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1.
Epstein-Barr virus (EBV) invasion of B-lymphocytes involves EBV gp350/220 binding to B-lymphocyte CR2. The anti-gp350 monoclonal antibody (mAb)-72A1 Fab inhibits this binding and therefore blocks EBV invasion of target cells. However, gp350/220 regions interacting with mAb 72A1 and involved in EBV invasion of target cells have not yet been identified. This work reports three gp350/220 regions, defined by peptide 11382, 11389, and 11416 sequences, that are involved in EBV binding to B-lymphocytes. Peptides 11382, 11389, and 11416 bound to CR2(+) but not to CR2(-) cells, inhibited EBV invasion of cord blood lymphocytes (CBLs), were recognized by mAb 72A1, and inhibited mAb 72A1 binding to EBV. Peptides 11382 and 11416 binding to peripheral blood lymphocytes (PBLs) induced interleukin-6 protein synthesis in these cells, this phenomenon being inhibited by mAb 72A1. The same behavior has been reported for gp350/220 binding to PBLs. Anti-peptide 11382, 11389, and 11416 antibodies inhibited EBV binding and EBV invasion of PBLs and CBLs. Peptide 11382, 11389, and 11416 sequences presented homology with the C3dg regions coming into contact with CR2 (C3dg and gp350 bound to similar CR2 regions). These peptides could be used in designing strategies against EBV infection.  相似文献   

2.
Epstein-Barr virus lacking glycoprotein gp85 cannot infect B-cells and epithelial cells. The gp85 belongs to the molecular complex required for virus invasion of B-lymphocyte or epithelial cells. Moreover, there is evidence that gp85 is necessary for virus attachment to epithelial cells. Thirty-six peptides from the entire gp85-sequence were tested in epithelial and lymphoblastoid cell line binding assays to identify gp85-regions involved in virus-cell interaction. Five of these peptides presented high binding activity to Raji, Ramos, P3HR-1, and HeLa cells, but not to erythrocytes; Raji-cell affinity constants were between 80 and 140nM. Of these five peptides, 11435 ((181)TYKRVTEKGDEHVLSLVFGK(200)), 11436 ((201)TKDLPDLRGPFSYPSLTSAQ(220)), and 11438 ((241)YFVPNLKDMFSRAVTMTAAS(260)) bound to a 65kDa protein on Raji-cell surface. These peptides and antibodies induced by them (recognising live EBV-infected cells) inhibited Epstein-Barr virus interaction with cord blood lymphocytes. It is thus probable that gp85-regions defined by peptides 11435, 11436, and 11438 are involved in EBV invasion of B-lymphocytes.  相似文献   

3.
The Epstein-Barr virus (EBV) envelope glycoprotein 350/220 (gp350/220) is the most abundant molecule on the viral surface and it is responsible for the initial viral attachment to cell surface of the host. As many other viral envelope proteins, it is highly glycosylated, not least with O-linked glycans, most of which essential for EBV life cycle. EBV gp350/220 is also a primary target for neutralizing antibodies in the human hosts and a promising candidate for an EBV vaccine. Here we showed that recombinant GalNAc transferases can glycosylate scan peptides of the EBV gp350/220 envelope protein immobilized on microarray glass slides. We also identified serum IgG antibodies to a selection of peptides and O-glycopeptides, whereas sera from EBV-IgG negative individuals remained negative. We here describe novel glycopeptide epitopes present within immunodominant stretches of EBV gp350/220 and demonstrate a remarkable variability between individual samples with respect to their reactivity patterns to peptides and glycopeptides. The study provides additional insights into the complex B-cell response towards the EBV gp350/220 envelope protein, which may have implications for diagnostic and vaccine developments.  相似文献   

4.
Epstein-Barr virus (EBV) is a tumor virus with marked B lymphotropism. After crossing the B-cell membrane, the virus enters cytoplasmic vesicles, where decapsidation takes place to allow transfer of the viral DNA to the cell nucleus. BNRF1 has been characterized as the EBV major tegument protein, but its precise function is unknown. We have constructed a viral mutant that lacks the BNRF1 gene and report here its in vitro phenotype. A recombinant virus devoid of BNRF1 (DeltaBNRF1) showed efficient DNA replication and production of mature viral particles. B cells infected with the DeltaBNRF1 mutant presented viral lytic antigens as efficiently as B cells infected with wild-type or BNRF1 trans-complemented DeltaBNRF1 viruses. Antigen presentation in B cells infected with either wild-type (EBV-wt) or DeltaBNRF1 virus was blocked by leupeptin addition, showing that both viruses reach the endosome/lysosome compartment. These data were confirmed by direct observation of the mutant virus in endosomes of infected B cells by electron microscopy. However, we observed a 20-fold reduction in the number of B cells expressing the nuclear protein EBNA2 after infection with a DeltaBNRF1 virus compared to wild-type infection. Likewise, DeltaBNRF1 viruses transformed primary B cells much less efficiently than EBV-wt or BNRF1 trans-complemented viruses. We conclude from these findings that BNRF1 plays an important role in viral transport from the endosomes to the nucleus.  相似文献   

5.
Epstein-Barr virus (EBV) is a human pathogen that is involved in numerous diseases and tumors. Since the EBV infection occurs in the early ages of life, and most of the population is subsequently exposed to EBV, the conventional method of vaccination to induce the prophylactic immunity cannot be considered effective in coping with the virus infection. In this study, we tested whether the injection of a plasmid vector that contained the gene for glycoprotein 350 (gp350), which had been identified as a ligand for virus' adsorption and a target for virus neutralizing antibodies, could induce effective immune responses against the antigen. As a result, the injection of the constructed plasmid vector into mice induced the production of gp350-specific antibodies. A major isotype of the gp350-specific antibodies was IgG1. The antibodies efficiently mediated the antibody-dependent cellular cytotoxicity against the cells expressing the gp350 antigen. In addition, the injection of the constructed plasmid vector stimulated the precursor T cell population that was specific to the gp350 antigen. In addition, gp350-specific cytotoxic T lymphocytes were efficiently stimulated by the injection of the constructed plasmid vector. These results suggested that the injection of the plasmid vector, containing the gp350 gene of Epstein-Barr virus, could be one of the most effective ways to induce both prophylactic and therapeutic vaccinations against the virus infection.  相似文献   

6.
Broadly cross-reactive human immunodeficiency virus (HIV)-neutralizing antibodies are infrequently elicited in infected humans. The two best-characterized gp41-specific cross-reactive neutralizing human monoclonal antibodies, 4E10 and 2F5, target linear epitopes in the membrane-proximal external region (MPER) and bind to cardiolipin and several other autoantigens. It has been hypothesized that, because of such reactivity to self-antigens, elicitation of 2F5 and 4E10 and similar antibodies by vaccine immunogens based on the MPER could be affected by tolerance mechanisms. Here, we report the identification and characterization of a novel anti-gp41 monoclonal antibody, designated m44, which neutralized most of the 22 HIV type 1 (HIV-1) primary isolates from different clades tested in assays based on infection of peripheral blood mononuclear cells by replication-competent virus but did not bind to cardiolipin and phosphatidylserine in an enzyme-linked immunosorbent assay and a Biacore assay nor to any protein or DNA autoantigens tested in Luminex assays. m44 bound to membrane-associated HIV-1 envelope glycoproteins (Envs), to recombinant Envs lacking the transmembrane domain and cytoplasmic tail (gp140s), and to gp41 structures containing five-helix bundles and six-helix bundles, but not to N-heptad repeat trimers, suggesting that the C-heptad repeat is involved in m44 binding. In contrast to 2F5, 4E10, and Z13, m44 did not bind to any significant degree to denatured gp140 and linear peptides derived from gp41, suggesting a conformational nature of the epitope. This is the first report of a gp41-specific cross-reactive HIV-1-neutralizing human antibody that does not have detectable reactivity to autoantigens. Its novel conserved conformational epitope on gp41 could be helpful in the design of vaccine immunogens and as a target for therapeutics.  相似文献   

7.
Complement receptor type 2 (CR2/CD21) is essential for the attachment of Epstein-Barr virus (EBV) to the surface of B-lymphocytes in an interaction mediated by the viral envelope glycoprotein gp350. The heavily glycosylated structure of EBV gp350 has recently been elucidated by x-ray crystallography, and the CR2 binding site on this protein has been characterized. To identify the corresponding gp350 binding site on CR2, we have undertaken a site-directed mutagenesis study targeting regions of CR2 that have previously been implicated in the binding of CR2 to the C3d/C3dg fragments of complement component C3. Wild-type or mutant forms of CR2 were expressed on K562 cells, and the ability of these CR2-expressing cells to bind gp350 was measured using flow cytometry. Mutations directed toward the two N-terminal extracellular domains of CR2 (SCR1-2) reveal that a large contiguous surface of CR2 SCR1-2 is involved in gp350 binding, including a number of positively charged residues (Arg-13, (Arg-28, (Arg-36, Lys-41, Lys-57, Lys-67, and Arg-83). These data appear to complement the CR2 binding site on gp350, which is characterized by a preponderance of negative charge. In addition to identifying the importance of charge in the formation of a CR2-gp350 complex, we also provide evidence that both SCR1 and SCR2 make contact with gp350. Specifically, two anti-CR2 monoclonal antibodies, designated as monoclonal antibodies 171 and 1048 whose primary epitopes are located within SCR2, inhibit binding of wild-type CR2 to EBV gp350; with regard to SCR1, both K562 cells expressing an S15P mutation and recombinant S15P CR2 proteins exhibit diminished gp350 binding.  相似文献   

8.
The Epstein-Barr virus (EBV) major envelope glycoprotein gp340 is the subject of current efforts to develop an EBV subunit vaccine. The importance of gp340-specific humoral immunity has been highlighted by studies of natural infection in humans and gp340 immunization of experimental animals. The former studies have demonstrated the presence of gp340-specific serum antibodies which mediate EBV neutralization, complement fixation, and antibody-dependent cellular cytotoxicity. The latter studies have often shown a correlation between the induction of gp340-specific EBV-neutralizing antibodies and protection from virus challenge. We have used a series of bacterial beta-galactosidase-gp340 fusion proteins and overlapping synthetic peptides from the gp340 open reading frame to map the positions of B-cell epitopes within the gp340 primary amino acid sequence. The data reported here indicate the presence of B-cell epitopes within the carboxy-terminal third of the gp340 polypeptide chain. These epitopes could not be detected with a peptide enzyme-linked immunosorbent assay, thereby suggesting that they are discontinuous. Affinity purification of antibodies with a gp340 fusion protein from the carboxy terminus of the gp340 polypeptide chain has been used to show that these antibodies are not EBV neutralizing in vitro. The consequences of these findings for future EBV vaccine development are considered.  相似文献   

9.
Entry of an enveloped virus such as Epstein-Barr virus (EBV) into host cells involves fusion of the virion envelope with host cell membranes either at the surface of the cell or within endocytic vesicles. Previous work has indirectly implicated the EBV glycoprotein gp85 in this fusion process. A neutralizing monoclonal antibody to gp85, F-2-1, failed to inhibit binding of EBV to its receptor but interfered with virus fusion as measured with the self-quenching fluorophore octadecyl rhodamine B chloride (R18) (N. Miller and L. M. Hutt-Fletcher, J. Virol. 62:2366-2372, 1988). To test further the hypothesis that gp85 functions as a fusion protein, EBV virion proteins including or depleted of gp85 were incorporated into lipid vesicles to form virosomes. Virosomes were labeled with R18, and those that were made with undepleted protein were shown to behave in a manner similar to that of R18-labeled virus. They bound to receptor-positive but not to receptor-negative cells and fused with Raji cells but not with receptor-positive, fusion-incompetent Molt 4 cells; monoclonal antibodies that inhibited binding or fusion of virus inhibited binding and fusion of virosomes, and virus competed with virosomes for attachment to cells. In contrast, virosomes made from virus proteins depleted of gp85 by immunoaffinity chromatography remained capable of binding to receptor-positive cells but failed to fuse. These results are compatible with the hypothesis that gp85 is actively involved in the fusion of EBV with lymphoblatoid cell lines and suggest that the ability of antibody F-2-1 to neutralize infectivity of EBV represents a direct effect on the function of gp85 as a fusion protein.  相似文献   

10.
The Epstein-Barr virus gp350/220 envelope protein mediates virus attachment to the EBV/C3dg receptor (CR2) of human B lymphocytes. Synthetic peptides corresponding to two regions in gp350/220, which have a similar amino acid sequence with the complement C3dg protein, were used to identify a receptor binding epitope. A peptide corresponding to the N terminus of gp350/220, EDPGFFNVE, bound to purified CR2 and to CR2 positive but not CR2 negative B and T lymphoblastoid cell lines. Soluble monomeric gp350/220 peptide blocked CR2 binding to immobilized EBV, while multimeric forms of the N-terminal gp350/220 peptide conjugated to albumin efficiently blocked recombinant gp350/220 and C3dg binding to B cells as well as EBV-induced B cell proliferation and transformation. These studies indicate that the N-terminal region of gp350/220 plays a crucial role in mediating the earliest stages of EBV infection of B cells and provides a molecular basis for the restricted host cell EBV tropism.  相似文献   

11.
12.
13.
Herpesviruses require membrane-associated glycoproteins gB, gH, and gL for entry into host cells. Epstein-Barr virus (EBV) gp42 is a unique protein also required for viral entry into B cells. Key interactions between EBV gp42 and the EBV gH/gL complex were investigated to further elucidate their roles in membrane fusion. Deletion and point mutants within the N-terminal region of gp42 revealed residues important for gH/gL binding and membrane fusion. Many five-residue deletion mutants in the N-terminal region of gp42 that exhibit reduced membrane fusion activity retain binding with gH/gL but map out two functional stretches between residues 36 and 96. Synthetic peptides derived from the gp42 N-terminal region were studied in in vitro binding experiments with purified gH/gL and in cell-cell fusion assays. A peptide spanning gp42 residues 36 to 81 (peptide 36-81) binds gH/gL with nanomolar affinity, comparable to full-length gp42. Peptide 36-81 efficiently inhibits epithelial cell membrane fusion and competes with soluble gp42 to inhibit B-cell fusion. Additionally, this peptide at low nanomolar concentrations inhibits epithelial cell infection by intact virus. Shorter gp42 peptides spanning the two functional regions identified by deletion mutagenesis had little or no binding to soluble gH/gL and were also unable to inhibit epithelial cell fusion, nor could they complement gp42 deletion mutants in B-cell fusion. These studies identify key residues of gp42 that are essential for gH/gL binding and membrane fusion activation, providing a nanomolar inhibitor of EBV-mediated membrane fusion.  相似文献   

14.
Cell surface-associated viral glycoproteins are thought to play a major role as target antigens in cellular cytotoxicity and antiviral immunosurveillance. One such glycoprotein is the Epstein-Barr virus (EBV)-encoded glycoprotein 350 (gp350), which is expressed on both virion envelope and EBV producer cells and carries the virus attachment protein moiety. Although it is known that some antibodies to gp350 can neutralize the virus, the role of this glycoprotein in EBV-specific cellular cytotoxicity is not yet clear. We describe here a study in which we successfully used a new approach to demonstrate that gp350 is a target antigen for EBV-specific antibody-dependent cellular cytotoxicity (ADCC). Transfection of gp350-negative cells resistant to natural killer (NK) cell activity (i.e., Raji) with a recombinant vector (pZIP-MA) containing the gene encoding the EBV-gp350 and the neomycin resistance gene enabled us to isolate cell clones with a stable and strong expression of gp350 on their surface membranes. ADCC determined by using two clones clearly demonstrated that gp350 is the target of the EBV ADCC. Interestingly, this ADCC was comparable to that obtained against the EBV-superinfected (coated) Raji cell expressing the same percentage of gp350 positivity as the two clones. No cytotoxic activity was detected against either nontransfected (gp350-negative) Raji cells or cells transfected with the vector [pZIP-neo-SV(X)1] lacking the gp350 gene. In addition to demonstrating that gp350 is a target molecule for EBV-specific ADCC, our approach in using NK-resistant transfectants provides a lead for probing the role of cell surface-associated viral antigens in specific cellular killing and immunosurveillance.  相似文献   

15.
Human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein gp140 interacts with its specific receptors on the surface of the target cells leading to cellular activation through various signaling pathways. The effect of blocking the chemokine repertoire in human brain microvascular endothelial cells in HIV dementia (HAD) disease has not been reported. Characterizing the nature of HIV-1 envelope protein gp140 (T-tropic, HXBc2) receptor binding conditions to HBMEC is critical to gain insight into the HIV dementia, and eventually to rationally design the agents to block envelope protein receptor interactions. HIV-1 gp140 oligomers were purified and separated to monomers, dimers, and trimers. The binding conditions of gp140 to HBMEC chemokine receptor, CXCR4, were optimized with an aim of understanding the structural interactions in HAD. Analysis of the interaction between HIV-1 gp140 and CXCR4 of HBMEC by saturation binding, cross-competition analysis with radiolabeled SDF and gp140, revealed a strong interaction, specificity between HIV-1 gp140 and CXCR4. Our binding data demonstrate that HIV-1 envelope protein gp140 enters cells by protein receptor mediated interactions that are regulated by the conformational state of the gp140 at physiological environment (pH and temperature). The CXCR4 antibody 12G5 inhibited SDF-1 binding to HBMEC indicating the specificity of gp140 binding to HBMEC. Scatchard analysis revealed the presence of approximately 70250 gp140 binding sites per cell with a K(d) of 4.5 nM. Cross-competition experiments using labeled SDF-1 and gp140 revealed that both unlabeled SDF-1 and gp140 are capable of displacing their radiolabeled counterparts. The binding assay conditions and radioligand binding assay are highly valuable to identify and design better HIV inhibitors for HAD.  相似文献   

16.
17.
Murine gammaherpesvirus 68 (MHV-68) is a naturally occurring virus of murid rodents which displays pathobiological characteristics similar to those of other gammaherpesviruses, including Epstein-Barr virus (EBV). However, unlike EBV and many other gammaherpesviruses, MHV-68 replicates in epithelial cells in vitro and infects laboratory strains of mice and therefore provides a good model for the study of gammaherpesviruses. Studies of sequences around the center of the MHV-68 genome identified a gene (designated BPRF1 for BamHI P fragment rightward open reading frame 1) whose putative product had motifs reminiscent of a transmembrane glycoprotein. All other gammaherpesviruses have a glycoprotein in this genomic position, but the BPRF1 gene showed sequence homology with only the EBV membrane antigen gp340/220. Biochemical analysis showed that the product of BPRF1 was a glycoprotein present on the surface of infected cells, and immunoelectron microscopy showed that it was present in the virus particle. In addition, antibodies to the BPRF1 product raised by using a bacterial fusion protein neutralized the virus in the absence of complement. The predominant molecular weights of the protein were 150,000 and 130,000. Pulse-chase analysis and endoglycosidase-H digestion showed that the 130,000-molecular-weight form was a precursor of the 150,000-molecular-weight form, and cell surface labelling showed that the 150,000-molecular-weight form alone was on the cell surface. We therefore named the protein gp150. Since gp150 is the first virion-associated glycoprotein and neutralizing determinant of MHV-68 to be characterized, it provides a valuable tool for the future study of virus-host interactions.  相似文献   

18.
We tested the hypothesis that stabilizing α-helix of Epstein–Barr virus gH-derived peptide 11438 used for binding human cells will increase its biological activity. Non-stable α-helix of peptide 11438 was unfolded in an entropy-driven process, despite the opposing effect of the enthalpy factor. Adding and/or changing amino acids in peptide 11438 allowed the designing of peptides 33207, 33208 and 33210; peptides 33208 and 33210 displayed higher helical content due to a decreased unfolding entropy change as was determined by AGADIR, molecular dynamics and circular dichroism analysis. Peptides 33207, 33208 and 33210 inhibited EBV invasion of peripheral blood mononuclear cells and displayed epitopes more similar to native protein than peptide 11438; these peptides could be useful for detecting antibodies induced by native gH protein since they displayed high reactivity with anti-EBV antibodies. Anti-peptide 33207 antibodies showed higher reactivity with EBV than anti-peptide 11438 antibodies being useful for inducing antibodies against EBV. Anti-peptide 33210 antibodies inhibit EBV invasion of epithelial cells better than anti-peptide 11438 antibodies. Peptide 33210 bound to normal T lymphocytes and Raji cells stronger than peptide 11438 and also induced apoptosis of monocytes and Raji cells but not of normal T cells in a similar way to EBV-gH. Peptide 33210 inhibited the monocytes’ development toward dendritic cells better than EBV and peptide 11438. In conclusion, stabilizing the α-helix in peptides 33208 and 33210 designed from peptide 11438 increased the antigenicity and the ability of the antibodies induced by peptides of inhibiting EBV invasion of host cells.  相似文献   

19.
The presence of a high number of activated T cells in the bloodstream and spontaneous proliferation of peripheral blood mononuclear cells in vitro are striking characteristics of human T-cell leukemia virus type I (HTLV-I) infection. The HTLV-I regulatory protein Tax and the envelope protein gp46 have been implicated in mediating the activation process. In this study, HTLV-I-producing cell lines and purified virus from the cell lines were examined for the ability to activate peripheral blood lymphocytes (PBLs) and Jurkat cells. Antisera and monoclonal antibodies against several cellular adhesion proteins involved in T-cell activation and against viral proteins were used to identify which molecules may be participating in the activation process. First, neither virus from a T-cell line, MT2, nor virus produced from the human osteosarcoma cell line HOS/PL was able to induce PBLs to proliferate. In contrast, both fixed and irradiated HTLV-I-producing T-cell lines induced proliferation of PBLs; HOS/PL cells did not activate PBLs. Second, HTLV-I-positive T-cell lines were capable of activating interleukin-2 mRNA expression in Jurkat cells. Induction of interleukin-2 expression was inhibited by anti-CD2 and anti-lymphocyte function-associated antigen 3 (LFA-3) monoclonal antibodies but not anti-human leukocyte antigen-DR, anti-CD4, anti-LFA-1, or anti-intercellular adhesion molecule 1. Similar results were obtained with PBLs as the responder cells. Furthermore, monoclonal antibodies and antisera against various regions of the HTLV-I envelope proteins gp46 and gp21 as well as p40tax did not block activation. These data indicate that HTLV-I viral particles are not intrinsically mitogenic and that infection of target T cells is not necessary for activation. Instead, the mitogenic activity is restricted to virus-producing T cells, requires cell-to-cell contact, and may be mediated through the LFA-3/CD2 activation pathway.  相似文献   

20.
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.  相似文献   

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