Adenoviruses (Ads) are a promising tool for gene and vaccine delivery. Though known for over 50 years, details about Ad structure and intermediates of its lifecycle remain elusive. CryoEM, a technique suitable for studying this large non-enveloped virus (>900Å in diameter), has increased our understanding of Ad structure, in particular of the components for which we do not have atomic resolutions structures. The cryoEM structure of a temperature-sensitive mutant, Ad2ts1, was determined to structurally characterize this intermediate in the Ad maturation process. Ad2ts1 fails to incorporate the viral protease and contains the precursor forms of multiple structural proteins when produced at non-permissive temperatures. It also has a cell entry defect and is defective in endosomal escape. The 10.5Å resolution cryoEM structure of Ad2ts1 shows that the core of the virus remains connected to the capsid via preproteins IIIa and VI, which are normally cleaved by the protease. In a second study, a cryoEM structure of an Ad vector, Ad35f, complexed with human ¦Á-defensin HD5, was determined to visualize the binding sites for defensin on the Ad capsid and to characterize the mechanism of neutralization. HD5 binds at negatively charged regions of the hexon, penton base, and fiber. Based on sequence analysis and information on which Ad types are neutralized by HD5, a critical binding site for neutralization is proposed, comprising 4 polar and negatively charged residues in the fiber N-terminal region and additional residues in the RGD-loop of the penton base. The mechanism of defensin neutralization may involve bridging the fiber and penton base and blocking programmed disassembly. These cryoEM studies highlight the importance of the exquisitely timed disassembly of the virion and release of viral proteins during cell entry.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-07202009-180045 |
| Date | 04 August 2009 |
| Creators | Silvestry Ramos, Mariena |
| Contributors | Albert H. Beth, Hassane S. Mchaourab, Phoebe L. Stewart, Charles E. Cobb, Jens Meiler, Terence S. Dermody |
| Publisher | VANDERBILT |
| Source Sets | Vanderbilt University Theses |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.vanderbilt.edu/available/etd-07202009-180045/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto 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 Vanderbilt University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, 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. |
Page generated in 0.0137 seconds