| dc.contributor.author | Corcoran, Jennifer A. | en_US |
| dc.date.accessioned | 2014-10-21T12:37:31Z | |
| dc.date.available | 2004 | |
| dc.date.issued | 2004 | en_US |
| dc.identifier.other | AAINQ89820 | en_US |
| dc.identifier.uri | http://hdl.handle.net/10222/54628 | |
| dc.description | Reptilian reovirus (RRV) is one of a limited number of non-enveloped viruses capable of inducing cell-cell fusion. A small, hydrophobic, basic, 125 amino acid fusion protein termed p14, is encoded by the first open reading frame (orf) of a bicistronic viral mRNA. Sequence comparisons to previously characterized reovirus fusion proteins identified p14 as a new member of the reovirus fusion-associated small transmembrane (FAST) protein family. | en_US |
| dc.description | p14 contains several key structural and functional motifs. A C-proximal proline-rich motif and N-glycosylation consensus sequence are not required for fusion activity, whereas N-terminal myristylation of p14 is an essential component of the fusion mechanism. p14 is a Type III (Nexo/C cyt) integral membrane protein. This topology results in the co-translational translocation of the essential myristylated N-terminal domain of p14 across the membrane. Site-directed mutagenesis, circular dichroism and an in vitro liposome fusion assay identified an N-proximal hydrophobic patch (HP) as a fusion motif integral to the p14-fusion mechanism. Solution NMR spectroscopy identified a beta-structured loop near the N terminus, with the C-terminal residues remaining unstructured. Certain substitutions in the HP that resulted in a fusion-minus phenotype also exerted a dominant negative effect on authentic p14-induced syncytium formation, suggesting p14 may multimerize. A co-immune precipitation assay indicated that p14 forms homomultimers. In vitro chemical cross-linking confirmed the close association of p14 monomers, and suggested p14 may form dimers. The mechanism of p14-mediated fusion depends on the presence of cholesterol in the plasma membrane. p14 localizes to two types of detergent-resistant membrane microdomains; however, fluorescence microscopy suggested that a greater population of the protein resided in cholesterol-dependent Lubrol-resistant membranes. It is possible that p14 may utilize Lubrol-rafts to target cell membrane protrusions as its preferred site of fusion initiation. | en_US |
| dc.description | Based on these studies, the following model for p14-mediated membrane fusion is proposed. RRV p14 adopts a membrane topology that externalizes its myristylated N-terminal domain, allowing the fatty acid to associate with the exoplasmic leaflet of the membrane. A critical number of p14 multimers localize to cholesterol-rich Lubrol-resistant membrane microdomains in non-planar regions of the plasma membrane. The hydrophobic patch dehydrates the intercellular space and/or interacts with one or both membranes, mediating membrane perturbation. The membrane-embedded myristic acid reversibly dissociates from the donor membrane and/or interacts with the target membrane, to increase membrane perturbation. The membrane-interacting regions of a critical number of p14 multimers mediate sufficient alteration of lipid bilayer structure that membrane merger is the result. This study accentuates the diversity and unusual properties of p14 and the FAST protein family as a third distinct class of viral membrane fusion proteins.* | en_US |
| dc.description | *This dissertation is a compound document (contains both a paper copy and a CD as part of the dissertation). The CD requires the following system requirements: Windows MediaPlayer or RealPlayer. | en_US |
| dc.description | Thesis (Ph.D.)--Dalhousie University (Canada), 2004. | en_US |
| dc.language | eng | en_US |
| dc.publisher | Dalhousie University | en_US |
| dc.publisher | | en_US |
| dc.subject | Biology, Microbiology. | en_US |
| dc.subject | Health Sciences, Immunology. | en_US |
| dc.title | Protein-mediated membrane fusion: New insights from the reptilian reovirusp14 fusion protein. | en_US |
| dc.type | text | en_US |
| dc.contributor.degree | Ph.D. | en_US |
