Model for studying virus attachment: identification and quantitation of Epstein-Barr virus-binding cells by using biotinylated virus in flow cytometry.
Epstein-Barr virus (EBV) was purified and biotinylated without significant loss of its cell-transforming activity. The use of biotinylated virus in conjunction with antibodies specific for selected cell surface molecules and flow cytometric analysis allowed for the positive identification of the virus-binding lymphocytes among a heterogeneous mononuclear cell population. Biotinylated EBV efficiently bound to all B lymphocytes, including those bearing surface mu, delta, gamma, and alpha immunoglobulin heavy chains or the surface CD5 (Leu-1) marker, but not to T lymphocytes, natural killer cells, or monocytes. By using biotinylated EBV and specific monoclonal antibodies in competitive inhibition experiments, it was also found that the virus attaches to an epitope on the CR2 molecule (the receptor for C3d and EBV), which is close to or identical with the one recognized by OKB7 monoclonal antibody, and that cell surface structures other than CR2 cannot mediate attachment of EBV. Moreover, studies on the binding of the virus to induced B lymphocytes (cells in S through G2 phase), and this was associated with the disappearance of the surface CR2 molecule and the inability of the virus to attach to these cells. The approach described here should be useful in studying the attachment of other viruses, identifying the specific cell types involved, and analyzing the effect of the cell cycle on virus binding.
Inghirami
G
G
Laboratory of Oral Medicine, National Institute of Dental Research, Bethesda, Maryland 20892.
Nakamura
M
M
Balow
J E
JE
Notkins
A L
AL
Casali
P
P
D000911:Antibodies, Monoclonal / Q000276:immunology; D000954:Antigens, Surface / Q000032:analysis; D001402:B-Lymphocytes / Q000276:immunology / Q000378:metabolism*; D001710:Biotin; D002453:Cell Cycle; D002469:Cell Separation / Q000379:methods*; D002472:Cell Transformation, Viral; D005434:Flow Cytometry; D004854:Herpesvirus 4, Human / Q000378:metabolism*; D006801:Humans; D007962:Leukocytes / Q000276:immunology / Q000378:metabolism; D008954:Models, Biological; D011947:Receptors, Antigen, B-Cell / Q000032:analysis; D011951:Receptors, Complement / Q000276:immunology / Q000378:metabolism; D017464:Receptors, Complement 3d; D011991:Receptors, Virus / Q000276:immunology / Q000378:metabolism
D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D011947:Receptors, Antigen, B-Cell; D011951:Receptors, Complement; D017464:Receptors, Complement 3d; D011991:Receptors, Virus; D001710:Biotin