protein of the virus, and it revealed a fold nearly identical to the major capsid proteins of the eukaryotic adenoviruses and PRD-1, a virus that infects bacteria. These findings indicate a virus phylogeny that spans the 3 domains of life. Difference electron density maps in which the x-ray model is subtracted from the electron cryomicroscopy density clearly shows an internal membrane in which the capsid proteins are anchored.

Single-Stranded RNA Viruses

Flock House virus is a T = 3, single-stranded RNA virus that infects Drosophila. We are studying viral entry and early expression and assembly of the capsid protein. Recently, studies on viral entry indicated the presence of an “eluted” particle early in infection that has initiated its disassembly program but is then eluted back into the medium. We did a phenotypic characterization of the particles, and we are using electron cryomicroscopy to study them. For studies on the expression and assembly of the capsid protein, we are using tags inserted genetically in the capsid protein that allow the freshly made proteins to be optically visualized with a fluorophore and in the electron microscope with photoconversion of the fluorophore. Recently, high-pressure freezing of infected cells revealed exceptionally detailed features of viral entry and regions of replication within the cell.

Refined atomic models of tetravirus structures and structure-based mutagenesis combined with highly sensitive assays for defining phenotypes have revealed the electrostatic principals of maturation for the T = 4 tetraviruses.

Cowpea mosaic virus is a 30-nM reagent that we use for chemistry and nanomedicine. We found that particles of the virus with doxorubicin bound internally can be specifically targeted to tumor cells via peptides on the viral surface that recognize receptors for vascularization signals that are highly expressed on tumor cells.


du Plessis, L., Hendry, D.A., Dorrington, R.A., Hanzlik, T.N., Johnson, J.E., Appel, M. Revised RNA2 sequence of the tetravirus nudaurelia capensis ω virus (NωV): annotated sequence record. Arch. Virol. 150:2397, 2005.

Khayat, R., Tang, L., Larson, E.T., Lawrence, C.M., Young., M., Johnson, J.E. Structure of an archaeal virus capsid protein reveals a common ancestry to eukaryotic and bacterial viruses. Proc. Natl. Acad. Sci. U. S. A. 102:18944, 2005.

Lander, G.C., Tang, L., Casjens, S.R., Gilcrease, E.B., Prevelige, P., Poliakov, A., Potter, C.S., Carragher, B., Johnson, J.E. The structure of an infectious P22 virion shows the signal for headful DNA packaging. Science 312:1791, 2006.

Lee, K.K., Tsuruta, H., Hendrix, R.W., Duda, R.L., Johnson, J.E. Cooperative reorganization of a 420 subunit virus capsid. J. Mol. Biol. 352:723, 2005.

Lin, T., Lomonossoff, G.P., Johnson, J.E. Structure-based engineering of an icosahedral virus for nanomedicine and nanotechnology. In: Nanotechnology in Biology and Medicine: Methods, Devices, and Applications. Vo-Dinh. T. (Ed.). CRC Press, Boca Raton, FL, in press.

Medintz, I.L., Sapsford, K.E., Konnert, J.H., Chatterji, A., Lin, T., Johnson, J.E., Mattoussi, H. Decoration of discretely immobilized cowpea mosaic virus with luminescent quantum dots. Langmuir 21:5501, 2005.

Natarajan, P., Lander, G.C., Shepherd, C.M., Reddy, V.S., Brooks, C.L. III, Johnson, J.E. Exploring icosahedral virus structures with VIPER. Nat. Rev. Microbiol. 3:809, 2005.

Ochoa, W., Chatterji, A., Lin, T., Johnson, J.E. Generation and structural analysis of reactive empty particles derived from an icosahedral virus. Chem. Biol. 13:771, 2006.

Prasad, T., Turner, M., Falkner, J., Mittleman, D., Johnson, J.E., Lin, T., Colvin, V. Nanostructured virus crystals for x-ray optics. IEEE Trans. Nanotechnol. 5:93, 2006.

Reddy, V.S., Johnson, J.E. Structure-derived insights into virus assembly. Adv. Virus Res. 64:45, 2005.

Sapsford, K.E., Soto, C.M., Blum, A.S., Chatterji, A., Lin, T., Johnson, J.E., Ligler, F.S., Ratna, B.R. A cowpea mosaic virus nanoscaffold for multiplexed antibody conjugation: application as an immunoassay tracer. Biosens. Bioelectron. 21:1668, 2006.

Shepherd, C.M., Borelli, I.A., Lander, G., Natarajan, P., Siddavanahalli, V., Bajaj, C., Johnson, J.E., Brooks, C.L. III, Reddy, V.S. VIPERdb: a relational database for structural virology. Nucleic Acids Res. 34:386, 2006.

Soto, C.M., Blum, A.S., Vora, G.J., Lebedev, N., Meador, C.E., Won, A.P., Chatterji, A., Johnson, J.E., Ratna, B.R. Fluorescent signal amplification of carbocyanine dyes using engineered viral nanoparticles. J. Am. Chem. Soc. 128:5184, 2006.

Speir, J.A., Bothner, B., Qu, C., Willits, D.A., Young, M.J., Johnson, J.E. Enhanced local symmetry interactions globally stabilize a mutant virus capsid that maintains infectivity and capsid dynamics J. Virol. 80:3582, 2006.

Tang, J., Johnson, J.M., Dryden, K.A., Young, M.J., Zlotnick, A., Johnson, J.E. The role of subunit hinges and molecular “switches” in the control of viral capsid polymorphism. J. Struct. Biol. 154:59, 2006.

Tang, L., Gilcrease, E.B., Casjens, S.R., Johnson, J.E. Highly discriminatory binding of capsid-cementing proteins in bacteriophage L. Structure 14:837, 2006.

Taylor, D.J., Speir, J.A., Reddy, V., Cingolani, G., Pringle, F.M., Ball, L.A., Johnson, J.E. Preliminary x-ray characterization of authentic providence virus and attempts to express its coat protein gene in recombinant baculovirus. Arch. Virol. 151:155, 2006.

Walukiewicz, H.E., Johnson, J.E., Schneemann, A. Morphological changes in the T = 3 capsid of Flock House virus during cell entry. J. Virol. 80:615, 2006.

Wikoff, W.R., Conway, J.F., Tang, J., Lee, K.K., Gan, L., Cheng, N., Duda, R.L., Hendrix, R.W., Steven, A.C., Johnson, J.E. Time-resolved molecular dynamics of HK97 capsid maturation interpreted by electron cryo-microscopy and x-ray crystallography. J. Struct. Biol. 153:300, 2006.


John E. Johnson, Ph.D.

Molecular Biology Reports

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