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PubMed1987

Identification of gp350 as the viral glycoprotein mediating attachment of Epstein-Barr virus (EBV) to the EBV/C3d receptor of B cells: sequence homology of gp350 and C3 complement fragment C3d.

The major Epstein-Barr virus (EBV) envelope glycoprotein, gp350, was purified from the B95-8 cell line and analyzed for its ability to mediate virus attachment to the isolated EBV/C3d receptor (CR2) of human B lymphocytes. Purified gp350 and EBV, but not cytomegalovirus, exhibited dose-dependent binding to purified CR2 in dot blot immunoassays. Binding was inhibited by certain monoclonal antibodies to CR2 and to gp350. Liposomes bearing incorporated gp350 bound to CR2-positive B-cell lines but not to CR2-negative lines. Liposome binding was also inhibited by the OKB7 anti-CR2 monoclonal antibody. A computer-generated comparison of the deduced gp350 amino acid sequence with that of the human C3d complement fragment revealed two regions of significant primary sequence homology, a finding which suggests that a common region on these two unrelated proteins may be involved in CR2 binding.

Amino Acid Sequence↗

PubMed1988

Characterization of a T-lymphocyte Epstein-Barr virus/C3d receptor (CD21).

The Epstein-Barr virus/C3d receptor (EBVR-CR2) was detected on three T-lymphoblastoid cell lines. The apparent Mrs of purified EBVR-CR2 of T-cell and B-cell origin were identical. The N-terminal amino acid sequence from the T-cell EBVR-CR2 confirmed the placement of this receptor in a multigene family of complement regulatory proteins. All EBVR-CR2-positive T-cell lines were T6 and T4-T8 antigen positive.

Amino Acid Sequence↗

PubMed1988

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.

Antibodies, Monoclonal↗

PubMed1989

Epstein-Barr virus (EBV)-containing nasopharyngeal carcinoma cells express the B-cell activation antigen blast2/CD23 and low levels of the EBV receptor CR2.

Anaplastic nasopharyngeal carcinoma (NPC) cells invariably harbor the Epstein-Barr virus (EBV) genome, an association that is unique among human virus-associated cancers. Although EBV is able to replicate in epithelial cells, results with expression of the EBV receptor (complement receptor type 2 [CR2]; also called CD21) in normal and malignant epithelial cells are conflicting. We grew five different EBV-associated NPC tumors in nude mice, and by using a sensitive transcriptional assay, we detected a very weak transcription signal of the EBV receptor CR2 gene in these cells. This suggests that low levels of EBV receptor may be expressed by malignant epithelial nasopharyngeal cells. The gene coding for Blast2/CD23, a B-cell activation molecule induced by EBV, was transcribed in three of the transplanted NPC tumors. The soluble form of the Blast2/CD23 protein was also detected in medium taken from short-term cultures of the same NPC cell lines. In contrast to the lymphoid system, in which Blast2/CD23 expression is associated with EBV nuclear antigen (EBNA2) expression, no EBNA2 protein could be detected in these NPC epithelial cells. Our study represents the first demonstration of Blast2/CD23 expression in epithelial cells. As the soluble form of the Blast2/CD23 protein possesses growth factor activity associated with EBV-induced B-cell immortalization, these results suggest a possible role for this molecule in the pathogenesis of NPC.

Animals↗

PubMed1990

Stable transfection of Epstein-Barr virus (EBV) nuclear antigen 2 in lymphoma cells containing the EBV P3HR1 genome induces expression of B-cell activation molecules CD21 and CD23.

A set of B-cell activation molecules, including the Epstein-Barr virus (EBV) receptor CR2 (CD21) and the B-cell activation antigen CD23 (Blast2/Fc epsilon RII), is turned on by infecting EBV-negative B-lymphoma cell lines with immortalizing strains of the viruslike B95-8 (BL/B95 cells). This up regulation may represent one of the mechanisms involved in EBV-mediated B-cell immortalization. The P3HR1 nonimmortalizing strain of the virus, which is deleted for the entire Epstein-Barr nuclear antigen 2 (EBNA2) protein open reading frame, is incapable of inducing the expression of CR2 and CD23, suggesting a crucial role for EBNA2 in the activation of these molecules. In addition, lymphoma cells containing the P3HR1 genome (BL/P3HR1 cells) do not express the viral latent membrane protein (LMP), which is regularly expressed in cells infected with immortalizing viral strains. Using electroporation, we have transfected the EBNA2 gene cloned in an episomal vector into BL/P3HR1 cells and have obtained cell clones that stably express the EBNA2 protein. In these clones, EBNA2 expression was associated with an increased amount of CR2 and CD23 steady-state RNAs. Of the three species of CD23 mRNAs described, the Fc epsilon RIIa species was preferentially expressed in these EBNA2-expressing clones. An increased cell surface expression of CR2 but not of CD23 was observed, and the soluble form of CD23 molecule (SCD23) was released. We were, however, not able to detect any expression of LMP in these cell clones. These data demonstrate that EBNA2 gene is able to complement P3HR1 virus latent functions to induce the activation of CR2 and CD23 expression, and they emphasize the role of EBNA2 protein in the modulation of cellular gene implicated in B-cell proliferation and hence in EBV-mediated B-cell immortalization. Nevertheless, EBNA2 expression in BL/P3HR1 cells is not able to restore the level of CR2 and CD23 expression observed in BL/B95 cells, suggesting that other cellular or viral proteins may also have an important role in the activation of these molecules: the viral LMP seems to be a good candidate.

Antigens, CD↗

PubMed1990

Soluble recombinant CR2 (CD21) inhibits Epstein-Barr virus infection.

Epstein-Barr virus (EBV), an oncogenic herpesvirus of humans, displays selective tropism for B lymphocytes and epithelial cells. EBV tropism is thought to be determined in part by a unique host cell receptor termed CR2 (CD21). Although previous studies have demonstrated that CR2 mediates EBV binding to B cells, its role in initiating EBV infection and B-cell transformation is less certain. In the studies reported here, soluble recombinant CR2 was shown to cause substantial inhibition of EBV infection of B cells in vitro, indicating that CR2 binding initiates EBV infection. Soluble CR2 may represent a therapeutic agent for acute and chronic EBV infections in humans.

Antigens, CD↗

PubMed1991

Epstein-Barr virus nuclear protein 2 mutations define essential domains for transformation and transactivation.

Epstein-Barr virus (EBV) nuclear protein 2 (EBNA-2) is essential for B-lymphocyte growth transformation. EBNA-2 transactivates expression of the EBV latent membrane protein (LMP-1) and also transactivates expression of the B-lymphocyte proteins CD21 and CD23. In order to analyze the functional domains of EBNA-2, we constructed 11 linker-insertion and 15 deletion mutations. Each of the mutant EBNA-2 proteins localized to the nucleus, and each was expressed at levels similar to wild-type EBNA-2. Deletion of both EBNA-2 basic domains was required to prevent nuclear localization, indicating that either is sufficient for nuclear translocation. The mutant EBNA-2 genes were assayed for lymphocyte transformation after recombination with the EBNA-2-deleted P3HR-1 EBV genome and for LMP-1 transactivation following transfection into P3HR-1-infected B-lymphoma cells. Cell lines transformed by recombinant EBV carrying EBNA-2 mutations were assayed for growth properties and LMP-1, CD21, and CD23 expression. The mutational analysis indicates that at least four separate EBNA-2 domains are essential for lymphocyte transformation. Two other domains are necessary for the full transforming phenotype. Two deletion and eight linker-insertion mutations did not reduce transforming activity. Mutations which diminish or abolish lymphocyte transformation also diminish or abolish LMP-1 transactivation, respectively. Cells transformed by recombinant EBV carrying EBNA-2 genes with diminished or normal transforming activity all expressed high levels of LMP-1, CD23, and CD21. These findings suggest that transactivation of these viral and cellular genes by EBNA-2 plays a critical role in lymphocyte transformation by EBV. Furthermore, these results indicate that the transformation and transactivation functions of EBNA-2 may not be separable.

Amino Acid Sequence↗

PubMed1991

Inhibition of Epstein-Barr virus infection in vitro and in vivo by soluble CR2 (CD21) containing two short consensus repeats.

The extracellular domain of CR2, the Epstein-Barr virus (EBV)/C3d receptor of B lymphocytes, contains 15 or 16 tandemly arranged short consensus repeat elements (SCR). Recombinant CR2 proteins containing SCR 1 and 2 fused to Staphylococcus aureus protein A (PA-CR2) and to murine complement factor H SCR 20 (CR2FH) were expressed in Escherichia coli and in insect cells, respectively. These recombinant CR2 molecules retained functional activity as indicated by their ability to bind to C3dg in an enzyme-linked immunosorbent assay and to inhibit EBV gp350/220 binding to B cells. PA-CR2 and CR2FH were as efficient in blocking EBV gp350/220 binding as the full-length CR2 extracellular domain, indicating that the first two SCR of CR2 contain the majority of the ligand binding activity of the receptor. PA-CR2 and CR2FH inhibited EBV-induced B-cell proliferation in vitro and blocked the development of EBV-induced lymphoproliferative disease in severe combined immunodeficient mice reconstituted with human lymphocytes. These studies indicate that soluble forms of truncated CR2 proteins may have potential therapeutic value in the treatment of EBV-induced lymphoproliferative disorders in humans that involve viral replication.

Amino Acid Sequence↗

PubMed1993

Complement-mediated binding of naturally glycosylated and glycosylation-modified human immunodeficiency virus type 1 to human CR2 (CD21).

Particulate glycoproteins lacking sialic acid, such as desialylated enveloped viruses, readily activate complement through the alternative pathway. Human immunodeficiency virus type 1 (HIV-1) contains two heavily glycosylated and partially sialylated envelope glycoproteins: a surface gp120 and a transmembrane gp41. The abilities of naturally glycosylated HIV-1 and glycosylation-modified HIV-1 to interact with the complement system were examined with a biological assay which measured the binding of whole virus particles to cells expressing CR2 (CD21), the complement receptor found naturally in abundance on follicular dendritic cells and immature B cells. HIV-1 IIIB was synthesized in the presence or absence of the mannosidase II inhibitor, swainsonine, to give rise to high-mannose-type, nonsialylated, nonfucosylated carbohydrate moieties. The virus also was treated with neuraminidase or endo-beta-galactosidase to remove terminal sialic acids. An enzyme immunoassay specific for HIV-1 p24 core protein was used to quantitate the amount of virus bound to cell surfaces. Virus particles incubated with 1:3-diluted, fresh HIV-1-negative human serum as a source of complement readily bound to MT-2 (CD4+ CR2+) and Raji-3 (CD4- CR2+) cells but not to CEM (CD4+ CR2-) cells, suggesting that the virus bound to CR2 independently of CD4. Compared with heat-inactivated or C3-deficient sera, fresh complement increased binding by as much as 62 times for naturally glycosylated virus, and 5 times more than this for glycosylation-modified virus. Similar observations were made with freshly isolated, non-mitogen-stimulated peripheral blood mononuclear cells. Additional evidence that HIV-1 bound to CR2 independently of CD4 was provided by the fact that binding was blocked by monoclonal antibody OKB7 (anti-CR2) but not by OKT4a (anti-CD4). Also, the virus bound to transfected K562 cells (CD4-) which expressed recombinant human CR2 but did not bind to untransfected K562 cells. Results obtained with complement component-deficient sera indicated that binding required the alternative complement pathway. Raji-3 and transfected K562 cells could not be infected with HIV-1 in the presence of complement, suggesting that utilization of CR2 as a receptor in the absence of CD4 does not allow virus entry. The demonstration of CR2 as a receptor for HIV-1 in the presence of complement, together with the ability to enhance binding by desialylation, provides new insights into mechanisms of HIV-1-induced immunity and immunopathogenesis.

Acquired Immunodeficiency Syndrome↗

PubMed1994

Determination of the role for CD21 during Epstein-Barr virus infection of B-lymphoblastoid cells.

Epstein-Barr virus (EBV), a herpesvirus with oncogenic potential, is camouflaged with glycoprotein 350/220, which mimics the human ligand C3dg and thereby binds to and exploits complement receptor type 2 (CR2; CD21), the EBV receptor. It has not been possible to determine the role of CR2 during postbinding events of viral infection because all B lymphocytes express endogenous CR2, precluding an informative study of receptor mutants. We have overcome this obstacle through creation of a novel experimental system based on molecular dissection of the ligand-binding domains of human CR2 and murine CR2. Our results demonstrate first, that two discontinuous amino acid substitutions within the ligand-binding domain of murine CR2 render it capable of mediating EBV infection of human B-lymphoblastoid cells, and second, that the specific role of CR2 during EBV infection is to capture virions at the cell surface, after which cofactors not associated with CR2 mediate postbinding events. These are the first studies to be described in which a cell that is normally susceptible to viral infection can be manipulated so as to direct entry of virions via recombinant or endogenous receptors.

Amino Acid Sequence↗

PubMed1997

Epstein-Barr virus infection of human gastric carcinoma cells: implication of the existence of a new virus receptor different from CD21.

Recombinant Epstein-Barr virus (EBV) with a selectable marker successfully infected the human gastric carcinoma cell lines AGS, MKN28, and MKN74. Following incubation in selective media, drug-resistant cell clones were isolated and proved to be infected with EBV. All gastric carcinoma cell clones were positive for EBNA 1 but negative for EBNA 2. LMP 1 expression was negative in most clones, but there were a few exceptions. Gastric carcinoma cells were negative for the EBV receptor CD21, and infection was not inhibited by pretreatment of cells with the anti-CD21 monoclonal antibody OKB7. The results indicate that gastric carcinoma cells are susceptible to EBV infection and that infection is mediated via a new receptor different from CD21.

Cell Line↗

PubMed1999

Epstein-Barr virus infection of human astrocyte cell lines.

Epstein-Barr virus (EBV) is implicated in different central nervous system syndromes. The major cellular receptor for EBV, complement receptor type 2 (CR2) (CD21), is expressed by different astrocyte cell lines and human fetal astrocytes, suggesting their susceptibility to EBV infection. We demonstrated the infection of two astrocyte cell lines, T98 and CB193, at low levels. As infection was mediated by CR2, we used two stable CR2 transfectant astrocyte cell lines (T98CR2 and CB193CR2) to achieve a more efficient infection. We have monitored EBV gene expression for 2 months and observed the transient infection of T98 and T98CR2 cells and persistent infection of CB193 and CB193CR2 cells. The detection of BZLF1, BALF2, and BcLF1 mRNA expression suggests that the lytic cycle is initiated at early time points postinfection. At later time points the pattern of mRNA expressed (EBER1, EBNA1, EBNA2, and LMP1) differs from latency type III in the absence of LMP2A transcription and in the expression of BALF2 and BcLF1 but not BZLF1. A reactivation of the lytic cycle was achieved in CB193CR2 cells by the addition of phorbol esters. These studies identify astrocyte cell lines as targets for EBV infection and suggest that this infection might play a role in the pathology of EBV in the brain.

Astrocytes↗

PubMed2000

Activated Notch1 modulates gene expression in B cells similarly to Epstein-Barr viral nuclear antigen 2.

Both Epstein-Barr viral nuclear antigen 2 (EBNA2) and activated Notch transactivate genes by interacting with the transcription factor RBP-Jkappa. The viral protein EBNA2 may hence be regarded as a functional equivalent of an activated Notch receptor. Until now, nothing has been known about the physiological role of Notch signaling in B cells. Here we investigated whether activated Notch can induce the same phenotypic changes as EBNA2 in Burkitt's lymphoma cells. An estrogen receptor fusion protein of the intracellular part of mouse Notch 1 (mNotch1-IC), mimicking in the presence of estrogen a constitutively active Notch receptor, was stably transfected into the Burkitt's lymphoma cell lines BL41-P3HR1 and HH514. Northern blot analysis revealed that the LMP2A gene is induced by Notch-IC in the presence of estrogen, whereas increased expression of LMP1 could be detected only if cycloheximide was simultaneously added. Concerning the cellular genes regulated by EBNA2, Notch-IC was able to upregulate CD21 but not CD23 expression. Immunoglobulin mu (Igmu) expression, which is downregulated by EBNA2, was also negatively regulated by Notch-IC. Similarly to EBNA2, Notch-IC was able to repress c-myc expression, which is under the control of the immunoglobulin heavy-chain locus in Burkitt's lymphoma cells with a t(8;14) translocation. The data show that Notch-IC is able to participate in gene regulation in B cells.

Animals↗

PubMed2000

Epstein-Barr virus entry utilizing HLA-DP or HLA-DQ as a coreceptor.

Epstein-Barr virus (EBV) glycoprotein gp350/gp220 association with cellular CD21 facilitates virion attachment to B lymphocytes. Membrane fusion requires the additional interaction between virion gp42 and cellular HLA-DR. This binding is thought to catalyze membrane fusion through a further association with the gp85-gp25 (gH-gL) complex. Cell lines expressing CD21 but lacking expression of HLA class II molecules are resistant to infection by a recombinant EBV expressing enhanced green fluorescent protein. Surface expression of HLA-DR, HLA-DP, or HLA-DQ confers susceptibility to EBV infection on resistant cells that express CD21. Therefore, HLA-DP or HLA-DQ can substitute for HLA-DR and serve as a coreceptor in EBV entry.

Cell Line↗

PubMed2003

Epstein-Barr Virus nuclear protein EBNA3A is critical for maintaining lymphoblastoid cell line growth.

To evaluate the role of Epstein-Barr Virus (EBV) nuclear antigen 3A (EBNA3A) in the continuous proliferation of EBV-infected primary B lymphocytes as lymphoblastoid cell lines (LCLs), we derived LCLs that are infected with a recombinant EBV genome that expresses EBNA3A fused to a 4-hydroxy-tamoxifen (4HT)-dependent mutant estrogen receptor hormone binding domain (EBNA3AHT). The LCLs grew similarly to wild-type LCLs in medium with 4HT despite a reduced level of EBNA3AHT fusion protein expression. In the absence of 4HT, EBNA3AHT moved from the nucleus to the cytoplasm and was degraded. EBNA3AHT-infected LCLs were unable to grow in medium without 4HT. The precise time to growth arrest varied inversely with cell density. Continued maintenance in medium without 4HT resulted in cell death, whereas readdition of 4HT restored cell growth. Expression of other EBNAs and LMP1, of CD23, and of c-myc was unaffected by EBNA3A inactivation. Wild-type EBNA3A expression from an oriP plasmid transfected into the LCLs protected the EBNA3AHT-infected LCLs from growth arrest and death in medium without 4HT, whereas EBNA3B or EBNA3C expression was unable to protect the LCLs from growth arrest and death. These experiments indicate that EBNA3A has a unique and critical role for the maintenance of LCL growth and ultimately survival. The EBNA3AHT-infected LCLs are also useful for genetic and biochemical analyses of the role of EBNA3A domains in LCL growth.

B-Lymphocytes↗

PubMed2004

Use of gHgL for attachment of Epstein-Barr virus to epithelial cells compromises infection.

Epstein-Barr virus (EBV) is a lymphotropic herpesvirus. However, access to B lymphocytes during primary infection may be facilitated by replication in mucosal epithelial cells. Attachment and penetration of EBV into these two cell types are fundamentally different. Both the distribution of receptors and the cellular origin of the virus impact the efficiency of infection. Epithelial cells potentially offer a wide range of receptors with which virus can interact. We report here on analyses of epithelial cells expressing different combinations of receptors. We find that the stoichiometry of the virus glycoprotein complex that includes gHgL and gp42 affects the use of gHgL not just for entry into epithelial cells but also for attachment. Penetration can be mediated efficiently with either a coreceptor for gp42 or gHgL, but the use of gHgL for attachment as well as penetration greatly compromises its ability to mediate entry.

Cell Line, Tumor↗

PubMed2005

Activation of CD21 and CD23 gene expression by Kaposi's sarcoma-associated herpesvirus RTA.

Epstein-Barr virus (EBV) EBNA2 and Kaposi's sarcoma-associated herpesvirus (KSHV) replication and transcription activator (RTA) are recruited to their responsive elements through interaction with a Notch-mediated transcription factor, RBP-Jkappa. In particular, RTA and EBNA2 interactions with RBP-Jkappa are essential for the lytic replication of KSHV and expression of B-cell activation markers CD21 and CD23a, respectively. Here, we demonstrate that like EBV EBNA2, KSHV RTA strongly induces CD21 and CD23a expression through RBP-Jkappa binding sites in the first intron of CD21 and in the CD23a core promoter, respectively. However, unlike EBV EBNA2, which alters immunoglobulin mu (Igmu) and c-myc gene expression, RTA did not affect Igmu and c-myc expression, indicating that KSHV RTA targets the Notch signal transduction pathway in a manner similar to but distinct from that of EBV EBNA2. Furthermore, RTA-induced expression of CD21 glycoprotein, which is an EBV receptor, efficiently facilitated EBV infection. In addition, RTA-induced CD23 glycoprotein underwent proteolysis and gave rise to soluble CD23 (sCD23) molecules in B lymphocytes and KSHV-infected primary effusion lymphocytes. sCD23 then stimulated primary human lymphocytes. These results demonstrate that cellular CD21 and CD23a are common targets for B lymphotropic gammaherpesviruses and that KSHV RTA regulates RBP-Jkappa-mediated cellular gene expression, which ultimately provides a favorable milieu for viral reproduction in the infected host.

Antigens, CD↗

PubMed1988

Expression of CD 21, CD 22, and the mouse erythrocyte receptor on peripheral B lymphocytes in rheumatoid arthritis.

The expression of the B cell antigens, CD 21, CD 22, and the mouse erythrocyte receptor (MER), on peripheral mononuclear cells (PMC) in 61 patients with rheumatoid arthritis (RA) and in 25 patients with various other forms of rheumatic disease was studied. Patients with RA showed significantly more peripheral B cells than control patients, whereas there was no difference between patients with RA and controls in resting B cells expressing the MER or resting and activated B cells expressing CD 21. Patients with active RA had significantly less MER positive and more CD 21 positive B cells than patients with inactive disease. The relation between disease activity and expression of MER and CD 21 was independent of drug treatment or production of classical rheumatoid factor. These data may be interpreted as a sign of B cell activation in RA. In addition, patients with seronegative RA receiving gold treatment showed significantly more MER positive cells than patients receiving different drugs, whereas patients receiving non-steroidal anti-inflammatory drugs (NSAIDs) alone had significantly more CD 21 positive cells. This may be the result of different immunomodulating effects of drugs on B cell subsets.

Antigens, CD↗