| Expression of herpes simplex virus type 1 glycoprotein D deletion mutants in mammalian cells. | |
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MedLine Citation:
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PMID: 2452897 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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Glycoprotein D (gD) is a viron envelope component of herpes simplex virus types 1 and 2. We have previously defined seven monoclonal antibody (MAb) groups which recognize distinct epitopes on the mature gD-1 protein of 369 amino acids. MAb groups VII, II, and V recognize continuous epitopes at residues 11-19, 272-279, and 340-356, respectively. MAb groups I, III, IV, and VI recognize discontinuous epitopes. Recent studies have focused on epitopes I, III, and VI. Using truncated forms of gD generated by recombinant DNA methods and proteolysis, epitopes III, IV, and VI were located within amino acids 1-233. A portion of discontinuous epitope I was located in a region within residues 233-275. For this study, we used recombinant DNA methods to create mutations in the gD-1 gene and studied the effects of those mutations on gD as expressed in mammalian cells. Plasmid pRE4, containing the coding sequence of gD-1 and the Rous sarcoma virus long terminal repeat promoter, was transfected into mammalian cells. The expressed protein, gD-1-(pRE4), was identical in size and antigenic properties to gD-1 from infected cells. Six in-frame deletion mutations were subsequently constructed by using restriction enzymes to excise portions of the gD-1 gene. Plasmids carrying these mutated forms were transfected into cells, and the corresponding proteins were examined at 48 h posttransfection for antigenicity and glycosylation patterns. Three deletions of varying size were located downstream of residue 233. Analysis of these mutants showed that amino acids within the region 234-244 were critical for binding of DL11 (group I), but not for other MAb groups. Three other deletion mutants lost all ability to bind MAbs which recognize discontinuous epitopes. In addition, much of the gD expressed by these mutants was observed to migrate as high-molecular-weight aggregated forms in nondenaturing gels. Each of these mutations involved the loss of a cysteine residue, suggesting that disulfide linkages play an essential role in the formation of discontinuous epitopes. The extent of glycosylation of the mutant gD molecules accumulated at 48 h posttransfection suggested altered carbohydrate processing. In one case, there was evidence for increased O-linked glycosylation. Those proteins which had lost a cysteine residue as part of the deletion did not accumulate molecules processed beyond the high-mannose stage. The results suggest that carbohydrate processing during synthesis of gD is very sensitive to alterations in structure, particularly changes involving cysteine residues. |
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Authors:
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G H Cohen; W C Wilcox; D L Sodora; D Long; J Z Levin; R J Eisenberg |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't; Research Support, U.S. Gov't, P.H.S. |
Journal Detail:
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Title: Journal of virology Volume: 62 ISSN: 0022-538X ISO Abbreviation: J. Virol. Publication Date: 1988 Jun |
Date Detail:
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Created Date: 1988-06-20 Completed Date: 1988-06-20 Revised Date: 2009-11-18 |
Medline Journal Info:
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Nlm Unique ID: 0113724 Medline TA: J Virol Country: UNITED STATES |
Other Details:
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Languages: eng Pagination: 1932-40 Citation Subset: IM |
Affiliation:
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Department of Microbiology, School of Dental Medicine, University of Pennsylvania, Philadelphia 19104-6003. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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Animals Antibodies, Monoclonal / immunology Antigens, Viral / genetics Cell Line Chromosome Deletion Cloning, Molecular Cricetinae Epitopes Gene Expression Regulation Genetic Vectors Protein Conformation Protein Processing, Post-Translational Simplexvirus / genetics*, immunology Viral Envelope Proteins / genetics*, immunology |
| Grant Support | |
ID/Acronym/Agency:
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AI-18289/AI/NIAID NIH HHS; DE-02623/DE/NIDCR NIH HHS; NS-07180/NS/NINDS NIH HHS |
| Chemical | |
Reg. No./Substance:
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0/Antibodies, Monoclonal; 0/Antigens, Viral; 0/Epitopes; 0/Viral Envelope Proteins; 0/glycoprotein D, Human herpesvirus 1 |
| Comments/Corrections | |
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