Entry of herpesviruses is mediated by the interactions between viral glycoproteins and cellular receptors. Among these glycoproteins, gB plays an important role. In this study, my major focus was to study gB's functions in the virus entry process and the structural requirements for gB to conduct its functions. The virus model in my study is bovine herpesvirus 1 (BHV-1), a member of the alphaherpesviruses. BHV-1 gB is a type I integral membrane protein with a potential transmembrane anchor at the C-terminal region. A cleavage site in the middle divides this molecule into two subunits, gBb and gBc. In this study, a truncated gB, gBt (residues 1 to 763), and N-terminal subunit, gBb (residues 1 to 505), were first expressed under the control of the bovine heat-shock protein 70A (hsp70A) gene promoter in stably transfected Madin Darby bovine kidney (MDBK) cells. Both forms of gB were secreted into the medium with apparent molecular weights as anticipated, and they were reactive to all gB-specific monoclonal antibodies used in this study. Affinity-purified gBt and gBb were able to elicit antibody responses in mice to an extent comparable to those induced by authentic gB. These results suggest that gBt and gBb retain the structural and antigenic properties of authentic gB. Furthermore, the intracellular processing of gBt and gBb was similar to that of authentic gB in virus-infected cells. Finally, gBt was proteolytically cleaved after conversion of the high mannose-containing precursor to the mature form. These truncated gBs that were prepared served as reagents for the core of my studies. BHV-I gB can bind to heparin sulfate (HS) and another non-HS receptor on MDBK cells. We assume that high-affinity binding to the non-HS receptor is important for BHV-1 infectivity. BHV-1 gB forms dimers in infected cells and in virions, and its dimerization domain may be located between residues 506 to 763. The cytoplasmic domain of BHV-1 gB is important for the existence of the high-affinity binding site. Without the cytoplasmic domain, the truncated gB derivatives exhibit conformational changes and loss of the high-affinity binding site. By comparing the expression of different gB derivatives in MDBK cells, it was found that in the putative transmembrane region, segment 3 is the real membrane anchor, whereas segment 2 is the fusogenic domain. (Abstract shortened by UMI.)
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-10212004-000820 |
| Date | 01 January 1996 |
| Creators | Li, Yuanhao |
| Contributors | Babiuk, Lorne A. |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-10212004-000820 |
| 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 Saskatchewan 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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