Structural, functional, and immunological characterization of bovine herpesvirus-1 glycoprotein gl expressed by recombinant baculovirus.
The major glycoprotein complex gl of bovine herpesvirus-1 was expressed at high levels (36 micrograms per 1 x 10(6) cells) in insect cells using a recombinant baculovirus. The recombinant gl had an apparent molecular weight of 116 kDa and was partially cleaved to yield 63-kDa (glb) and 52-kDa (glc) subunits. This processing step was significantly less efficient in insect cells than the analogous step in mammalian cells, even though the cleavage sites of authentic and recombinant gl were shown to be identical. The oligosaccharide linkages were mostly endoglycosidase-H-sensitive, in contrast to those of authentic gl, which has mostly endoglycosidase-H-resistant linkages and an apparent molecular weight of 130/74/55 kDa. Despite the reduced cleavage and altered glycosylation, the recombinant glycoprotein was transported and expressed on the surface of infected insect cells. These surface molecules were biologically active as demonstrated by their ability to induce cell-cell fusion. Fusion was inhibited by three monoclonal antibodies specific for antigenic domains I and IV on gl. Domain I maps to the extracellular region of the carboxy terminal fragment glc and domain IV to the very amino terminus of the glb fragment, indicating that domains mapping in two distinct regions of gl function in cell fusion. Monoclonal antibodies specific for eight different epitopes recognized recombinant gl, indicating that the antigenic characteristics of the recombinant and authentic glycoproteins are similar. In addition, the recombinant gl was as immunogenic as the authentic gl, resulting in the induction of gl-specific antibodies in cattle.
van Drunen Littel-van den Hurk
S
S
Veterinary Infectious Disease Organization, University of Saskatchewan, Saskatoon, Canada.
Parker
M D
MD
Fitzpatrick
D R
DR
van den Hurk
J V
JV
Campos
M
M
Babiuk
L A
LA
Zamb
T
T
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D015726:Giant Cells / Q000166:cytology; D007242:Herpesvirus 1, Bovine / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D014162:Transfection; D014415:Tunicamycin / Q000494:pharmacology; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000378:metabolism
D011994:Recombinant Proteins; D014764:Viral Proteins; C046998:bovine herpesvirus type-1 glycoproteins; D014415:Tunicamycin