Scientific Report 2005
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.
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. 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.
turreted icosahedral virus is an archaeal virus isolated from Sulfolobus,
which grows in the acidic hot sulfur springs (pH 24, 72°C92°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
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.
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.
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.
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.<
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.
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.
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.
A., Ochoa, W.F., Ueno, T., Lin, T., Johnson, J.E.
A virus-based nanoblock with tunable electrostatic properties. Nano Lett. 5:597,
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.
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.
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.
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.
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.
I., Mattoussi, H., Sapsford, K., Chatterji, A., Johnson, J.E.
Decoration of discretely immobilized cowpea mosaic virus with luminescent quantum
dots. Langmuir, in press.
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.
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.
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.
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.
E., Johnson, J.E., Finn, M.G.
Natural supramolecular building blocks: icosahedral virus particles organized by
attached oligonucleotides. Nano Lett. 4:1385, 2004.
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.
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,
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,
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.