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
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
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.
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,
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.
An atomic model of a plant reovirus: rice dwarf virus. Structure (Camb.) 11:1193, 2003.
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.
Virus particle dynamics. Adv. Protein Chem. 64:197, 2003.
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,
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,
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,
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
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
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.