Scientific Report 2006

Molecular Biology

Biology and Applications of Icosahedral Virus Capsids

A. Schneemann, B. Groschel, C. Hsu, J. Lee, D.J. Manayani, D. Marshall, J.E. Petrillo, M.E. Siladi, P.A. Venter

Coat proteins of nonenveloped, icosahedral viruses perform multiple functions during the course of viral infection, including capsid assembly, specific encapsidation of the viral genome, binding to a cellular receptor, and uncoating. In some viruses, a single type of protein is sufficient to carry out these functions; we are interested in the determinants that endow a polypeptide chain with such versatility. We seek to harness this versatility for novel applications of viruses in biotechnology and nanotechnology.

We focus on a structurally and genetically well-characterized virus family, the T = 3 nodaviruses. Nodaviruses are composed of 180 copies of a single coat protein and 2 strands of positive-sense RNA. Currently, we are elucidating the mechanism by which the 2 genomic RNAs are packaged into a single virion. Our long-term goal is to develop nodaviruses as RNA packaging and delivery vectors. Our data indicate that the 2 viral RNAs are recognized separately, but it is not yet known whether packaging occurs sequentially and whether one or more coat protein subunits are involved in this process. Interestingly, we found that RNA genome packaging is coupled to genome replication, suggesting potential approaches for packaging of foreign RNAs.

In other studies, we are investigating the mechanism by which nodaviral protein B2 suppresses RNA silencing in infected cells. In collaboration with J.R. Williamson, Department of Molecular Biology, we showed that B2 binds to double-stranded RNA in a sequence-independent manner and that it interferes with cleavage of double-stranded RNA substrates by the cellular protein Dicer. Moreover, in collaboration with J.L. Imler, University of Strasbourg, Strasbourg, France, we showed that B2 is critical for nodaviral infection of Drosophila and that Dicer plays an essential role in host defense against nodaviruses in vivo.

We are also collaborating with several investigators at Scripps Research, the Salk Institute, and Harvard University to develop nodaviruses as platforms for delivery of anthrax antitoxins. To this end, we are using particles to display the VWA domain of capillary morphogenesis protein 2, the cellular receptor for anthrax toxin, in a multivalent fashion on the surface of the virion. Two insertion sites yielding different patterns of 180 copies of the VWA domain were selected on the basis of computational modeling of the high-resolution crystal structure of the insect nodavirus Flock House virus. The resulting chimeric viruslike particles functioned as a potent anthrax antitoxin in cell culture and protected rats from challenge with lethal toxin. This research is important because it shows that protein domains containing more than 150 amino acids can be displayed on Flock House virus in a biologically functional form, suggesting numerous additional applications.

Flock House virus particles are also good candidates for novel materials in nanotechnology applications. The particles are stable, easily manipulated, biocompatible, and nontoxic in vivo and can be produced easily and in high quantities. The high-resolution x-ray structure of the virus revealed the potential for using chemical approaches to attach ligands to the surface of the virus and for using genetic strategies to modify the capsid. In collaboration with M. Manchester, Department of Cell Biology, and M. Ozkan, University of California, Riverside, we used conjugation chemistry to couple inorganic nanotubes and quantum dots to Flock House virus particles to produce an array of novel hybrid structures. This approach may one day be used to fabricate unique materials for a variety of applications, including biofilms with tunable pore size, 3-dimensional scaffolds for production of nanoelectronic devices, and drug delivery.

Publications

Chao, J.A., Lee, J.H., Chapados, B.R., Debler, E.W., Schneemann, A., Williamson, J.R. Dual modes of RNA-silencing suppression by Flock House virus protein B2. Nat. Struct. Mol. Biol. 12:952, 2005.

Destito, G., Schneemann, A., Manchester, M. Biomedical nanotechnology using virus-based nanoparticles. Curr. Top. Microbiol. Immunol., in press.

Galiana-Arnoux, D., Dostert, C., Schneemann, A., Hoffmann, J.A., Imler, J.L. Essential function in vivo for Dicer-2 in host defense against RNA viruses in Drosophila. Nat. Immunol. 7:590, 2006.

Hsu, C., Singh, P., Ochoa, W., Manayani, D.J., Manchester, M., Schneemann, A., Reddy, V.S. Characterization of polymorphism displayed by the coat protein mutants of tomato bushy stunt virus. Virology 349:222, 2006.

Schneemann, A. The structural and functional role of RNA in icosahedral virus assembly. Annu. Rev. Microbiol. 60:51, 2006.

Singh, P., Destito, G., Schneemann, A., Manchester, M. Canine parvovirus-like particles, a novel nanomaterial for tumor targeting. J. Nanobiotechnol. 4:2, 2006.

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.