Scientific Report 2007
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
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
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.
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.
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
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.
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.
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.
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.
J.E., Chiu, W. DNA
packaging and delivery machines in tailed bacteriophages. Curr. Opin. Struct. Biol.
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,
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.
A., van Duijn, E., Lander, G., Fu, C.Y., Johnson, J.E., Prevelige, P.E., Jr., Heck,
mass spectrometry and electron microscopy as complementary tools for investigation
of the heterogeneity of bacteriophage portal assemblies. J. Struct. Biol. 157:371,