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Scientific Report 2008

Molecular Biology

Structure, Function, and Applications of Virus Particles

J.E. Johnson, M. Banerjee, C.-Y. Fu, I. Gertsman, R. Huang, R. Khayat, G. Lander, J. Lanman, K.K. Lee, T. Matsui, A. Odegard, J. Speir, R. Taurog

We 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, nanochemistry, and nanobiology. 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 the particles' 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 the groups led by B. Carragher, M.G. Finn, M. Manchester, R.A. Milligan, C. Potter, V. Reddy, A. Schneemann, G. Siuzdak, and J.R. Williamson, 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 structure similar to that seen in armor of medieval knights.

In the past year, we focused on the structures of prohead I and prohead II, the first and second intermediates in the assembly pathway. The prohead II structure is at 3.7-Å resolution, and the subunit fold and the location of many side chains have been determined. The tertiary structure of the subunit in prohead II differs from that of the subunit in head II, an unexpected result. At lower resolution, the transition from prohead II to head II appeared to be rigid body motions. It is now clear that contacts near the 3-fold particle axes are fixed and that a dramatic change occurs in the subunit structure, with a twist about 3 β-strands and the bending of a long helix, although the domains remain largely rigid. The change in tertiary structure may be the energy storage mechanism that propels the maturation of the particle.

We used electron cryomicroscopy to study bacteriophage λ in the prohead and head states. The crystal structure of the HK97 bacteriophage capsid fits most of the T = 7 λ particle density with only minor adjustment. A prominent surface feature at the 3-fold axes corresponds to the cementing protein glycoprotein D, necessary for stabilization of the capsid shell. The position of the glycoprotein coincides with the location of the covalent cross-link formed in the docked HK97 crystal structure, suggesting an evolutionary replacement of this gene product in bacteriophage λ by autocatalytic chemistry in HK97.

Single-Stranded RNA Viruses

Flock House virus is a T = 3, single-stranded RNA virus that infects Drosophila. Infectivity of Flock House virus requires the autocatalytic cleavage of the capsid protein at residue 363, liberating the C-terminal 44-residue γ peptides that remain associated with the particle. In vitro studies indicated that the amphipathic-helical part (residues 364—385) is membrane active, suggesting its role in RNA membrane translocation during infection. We have now shown that a maturation-defective mutant of Flock House virus can be rescued by viruslike particles that lack the genome but undergo maturation cleavage in a baculovirus expression system. We propose that colocalization of the 2 defective particle types in an entry compartment allows the rescue by γ peptides.

We used time-resolved electron cryomicroscopy for structural studies of the T = 4 tetravirus Nudaurelia capensis ω virus. We found that a large-scale structural change induced by lowering the pH from 7 to 5 occurs in less than 100 milliseconds, but the annealing of the polypeptide chains to form active autocatalytic sites varies dramatically with the position of the subunit in the surface lattice. Subunits adjacent to 5- and 3-fold axes form active sites in less than 3 minutes, whereas the other 2 quasi-equivalent subunits are much slower. One of the latter subunits forms active sites in 30 minutes; the other requires more than 2 hours. These data explain well the unusual kinetics of the cleavage reaction.


Banerjee, M., Johnson, J.E. Activation, exposure and penetration of virally encoded, membrane-active, polypeptides during non-enveloped virus entry. Curr. Protein Pept. Sci. 9:16, 2008.

Gan, L., Johnson, J.E. An optimal exposure strategy for cryoprotected virus crystals with lattice constants greater than 1000 Å. J. Synchrotron Radiat. 15:223, 2008.

Johnson, J.E. Multi-disciplinary studies of viruses: the role of structure in shaping the questions and answers. J. Struct. Biol., in press.

Johnson, J.E., Chiu, W. DNA packaging and delivery machines in tailed bacteriophages. Curr. Opin. Struct. Biol. 17:237, 2007.

Johnson, J.E., Speir, J.A. Principles of virus structure. In: Encyclopedia of Virology, 3rd ed. Mahy, B.W.J., van Reganmortel, M.H.V. (Eds.). Academic Press/Elsevier, New York, 2008, Vol. 5, p. 393.

Kang, S., Lander, G., Johnson, J.E., Prevelige, P.E. Development of bacteriophage P22 as a platform for molecular display: genetic and chemical modifications of the procapsid exterior surface. Chembiochem 9:514, 2008.

Lanman, J., Crum, J., Deerinck, T.J., Gaietta, J.M., Schneemann, A., Sosinsky, G., Ellisman, M.H., Johnson, J.E. Visualizing Flock House virus infection in Drosophila cells with correlated fluorescence and electron microscopy. J. Struct. Biol. 161:439, 2008.

Lee, J., Doerschuk, P.C., Johnson, J.E. Exact reduced-complexity maximum likelihood reconstruction of multiple 3-D objects from unlabeled unoriented 2-D projections and electron microscopy of viruses. IEEE Trans. Image Process. 16:2865, 2007.

Lee, K.K., Gan, L., Tsuruta, H.C.M., Conway, J.F., Duda, R.L., Hendrix, R.W., Steven, A.C., Johnson, J.E. Virus capsid expansion driven by the capture of mobile surface loops. Structure, in press.

Sosinsky, G.E., Crum, J., Jones, Y.Z., Lanman, J., Smarr, B., Terada, M., Martone, M.E., Deerinck, T.J., Johnson, J.E., Ellisman, M.H. The combination of chemical fixation procedures with high pressure freezing and freeze substitution preserves highly labile tissue ultrastructure for electron tomography applications. J. Struct. Biol. 161:359, 2008.

Speir, J.A., Johnson, J.E. Nonenveloped virus structure. In: Encyclopedia of Virology, 3rd ed. Mahy, B.W.J., van Reganmortel, M.H.V. (Eds.). Academic Press/Elsevier, New York, 2008, Vol. 5, p. 380.Speir, J.A., Johnson, J.E. Tetravirus structure. In: Encyclopedia of Virology, 3rd ed. Mahy, B.W.J., van Reganmortel, M.H.V. (Eds.). Academic Press/Elsevier, New York, 2008, Vol. 5, p. 27.

Steinmetz, N.F., Lin, T., Lomonossoff, G. P., Johnson, J.E. Structure-based engineering of an icosahedral virus for nanomedicine and nanotechnology. In: Viruses as Nanomaterials for Biomedicine and Bioengineering. Manchester, M., Steinmetz, N.F. (Eds.). Springer, New York, in press.

Szymczyna, B.R., Gan, L., Johnson, J.E., Williamson, J.R. Solution NMR studies of the maturation intermediates of a 13 MDa viral capsid. J. Am. Chem. Soc. 129:7867, 2007.

Walukiewicz, H.E., Banerjee, M., Schneemann, A., Johnson, J.E. Rescue of maturation-defective Flock House virus infectivity with noninfectious, mature, viruslike particles. J. Virol. 82:2025, 2008.

Wickner, R.B., Tang, J., Gardner, N., Johnson, J.E. The yeast double-stranded RNA virus L-A resembles mammalian dsRNA virus cores. In: Segmented Double-Stranded RNA Viruses: Structure and Molecular Biology. Patton, J.T. (Ed.). Caister Academic Press, Portland, OR, 2007, p. 105.


John E. Johnson, Ph.D.

Molecular Biology Reports

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