Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells.
Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK- cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1-infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1-infected bovine cells. For gI, a deficiency in N-linked glycosylation of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected or BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes. Immunization of mice with the transfected cells elicited BHV-1-specific virus-neutralizing antibody, thus verifying the antigenic authenticity of the recombinant glycoproteins and the important role of gI and gIII as targets of the immune response to BHV-1 in this murine model system.
Fitzpatrick
D R
DR
Department of Veterinary Microbiology, University of Saskatchewan, Saskatoon, Canada.
Zamb
T
T
Parker
M D
MD
van Drunen Littel-van den Hurk
S
S
Babiuk
L A
LA
Lawman
M J
MJ
D000818:Animals; D002459:Cell Fusion; D002478:Cells, Cultured; D003165:Complement System Proteins / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D007242:Herpesvirus 1, Bovine / Q000235:genetics*; D007114:Immunization; D007150:Immunohistochemistry; D051379:Mice; D010957:Plasmids; D011233:Precipitin Tests / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis* / Q000502:physiology
D011994:Recombinant Proteins; D014764:Viral Proteins; C046998:bovine herpesvirus type-1 glycoproteins; D003165:Complement System Proteins