Herpes simplex virus type 1 replicates its DNA and builds progeny nucleocapsids in the nucleus of the infected cell. Replicated viral DNA is a concatemer that is cleaved to unit-length genomes and packaged into capsid precursors, and successful DNA packaging is required for the mature capsid to exit the nucleus and become incorporated into virions. The DNA cleavage and packaging machinery is highly conserved across herpesvirus subfamilies; they represent novel targets for anti-herpesviral treatments. Seven genes have been identified that are essential for the packaging reaction. Of these, the UL25 gene product is unique because it is required for the completion rather than the initiation of DNA packaging. In the absence of functional UL25, the nuclei of infected cells accumulate empty capsids and free, close to genome-length viral DNA. This phenotype differs from null mutations of the other packaging genes, which block cleavage of the concatemer and expulsion of the capsid scaffold protein. While the functions of several members of the packaging machinery have been deduced by analogy with the dsDNA bacteriophages, the role of UL25 protein (pUL25) in this process is unclear. pUL25 is stably associated with the capsid vertices, where it forms a heterodimer with the packaging protein UL17. The goal of this project is to elucidate the pUL25 capsid-binding mechanism and its significance for DNA packaging and virion assembly. Mutational analysis of UL25 mapped the capsid-binding domain to the pUL25 N-terminus. pUL25 interactions with the capsid surface proteins were visualized with cryo-electron microscopy and 3D image reconstructions of capsids containing pUL25 fusion proteins. Finally, we demonstrated that UL25 mutants aberrantly cleave viral DNA before the second packaging signal. These data support a model in which pUL25 provides structural support to capsid vertices without interacting directly with DNA during the packaging reaction.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-08252010-134239 |
| Date | 03 September 2010 |
| Creators | Cockrell, Shelley Kristen |
| Contributors | Tom Smithgall, Neal DeLuca, Roger Hendrix, Fred Homa, James Conway |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-08252010-134239/ |
| 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 University of Pittsburgh 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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