Structural Analysis Of The Sp Domain And A Putative Six-Helix Bundle In Rous Sarcoma Virus Assembly
| dc.contributor.author | Bush, Di | en_US |
| dc.contributor.chair | Vogt, Volker M | en_US |
| dc.contributor.committeeMember | Stipanuk, Martha Harney | en_US |
| dc.contributor.committeeMember | Whittaker, Gary R | en_US |
| dc.date.accessioned | 2013-09-16T16:42:37Z | |
| dc.date.available | 2018-08-20T06:01:13Z | |
| dc.date.issued | 2013-08-19 | en_US |
| dc.description.abstract | Purified retroviral Gag proteins can assemble in vitro to form immature virus -like particles (VLPs). By electron cryo-tomography, Rous sarcoma virus (RSV) VLPs show an organized hexameric lattice consisting chiefly of the capsid (CA) domain, with periodic stalk-like densities below the lattice. I hypothesize that the structure underlying these densities is formed by amino acid residues immediately downstream of the folded CA, namely the short spacer peptide SP, along with a dozen flanking residues. These 24 residues comprise the SP assembly (SPA) domain, and I propose that neighboring SPA units in a Gag hexamer coalesce to form a six-helix bundle. Using in vitro assembly, alanine scanning mutagenesis, and biophysical analyses, I have further characterized the structure and function of SPA. Most of the amino acid residues in SPA could not be mutated individually without abrogating assembly, with the exception of a few residues at the N- and C-termini as well as three hydrophilic residues within SPA. I interpret these results to mean that the amino acids that do not tolerate mutations contribute to higher-order structures in VLPs. Hydrogen-deuterium exchange analyses of unassembled Gag in comparison with assembled VLPs showed stron g protection at the SPA region, consistent with a higher-order structure. Circular dichroism revealed that a 29mer SPA peptide shifts from a random coil to a helix in a concentration dependent manner. Analytical ultracentrifugation showed concentration -dependent self- association of the peptide into a hexamer. Taken together, these results provide the first convincing evidence for the formation of a critical six-helix bundle (6HB) in RSV Gag assembly. I have modeled the RSV 6HB using structural and sequence homology with an existing synthetic 6HB and discovered a salt-bridge that may help stabilize the helical bundle. In an attempt to assess the structure of the immature retroviral core, which is made solely of Gag, I also crystallized several proteins containing the minimum regions of Gag required for immature assembly. Due to the inherent flexibility of Gag, which is a multi-domain protein, no crystal structure has been reported thus far of a Gag -like protein. I have shown that it is possible to crystallize a "minimal Gag", but that the crystals are difficult to optimize to the quality required for X-ray diffraction. I have also crystallized the aforementioned SPA peptide in order to determine the atomic -level structure of the putative 6HB. Unfortunately, my diffraction results show that the crystal packing is disordered. Although much more optimization will likely be required to produce crystals that can diffract to high resolution, my work has shown that it is possible to crystallize both a Gag-like protein and a peptide corresponding to the SP domain. | en_US |
| dc.identifier.other | bibid: 8267104 | |
| dc.identifier.uri | https://hdl.handle.net/1813/34225 | |
| dc.language.iso | en_US | en_US |
| dc.subject | retrovirus | en_US |
| dc.subject | assembly | en_US |
| dc.subject | virus structure | en_US |
| dc.title | Structural Analysis Of The Sp Domain And A Putative Six-Helix Bundle In Rous Sarcoma Virus Assembly | en_US |
| dc.type | dissertation or thesis | en_US |
| thesis.degree.discipline | Biochemistry | |
| thesis.degree.grantor | Cornell University | en_US |
| thesis.degree.level | Doctor of Philosophy | |
| thesis.degree.name | Ph. D., Biochemistry |
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