Scientific Report 2006
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
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
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 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 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
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.
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,
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.