Adenovirus (Ad) vectors are promising vehicles for vaccine delivery and gene therapy, but vector development has been hampered by the lack of an atomic structure of the intact virion. In this work, cryoEM single particle reconstruction methods have been applied to study the structure of an adenovirus vector, Ad35F. The 9 Å resolution cryoEM structure of Ad35F was analyzed by docking the crystal structures of two major capsid proteins, hexon and penton base. A long α-helix of ~ 40 residues was observed on the capsid surface and assigned to the C-terminal domain of protein IX recently used as a platform for vector retargeting. In the 6 Å resolution structure of Ad35F α-helices of 10 or more residues are clearly visualized for the first time. This in combination with secondary structure prediction for proteins IIIa, VI, VIII, and IX has led to new and more precise assignments for these capsid proteins. The coiled coil of protein IX is assigned to a tetrameric coiled coil now observed on the outer capsid surface. Protein IIIa, which is predicted to be highly α-helical, is assigned to a cluster of helices below the penton base on the inner capsid surface. Protein VI, a membrane lytic factor, is sequestered within the hexon cavities on the inner capsid surface.
The new protein assignments have important implications for vector design, virus assembly, and capsid disassembly during cell entry. First, since protein IIIa, VI, and VIII are all located on the inner capsid surface, our structural work indicates that the best candidate proteins for retargeting are hexon, penton base, fiber and protein IX. With the new interior position of protein IIIa, it is easier to envision how protein IIIa can facilitate DNA packaging during assembly. During cell entry in the low pH environment of the endosome, protein IIIa is well situated to allow rapid release of vertex proteins, including protein VI. The sequestration of protein VI is likely important to prevent membrane lysis until the appropriate time point in the viral life cycle.
| Identifer | oai:union.ndltd.org:VANDERBILT/oai:VANDERBILTETD:etd-10252006-093139 |
| Date | 01 November 2006 |
| Creators | Saban, Susan Dora |
| Contributors | Al Beth, Terence S. Dermody, Hassane Mchaourab, Dave Piston, Charles R. Sanders |
| 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-10252006-093139/ |
| 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. |
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