ABSTRACT
Early events of human B-lymphocyte infection by Epstein-Barr virus involve the virus binding to CD21, capping, and subsequent internalization of the virus-receptor complex. We show here that alpha interferon (IFN-alpha) inhibits the capping of Epstein-Barr virus-CD21 complexes. Synthetic peptides with the CD21 binding motif of IFN-alpha mimic IFN-alpha activity, suggesting that this effect may be mediated by IFN-alpha-CD21 interaction. Our findings demonstrate a novel and immediate mechanism of IFN-alpha action.
Subject(s)
B-Lymphocytes/microbiology , Herpesvirus 4, Human/drug effects , Interferon-alpha/pharmacology , Peptide Fragments/pharmacology , Receptors, Complement 3d/metabolism , Binding, Competitive , Herpesvirus 4, Human/growth & development , Herpesvirus 4, Human/metabolism , Humans , Interferon-alpha/immunology , Receptors, Complement 3d/immunologyABSTRACT
Interferon alpha contains a sequence motif similar to the complement receptor type two (CR2/CD21) binding site on complement fragment C3d. Antibodies against a peptide with the CR2 binding sequence on C3d react with a peptide carrying the IFN alpha CR2 binding motif (residues 92-99) and with recombinant IFN alpha. The IFN alpha-derived peptide, as well as recombinant IFN alpha, inhibits C3bi/C3d interaction with CR2 on the Burkitt lymphoma Raji. The direct interaction of IFN alpha and CR2 is inhibited by polyclonal anti-IFN alpha, anti-CR2 and anti-C3d peptide antibodies as well as by C3bi/C3d, EBV coat protein gp350/220 and IFN but not by IFN gamma. [125I]IFN alpha binding to Raji cells is inhibited by polyclonal anti-IFN alpha and anti-CR2 antibodies, by peptides with the CR2 binding motif and partially by C3bi/C3d. Monoclonal anti-CR2 antibody HB5, but not OKB-7, blocks IFN alpha binding to Raji cells. CR2 or CR2-like molecules may therefore be the major IFN alpha receptors on B lymphocytes.
Subject(s)
Herpesvirus 4, Human/physiology , Receptors, Complement/genetics , Receptors, Immunologic/genetics , Receptors, Virus/genetics , Amino Acid Sequence , Animals , Antibodies, Monoclonal/immunology , Antigens, Differentiation, B-Lymphocyte/genetics , Antigens, Differentiation, B-Lymphocyte/metabolism , B-Lymphocytes/immunology , Cell Line , Complement C3d/metabolism , Enzyme-Linked Immunosorbent Assay , Humans , Interferon Type I/metabolism , L Cells/immunology , Lymphocyte Activation , Mice , Molecular Sequence Data , Receptors, Complement/metabolism , Receptors, Complement 3d , Receptors, Immunologic/metabolism , Receptors, Interferon , Receptors, Virus/physiology , Recombinant Proteins , Rosette Formation , Sequence Homology, Nucleic Acid , TransfectionABSTRACT
We present herein the first evidence that interaction of specific EBV/C3dR ligands, as human C3bi/C3d and anti-EBV/C3dR MoAb, with EBV/C3dR enhanced significantly, in a dose dependent process, phosphorylation of EBV/C3dR and p120 RNP present in subcellular fractions, as purified plasma membranes and nuclei, of the human B lymphoma cell line, Raji. The use of kinase effectors allowed to detect some of the kinases involved in these phosphorylations. Pp60src-like phosphotyrosine kinase and protein kinase C were involved in the phosphorylation of plasma membrane or nuclear EBV/C3dR. An additional calcium/calmodulin-dependent kinase was also involved in nuclear EBV/C3dR phosphorylation. P120 RNP phosphorylation was under the control of protein kinase C and of CaCl2/Calmodulin-dependent kinase but also of casein kinase II.
Subject(s)
Cell Nucleus/metabolism , Protein Kinases/metabolism , Receptors, Virus/metabolism , Ribonucleoproteins/metabolism , Burkitt Lymphoma , Cell Line , Cell Membrane/metabolism , Herpesvirus 4, Human/physiology , Humans , Kinetics , Ligands , Phosphorylation , Receptors, Complement 3d , Receptors, Virus/isolation & purification , Ribonucleoproteins/isolation & purification , Subcellular Fractions/metabolismABSTRACT
Glycoprotein (gp) 140, the EBV/C3dR of B lymphocytes, is a membrane site involved in human cell regulation. To analyze the specificities of the binding sites for EBV and for C3d on the gp 140 molecule, two distinct approaches were used. First, anti-EBV/C3dR mAb were prepared against highly purified EBV/C3dR. Nine anti-EBV/C3dR mAb were obtained. Four of these anti-EBV/C3dR mAb inhibited C3d binding but not EBV binding on gp 140, whereas four others exerted an inverse effect. These differences could not be due to differences in isotype, antibody concentration, affinity constant, and number of molecules bound on cell surface, as these parameters were identical for the nine used mAb. Second, polyclonal anti-idiotypic antibodies (Ab2) were prepared against F(ab)'2 fragments of polyclonal anti-EBV/C3dR (Ab1). Ab2 recognized the variable portion of Ab1 as controlled by immunoblotting experiments. Ab2, which did not react with the cell surface, inhibited Ab1 binding on Raji cells. Ab2 mimicked the EBV/C3dR by its properties to bind to particle-bound C3d and EBV, preventing their binding on Raji cell surface. C3d binding specificities contained in Ab2 were isolated by affinity chromatography on C3b/C3bi-Sepharose. These specificities, being the internal image of C3d binding site of EBV/C3dR, reacted with Ab1 and inhibited particle-bound C3d binding on Raji cells but did not react with EBV. Taken together, these data support strongly that gp 140, the EBV/C3dR, carried two distinct binding sites, one for EBV and one for C3d.
Subject(s)
Antibodies, Anti-Idiotypic , Antibodies, Monoclonal , B-Lymphocytes/metabolism , Binding Sites, Antibody , Herpesvirus 4, Human/metabolism , Immunoglobulin Idiotypes/immunology , Receptors, Complement/immunology , Receptors, Virus/immunology , Animals , Antibodies, Anti-Idiotypic/analysis , Antibodies, Anti-Idiotypic/physiology , Antibodies, Monoclonal/analysis , Antibodies, Monoclonal/physiology , Antibody Specificity , Binding, Competitive , Cell Line , Humans , Immunoglobulin G/analysis , Immunoglobulin G/physiology , Membrane Glycoproteins/immunology , Membrane Glycoproteins/metabolism , Mice , Molecular Weight , Receptors, Complement 3dABSTRACT
gp140, the EB/C3d receptor (EBV/C3dR; CR2), is a membrane site involved in human B cell regulation. Cross-linking of this receptor on the cell surface by its specific ligands led to the enhancement of B cell proliferation in synergy with T cell factors. In vitro activation of human peripheral B lymphocytes by cross-linking membrane immunoglobulins with anti-mu antibody induced EBV/C3dR phosphorylation. These studies were pursued by analyzing cell-free phosphorylation of EBV/C3dR isolated from Raji cell fractions, and immobilized on OKB7, a monoclonal anti-EBV/C3dR antibody. Three EBV/C3dR-related antigens which could be cell-free phosphorylated were detected: gp140, the EBV/C3dR, p130 and p120. gp140, the mature form of EBV/C3dR, was isolated from plasma membrane and from purified nuclei. p130 was identified as an intracellular intermediate of EBV/C3dR glycosylation, localized in low-density microsomes. Phosphoamino acid analysis of EBV/C3dR allowed the detection of phosphotyrosine and phosphoserine residues. These data suggest that EBV/C3dR could carry an autophosphorylation activity and could be associated to serine kinases. Using polyclonal anti-p120 antibody and anti-120 kDa nuclear ribonucleoprotein monoclonal antibody (mAb), p120 was identified as a nuclear ribonucleoprotein antigenically not related to EBV/C3dR. Detection of p120 on EBV/C3dR, immobilized on OKB7, was due to interactions between both antigens, instead of anti-EBV/C3dR mAb cross-reactivity with p120. Cell-free phosphorylation of p120 was under the control of EBV/C3dR. However, it is not yet established whether other nuclear or membrane components were involved in the control of p120 cell-free phosphorylation by EBV/C3dR. From the data presented herein, we propose that phosphorylation of a 120-kDa nuclear ribonucleoprotein by EBV/C3dR-associated kinases could represent a crucial step in in vivo regulation of human B cell activation.
