The SARS-Coronavirus (SARS-CoV) is the etiological agent of the severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. Investigations, described herein, have focused on the genetic manipulation of the SARS-CoV S glycoprotein in order to delineate functional domains within the protein. This was accomplished by incorporating single point mutations, cluster-to-lysine and cluster-to-alanine mutations, as well as carboxyl terminal truncations into the protein and investigating these mutants in transient expression experiments. Mutagenesis of either the coiled-coil domain of the S glycoprotein amino terminal heptad repeat, the predicted fusion peptide, or adjacent but distinct regions, severely compromised S-mediated cell-to-cell fusion, while intracellular transport and cell-surface expression were not adversely affected. Surprisingly, a carboxyl terminal truncation of 17 amino acids substantially increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17 amino acids regulate the S fusogenic properties. In contrast, truncation of 26 or 39 amino acids eliminating either one or both of the two endodomain cysteine-rich motifs, respectively, inhibited cell fusion in comparison to the wild-type S. The cysteine rich domains were further studied by constructing cysteine cluster to alanine mutants in order to ascertain their importance in the function of the protein. Results showed that the two cysteine clusters proximal to the transmembrane region were vital in the functioning of the spike protein in mediating cell-to-cell fusion. Mutagenesis of the acidic amino acid cluster in the carboxyl terminus of the S glycoprotein as well as modification of a predicted phosphorylation site within the acidic cluster revealed that this amino acid motif may play a functional role in the retention of S at cell-surfaces. A panel of truncations for Bovine Coronavirus (BCoV) S was also constructed and compared to truncations made for the SARS-CoV S glycoprotein. It was found that the two sets of truncations had very little comparable effects on protein function when compared to one another. This genetic analysis reveals that the SARS-CoV S glycoprotein contains extracellular domains that regulate cell fusion as well as distinct endodomains that function in intracellular transport, cell-surface expression and cell fusion.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-11162005-142155 |
| Date | 01 December 2005 |
| Creators | Petit, Chad Michael |
| Contributors | Ding Shih, Patrick DiMario, Elmer Godeny, Dennis Ingram, Konstantin G. Kousoulas |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-11162005-142155/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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