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Scientific Report 2007


Molecular Biology




Structure, Function, and Applications of Virus Particles


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

We investigate model virus systems that provide insights for understanding viral 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 x-ray diffraction, 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 the 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 B. Carragher, M.G. Finn, M. Manchester, D.R. Millar, 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 bacteriophage 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. The late stage of maturation is a classic Brownian ratchet in which pentameric subunits fluctuate like a piston through a radial trajectory of 15 Å and are trapped at the top of the trajectory by formation of the covalent cross-link. If the cross-link can not form, the maturation stops with the pentamers still sampling the trajectory.

Bacteriophage P22 is the prototype of the Podoviridae that 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. Recently, structures of bacteriophage λ were determined at subnanometer resolution by electron cryomicroscopy. These structures showed that the fold of the capsid protein is the same as that of the HK97 subunit.

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. The x-ray structure of the major capsid protein of the virus revealed a fold nearly identical to the folds of the major capsid proteins of the eukaryotic adenoviruses and PRD-1, a virus that infects bacteria. These findings indicate a viral 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 show 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 tetra-cysteine 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. Tomographs prepared with the micrographs show that translation of the RNA encoding the capsid protein and the assembly of virions takes place within chambers created by remodeled mitochondria.

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

Publications

Cheung, C.L., Chung, S.W., Chatterji, A., Lin, T., Johnson, J.E., Hok, S., Perkins, J., De Yoreo, J.J. Physical controls on directed virus assembly at nanoscale chemical templates. J. Am. Chem. Soc. 128:10801, 2006.

Gan, L., Speir, J.A., Conway, J.F., Lander, G., Cheng, N., Firek, B.A., Hendrix, R.W., Duda, R.L., Liljas, L., Johnson, J.E. Capsid conformational sampling in HK97 maturation visualized by x-ray crystallography and cryo-EM. Structure 14:1655, 2006.

Johnson, J.E., Chiu, W. DNA packaging and delivery machines in tailed bacteriophages. Curr. Opin. Struct. Biol. 17:237, 2007.

Maia, L.F., Soares, M.R., Valente, A.P., Almeida, F.C., Oliveira, A.C., Gomes, A.M., Freitas, M.S., Schneemann, A., Johnson, J.E., Silva, J.L. Structure of a membrane-binding domain from a non-enveloped animal virus: insights into the mechanism of membrane permeability and cellular entry. J. Biol. Chem. 281:29278, 2006.

Martin, B.D., Soto, C.M., Blum, A.S., Sapsford, K.E., Whitley, J.L., Johnson, J.E., Chatterji, A., Ratna, B.R. An engineered virus as a bright fluorescent tag and scaffold for cargo proteins: capture and transport by gliding microtubules. J. Nanosci. Nanotechnol. 6:2451, 2006.

Poliakov, A., van Duijn, E., Lander, G., Fu, C.Y., Johnson, J.E., Prevelige, P.E., Jr., Heck, A.J. Macromolecular mass spectrometry and electron microscopy as complementary tools for investigation of the heterogeneity of bacteriophage portal assemblies. J. Struct. Biol. 157:371, 2007.

 

John E. Johnson, Ph.D.
Professor



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