Scientific Report 2005

Molecular Biology

Structure, Function, and Applications of Virus Particles

J.E. Johnson, L. Basumallic, A. Chatterji, W. Fernandez-Ochoa, L. Gan, I. Gertsman, R. Khayat, J. Lanman, K. Lee, T. Matsui, P. Natarajan, A. Odegard, J. Speir, L. Tang,
H. Walukiewicz, E. Wu

We investigate model virus systems that provide insights for understanding assembly, maturation, entry, localization, and replication of nonenveloped viruses. We also have developed viruses as reagents for applications in nanotechnology, chemistry, and biology. 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 Scripps Research, including groups led by C.L. Brooks, D.A. Case, B. Carragher, M.G. Finn, M.R. Ghadiri, T. Lin, M. Manchester, D.R. Millar, R.A. Milligan, C. Potter, V. Reddy, A. Schneemann, G. Siuzdak, K.F. Sullivan, 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 bacterial 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 the armor of medieval knights. We created a model of the procapsid on the basis of the 5-Å electron cryomicroscopy structure in which the coordinates from the head II particle were readily fitted. Recently, we used single-value decomposition analysis of time-resolved solution x-ray scattering data and single-molecule fluorescence to show that the initial maturation of prohead II (~470 Å in diameter) to expansion intermediate I (546 Å in diameter) occurs as a highly cooperative, stochastic event with no significantly populated intermediates and takes less than 1 second for an individual particle.

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 previously determined the icosahedrally averaged structure of the capsid at 20-Å resolution, the 10-Å structure of the connector protein, and the 20-Å structure of the tail machine. Recently, we did a reconstruction of the virus without imposing symmetry, enabling us to visualize the detailed relationship of all these components (Fig. 1).

Fig. 1. Electron cryomicroscopy reconstruction of the bacteriophage P22. The reconstruction was done with 1800 particles and no applications of symmetry. The reconstructed density required first generating an icosahedrally averaged electron density that ignored the tail assembly and then inserting the tail assembly (determined as a separate reconstruction) into the icosahedral density to create a tailed phage model. The model was then back projected in all icosahedral orientations onto each individual particle, and the orientation that gave the highest correlation coefficient (i.e., aligned the tails) was used to reconstruct the final density. Note that without any application of symmetry, both the tail machine and the T = 7 surface lattice are clearly defined in the reconstructed density.

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 revealed a fold nearly identical to the major capsid proteins of the eukaryotic adenoviruses; PBCV-1, a virus that infects fresh water algae; and PRD-1, a virus that infects bacteria. These findings indicate a virus phylogeny that spans the 3 domains of life (Eucarya, Bacteria, and Archaea) and suggests that these viruses may be related to a virus that preceded the division of life into 3 domains more than 3 billion years ago.

Single-Stranded RNA Viruses

Flock House virus is a 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 genetically inserted 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.

Tetraviruses are single-stranded RNA viruses that infect Lepidoptera. Expression of the capsid protein in the baculovirus system leads to spontaneous assembly of viruslike particles that we can investigate in vitro. The particles exist as procapsids (480 Å) at pH 7 and as capsids (410 Å) at pH 5. We used limited proteolysis and mass spectrometry to investigate the driving force of the transition, the mechanism of an autocatalytic cleavage, and the dynamic features of both forms.

Cowpea mosaic virus is a 30-nM reagent that we use for chemistry and nanotechnology. In collaboration with T. Lin, Department of Molecular Biology, 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.

Publications

Blum, A.S., Soto, C.M. Wilson, C.D., Brower, T.L., Pollack, S.K., Schull, T.L., Chatterji, A., Lin, T., Johnson, J.E., Amsinck, C., Franzon, P., Shashidhar, R., Ratna, B.R. An engineered virus as a scaffold for three-dimensional self-assembly on the nanoscale. Small 1:702, 2005.<

Blum, A.S., Soto, C.M., Wilson, C.D., Cole, J.D., Kim, M., Gnade, B., Chatterji, A., Ochoa, W.F., Lin, T., Johnson, J., Ratna, B.R. Cowpea mosaic virus as a scaffold for 3-D patterning of gold nanoparticles. Nano Lett. 4:867, 2004.

Bothner, B., Taylor, D., Jun, B., Lee, K.K., Siuzdak, G., Schultz, C.P., Johnson, J.E. Maturation of a tetravirus capsid alters the dynamic properties and creates a metastable complex. Virology 334:17, 2005.

Chatterji, A., Ochoa, W.F., Paine, M., Ratna, B.R., Johnson, J.E., Lin, T. New addresses on an addressable virus nanoblock; uniquely reactive Lys residues on cowpea mosaic virus. Chem. Biol. 11:855, 2004.

Chatterji, A., Ochoa, W.F., Ueno, T., Lin, T., Johnson, J.E. A virus-based nanoblock with tunable electrostatic properties. Nano Lett. 5:597, 2005.

Falkner, J.C., Turner, M.E., Bosworth, J.K., Trentler, T.J., Johnson, J.E., Lin, T., Colvin, V.L. Virus crystals as nanocomposite scaffolds. J. Am. Chem. Soc. 127:5274, 2005.

Girard, E., Kahn, R., Mezouar, M., Dhaussy, A.C., Lin, T., Johnson, J.E., Fourme, R. The first crystal structure of a complex macromolecular assembly under high pressure: CpMV at 330 MPa. Biophys. J. 88:3562, 2005.

Johnson, K.N., Tang, L., Johnson, J.E., Ball, L.A. Heterologous RNA encapsidated in Pariacoto virus-like particles forms a dodecahedral cage similar to genomic RNA in wild-type virions. J. Virol. 78:11371, 2004.

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.

Lin, T., Schildkamp, W., Brister, K., Doerschuk, P.C., Somayazulu, M., Mao, H.K., Johnson, J.E. The mechanism of high pressure induced ordering in a macromolecular crystal. Acta Crystallogr. D Biol. Crystallogr. 61:737, 2005.

Medintz, I., Mattoussi, H., Sapsford, K., Chatterji, A., Johnson, J.E. Decoration of discretely immobilized cowpea mosaic virus with luminescent quantum dots. Langmuir, in press.

Natarajan, P., Lander, G., Shepherd, C., Reddy, V., Brooks, C.L. III, Johnson, J.E. Virus Particle Explorer (VIPER), a Web-based repository of virus structural data and derived information. Nat. Microbiol. Rev., in press.

Reddy, V., Schneemann, A., Johnson, J.E. Nodavirus endopeptidase. In: Handbook of Proteolytic Enzymes, 2nd ed. Barret, A.J., Rawlings, N.D., Woessner, J.F. (Eds.). Academic Press, San Diego, 2004, Vol. 2, p. 198.

Reddy, V.S., Natarajan, P., Lander, G., Qu, C., Brooks, C.L. III, Johnson, J.E. Virus Particle Explorer (VIPER): a repository of virus capsid structures. In: Conformational Proteomics of Macromolecular Architecture: Approaching the Structure of Large Molecular Assemblies and Their Mechanisms of Action. Cheng, R.H., Hammar, L. (Eds.). World Scientific, River Edge, NJ, 2004, p. 403.

Schwarcz, W.D., Barroso, S.P., Gomes, A.M., Johnson, J.E., Schneemann, A., Oliveira, A.C., Silva, J.L. Virus stability and protein-nucleic acid interaction as studied by high-pressure effects on nodaviruses. Cell. Mol. Biol. (Noisy-le-grand) 50:419, 2004.

Strable, E., Johnson, J.E., Finn, M.G. Natural supramolecular building blocks: icosahedral virus particles organized by attached oligonucleotides. Nano Lett. 4:1385, 2004.

Tang, J., Naitow, H., Gardner, N.A., Kolesar, A., Tang, L., Wickner, R.B., Johnson, J.E. The structural basis of recognition and removal of cellular mRNA 7-methyl G “caps” by a viral capsid protein: a unique viral response to host defense. J. Mol. Recognit. 18:158, 2005.

Tang, L., Marion, W.R., Cingolani, G., Prevelige, P.E., Johnson, J.E. The three-dimensional structure of the bacteriophage P22 tail machine. EMBO J. 24:2087, 2005.

Taylor, D.J., Johnson, J.E. Folding and particle assembly are disrupted by single-point mutations near the autocatalytic cleavage site of nudaurelia capensis 4 virus capsid protein. Protein Sci. 14:401, 2005.

Taylor, D.J., Speir, J., Reddy, V., Cingolani, G., Pringle, F., 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., in press.