Incorporation of the purified Epstein Barr virus/C3d receptor (CR2) into liposomes and demonstration of its dual ligand binding functions.
The 145-kDa molecule that has been identified as the C3d receptor CR2 was isolated from lysates of Raji cells by affinity chromatography by using the monoclonal antibody (MoAb)HB-5. The purified protein was incorporated into 14C-phosphatidylcholine liposomes by deoxycholate dialysis followed by flotation on discontinuous sucrose gradients. Incorporation of the receptor was verified by testing the gradient fractions for CR2 by an enzyme-linked immunosorbent assay. Liposomes were shown to be unilamellar vesicles ranging in diameter from 25 to 100 nm by electron microscopy. The external orientation of CR2 in the membranes was demonstrated by immunoelectron microscopy. The functional activities of liposomes containing CR2 and liposomes without protein were compared. CR2 liposomes bound to EC3d, but not to E, and this binding was inhibited by the anti-CR2 MoAb OKB7 and by a MoAb specific for C3d. Control liposomes failed to bind to either E or EC3d. The ability of CR2 to function as a receptor for Epstein Barr virus (EBV) was tested in two ways. First, CR2 liposomes bound to B95-8, a cell line expressing EBV membrane antigens, but not to B95-8 cells treated with the viral DNA polymerase inhibitor phosphonoformic acid. Second, liposomes containing CR2 were shown by ultracentrifugal analyses to bind directly to purified EBV, and this binding was also inhibited by OKB7. Control liposomes did not bind to B95-8 cells or to EBV. These findings show that CR2 purified from detergent extracts of Raji cells can be reconstituted into lipid membranes with maintenance of its dual functions as a receptor for C3d and EBV.
D000818:Animals; D002460:Cell Line; D003176:Complement C3 / Q000378:metabolism*; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D006801:Humans; D008081:Liposomes / Q000276:immunology / Q000494:pharmacology; D051379:Mice; D008854:Microscopy, Electron; D011951:Receptors, Complement / Q000032:analysis / Q000276:immunology / Q000378:metabolism*; D017464:Receptors, Complement 3d; D011991:Receptors, Virus / Q000032:analysis / Q000276:immunology / Q000378:metabolism*
D003176:Complement C3; D008081:Liposomes; D011951:Receptors, Complement; D017464:Receptors, Complement 3d; D011991:Receptors, Virus