Biochemical and structural characterization of self-assembling viral particles

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DESCRIPTION (provided by applicant): The Sulfolobus islandicus rod-shaped virus (SIRV) is a fibrous virus that infects thermophilic, acidophilic Archaea found in thermal vents and hot springs around the world. The virus is composed of a coat protein wrapped around the linear dsDNA viral genome; at the termini of the virion are a plug and three tail fibers. The purified SIRV coat protein has been found to self-assemble into long, helical virus-like particles (VLPs) when incubated at low pH, in the absence of DMA. This assembly is reversible, and monomeric coat protein can be obtained by reintroducing VLPs into high pH buffer. The biochemical and structural bases of the pH- driven assembly of the virus particle will provide new insights into the requirements for survival in extreme environments. I propose to characterize the SIRV coat protein assembly and determine the structure of both the coat protein by X-ray crystallography and the VLP by cryoelectron microscopy (cryo-EM). The conditions under which the coat protein self-assembles into VLPs and the kinetics of this process will be investigated in detail. To investigate the role of dsDNA in viral assembly, the formation of the VLP in the presence of dsDNA will also be studied. The structure of the monomeric SIRV coat protein will be determined by X-ray crystallography. Due to the difficulty of crystallizing fibrous virus particles, the structure of the VLP will be determined by cryo-EM, using the helical symmetry of the VLP to aid in the reconstruction of the three dimensional structure. The X-ray crystal structure of the coat protein will then be combined with the cryo- EM reconstruction of the VLP to create a quasi-atomic model of the VLP. This model will be used to identify the intermolecular interactions that drive viral assembly, which will form the basis of further biochemical studies of VLP assembly. Self-assembling viral particles have become a target for many pharmaceutical and nanotechnology applications, due to their regular structure and the ability to package small molecules in their hollow interiors. The SIRV VLPs are fibrous like the tobacco mosaic virus, which has been used as a platform for synthesis of nanowires. Due to its thermophilic, acidophilic nature, SIRV may be useful in nanotechnology applications that require high heat or low pH. In addition, the reversibility of the SIRV VLP assembly may be utilized as a mechanism for delivering therapeutics or imaging agents inside the VLP to a specific organ.