Scientific Report 2005

Molecular Biology

Biology and Applications of Capsids of Icosahedral Viruses

A. Schneemann, B. Groschel, J. Lee, D. Manayani, M. 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 recently discovered that packaging of the RNA genome is directly coupled to replication of the genome, 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. Preliminary data indicate that protein B2 binds to double-stranded RNA and that it interferes with cleavage of double-stranded RNA substrates by the cellular protein Dicer.

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 protect cultured cells from the toxic effects of protective antigen and lethal factor, 2 of the 3 proteins that make up anthrax toxin. Experiments in animals are currently under way to show that these particles also function as antitoxins in vivo. This research is important because it illustrates 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, California, 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 sizes, 3-dimensional scaffolds for production of nanoelectronic devices, and drug delivery.

Publications

Portney, N.G., Singh, K., Chaudhary, S., Destito, G., Schneemann, A., Manchester, M., Ozka, M. Organic and inorganic nanoparticle hybrids. Langmuir 21:2098, 2005.

Schwarcz, W.D., Barroso, S.P., Gomes, S.M., Johnson, J.E., Schneemann, A., Oliveira, A.C., Silva, J.L. Virus stability and protein-nucleic acid interaction as studied by high-pressure effects on nodaviruses. Cell. Mol. Biol. (Noisy-le-grand). 50:419, 2004.

Venter, P.A., Krishna, N.K., Schneemann, A. Capsid protein synthesis from replicating RNA directs specific packaging of the genome of a multipartite, positive-strand RNA virus. J. Virol. 79:6239, 2005.