The predominant mechanism of herpes simplex virus type 1 (HSV-1) entry into permissive cells involves initial virus attachment to the cells by the interaction of envelope glycoproteins gC and gB with cell surface glycosaminoglycans (GAGs), binding of envelope glycoprotein D to one of several dissimilar co-receptors, and fusion of the virus envelope with the cell membrane requiring the combined essential functions of glycoproteins gD, gB and gH/gL. The binding of gD to its cognate receptor appears to result in emission of an activating signal to the fusion apparatus which minimally consists of the other essential glycoproteins. To gain a better understanding of gDs involvement in the fusion-activating process, we took the approach of separating gD from the virus envelope to determine whether a soluble form of gD (sgD) could mediate entry of gD-deficient virus. The results showed that sgD enabled entry of gD-deficient HSV-1 into CHO-K1 cells bearing the gD receptors HVEM or nectin-1. Using mutant forms of sgD that selectively bind to one or the other receptor, we demonstrated that entry by this mechanism is receptor specific.
Investigation of the mechanism of sgD-mediated entry demonstrated that the presence of virus at the cell surface was required at the time of sgD-receptor binding, which could be explained in part by our observation that sgD rapidly dissociated from the receptor under our experimental conditions. In addition, entry was not eliminated instantaneously when receptor-bound sgD was exposed to 37ºC, suggesting that the active conformation of receptor-bound sgD is not highly unstable. sgD was not stabilized at the cell surface or internalized in the presence of gD-deficient virus. Using lysosomotropic agents as well as protease protection assays, we obtained no reproducible evidence that sgD-mediated entry takes place by endocytosis.
Surprisingly, virus attachment to cell-surface GAGs was not required for sgD-mediated entry. Furthermore, gD-deficient virus attached to GAG-deficient cells in the absence of sgD, revealing a previously unknown binding interaction between the HSV virion and the cell. This interaction was shown to be of a less stable nature than the virus-GAG interaction, and may play a role in normal virus entry. Our results provide new tools and directions to unravel the still incompletely understood events set in motion by gD binding to its receptor.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-08032006-110604 |
| Date | 17 August 2006 |
| Creators | Tsvitov, Marianna |
| Contributors | Dr. Joseph C. Glorioso, Dr. Stephen L. Phillips, Dr. Ora A. Weisz, Dr. Fred L. Homa, Dr. Paul D. Robbins, Dr. Paul R. Kinchington |
| 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-08032006-110604/ |
| 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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