Tropism Of Epstein-Barr Virus Research Articles

Memory B cells producing somatically mutated antiphospholipid antibodies are present in healthy individuals

AbstractAntiphospholipid antibodies (aPLs) are associated with thrombosis and recurrent abortions during autoimmune pathologies, but they are also produced in healthy individuals and during infectious diseases. To analyze the possible links between physiologic and pathologic aPLs, it is of importance to characterize normal aPL production. We took advantage of the known tropism of Epstein-Barr virus (EBV) for B cells in general, and memory B cells in particular, during primary infectious mononucleosis (IMN) in 3 patients to get access to anticardiolipin (aCL)-producing B cells. Flow cytometry analysis of these cells showed that, depending on the patient, 10% to 60% of immunoglobulin M (IgM) aCL-producing B cells express the CD27 marker of memory B cells. Single cell sorting of aCL B cells, followed by single cell reverse transcription-polymerase chain reaction (RT-PCR) amplification of their immunoglobulin variable region genes, showed that some of these cells produce mutated forms of aCL antibodies, confirming their memory B-cell origin. Considering that, during primary IMN, EBV infects and expands already pre-existing memory B cells, we conclude that healthy individuals have a discrete pool of aCL memory cells able to produce mutated forms of antibodies. The implications of this new information are discussed in light of different hypotheses regarding the origin of aCL. (Blood. 2003;102:2459-2465)

Infection and immune control of human oncogenic γ-herpesviruses in humanized mice.

Epstein-Barr virus (EBV) and Kaposi sarcoma-associated herpesvirus (KSHV) comprise the oncogenic human γ-herpesvirus family and are responsible for 2-3% of all tumours in man. With their prominent growth-transforming abilities and high prevalence in the human population, these pathogens have probably shaped the human immune system throughout evolution for near perfect immune control of the respective chronic infections in the vast majority of healthy pathogen carriers. The exclusive tropism of EBV and KSHV for humans has, however, made it difficult in the past to study their infection, tumourigenesis and immune control in vivo. Mice with reconstituted human immune system components (humanized mice) support replication of both viruses with both persisting latent and productive lytic infection. Moreover, B-cell lymphomas can be induced by EBV alone and KSHV co-infection with gene expression hallmarks of human malignancies that are associated with both viruses. Furthermore, cell-mediated immune control by primarily cytotoxic lymphocytes is induced upon infection and can be probed for its functional characteristics as well as putative requirements for its priming. Insights that have been gained from this model and remaining questions will be discussed in this review. This article is part of the theme issue 'Silent cancer agents: multi-disciplinary modelling of human DNA oncoviruses'.

Prevalence of Epstein–Barr Virus Genotypes in Pakistani Lymphoma Patients

The Epstein-Barr virus (EBV) is a herpesvirus infecting more than 90% of the human population. The tropism of EBV for B lymphocytes is evidenced in its association with many lymphoproliferative disorders. Different types of EBV (EBV-1 and EBV-2), classified on the basis of EBV nuclear antigen-2 (EBNA-2) genotyping, have been reported in benign and malignant pathologies, but there is almost no information about their frequency in the Pakistani population. The aim of this study was to determine the frequency and distribution of EBNA-2-based EBV genotypes in lymphoma patients. Genomic DNA was extracted from formalin-fixed paraffin embedded (FFPE) tissue samples obtained from 73 EBV-DNA-positive lymphoma patients. The β-globin gene was amplified to assess the presence and quality of cellular DNA from all samples. EBER-1 DNA was detected by PCR to confirm EBV presence in tissue samples. EBNA-1 mRNA relative quantification done by quantitative PCR substantiated EBNA-1 mRNA overexpression in 43.8% of EBV-positive cases in comparison to EBV-positive control cell line. EBNA-2 genotyping was done by nested PCR. Among typable samples, EBV-1 was found in 90.7% of samples while EBV-2 was present in 9.3% cases. These results show that EBV-1 was the most prevalent type in the lymphoma population of Pakistan. This epidemiology of EBV in Pakistani lymphoma patients represents an important first step in using EBV for prognosis and monitoring treatment response.

Epstein-Barr Virus Fusion with Epithelial Cells Triggered by gB Is Restricted by a gL Glycosylation Site.

Epstein-Barr virus (EBV) entry into epithelial cells is mediated by the conserved core fusion machinery, composed of the fusogen gB and the receptor-binding complex gH/gL. The heterodimeric gH/gL complex binds to the EBV epithelial cell receptor or gp42, which binds to the B-cell receptor, triggering gB-mediated fusion of the virion envelope with cellular membranes. Our previous study found that the gL glycosylation mutant N69L/S71V had an epithelial cell-specific hyperfusogenic phenotype. To study the influence of this gL mutant on the initiation and kinetics of gB-driven epithelial cell fusion, we established a virus-free split-green fluorescent protein cell-cell fusion assay that enables real-time measurements of membrane fusion using live cells. The gL_N69L/S71V mutant had a large increase in epithelial cell fusion activity of up to 300% greater than that of wild-type gL starting at early time points. The hyperfusogenicity of the gL mutant was not a result of alterations in complex formation with gH or alterations in cellular localization. Moreover, the hyperfusogenic phenotype of the gL mutant correlated with the formation of enlarged syncytia. In summary, our present findings highlight an important role of gL in the kinetics of gB-mediated epithelial cell fusion, adding to previous findings indicating a direct interaction between gL and gB in EBV membrane fusion.IMPORTANCE EBV predominantly infects epithelial cells and B lymphocytes, which are the cells of origin for the EBV-associated malignancies Hodgkin and Burkitt lymphoma as well as nasopharyngeal carcinoma. Contrary to the other key players of the core fusion machinery, gL has the most elusive role during EBV-induced membrane fusion. We found that the glycosylation site N69/S71 of gL is involved in restricting epithelial cell fusion activity, strongly correlating with syncytium size. Interestingly, our data showed that the gL glycosylation mutant increases the fusion activity of the hyperfusogenic gB mutants, indicating that this gL mutant and the gB mutants target different steps during fusion. Our studies on how gL and gB work together to modulate epithelial cell fusion kinetics are essential to understand the highly tuned tropism of EBV for epithelial cells and B lymphocytes and may result in novel strategies for therapies preventing viral entry into target host cells. Finally, making our results of particular interest is the absence of gL syncytial mutants in other herpesviruses.

Structural basis for Epstein-Barr virus host cell tropism mediated by gp42 and gHgL entry glycoproteins.

Herpesvirus entry into host cells is mediated by multiple virally encoded receptor binding and membrane fusion glycoproteins. Despite their importance in host cell tropism and associated disease pathology, the underlying and essential interactions between these viral glycoproteins remain poorly understood. For Epstein–Barr virus (EBV), gHgL/gp42 complexes bind HLA class II to activate membrane fusion with B cells, but gp42 inhibits fusion and entry into epithelial cells. To clarify the mechanism by which gp42 controls the cell specificity of EBV infection, here we determined the structure of gHgL/gp42 complex bound to an anti-gHgL antibody (E1D1). The critical regulator of EBV tropism is the gp42 N-terminal domain, which tethers the HLA-binding domain to gHgL by wrapping around the exterior of three gH domains. Both the gp42 N-terminal domain and E1D1 selectively inhibit epithelial-cell fusion; however, they engage distinct surfaces of gHgL. These observations clarify key determinants of EBV host cell tropism.

Structural and Mechanistic Insights into the Tropism of Epstein-Barr Virus.

Epstein-Barr virus (EBV) is the prototypical γ-herpesvirus and an obligate human pathogen that infects mainly epithelial cells and B cells, which can result in malignancies. EBV infects these target cells by fusing with the viral and cellular lipid bilayer membranes using multiple viral factors and host receptor(s) thus exhibiting a unique complexity in its entry machinery. To enter epithelial cells, EBV requires minimally the conserved core fusion machinery comprised of the glycoproteins gH/gL acting as the receptor-binding complex and gB as the fusogen. EBV can enter B cells using gp42, which binds tightly to gH/gL and interacts with host HLA class II, activating fusion. Previously, we published the individual crystal structures of EBV entry factors, such as gH/gL and gp42, the EBV/host receptor complex, gp42/HLA-DR1, and the fusion protein EBV gB in a postfusion conformation, which allowed us to identify structural determinants and regions critical for receptor-binding and membrane fusion. Recently, we reported different low resolution models of the EBV B cell entry triggering complex (gHgL/gp42/HLA class II) in “open” and “closed” states based on negative-stain single particle electron microscopy, which provide further mechanistic insights. This review summarizes the current knowledge of these key players in EBV entry and how their structures impact receptor-binding and the triggering of gB-mediated fusion.

Sequence Variation Analysis of Epstein-Barr Virus Nuclear Antigen 1 Gene in the Virus Associated Lymphomas of Northern China.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is the only viral protein expressed in all EBV-positive tumors as it is essential for the maintenance, replication and transcription of the virus genome. According to the polymorphism of residue 487 in EBNA1 gene, EBV isolates can be classified into five subtypes: P-ala, P-thr, V-val, V-leu and V-pro. Whether these EBNA1 subtypes contribute to different tissue tropism of EBV and are consequently associated with certain malignancies remain to be determined. To elucidate the relationship, one hundred and ten EBV-positive lymphoma tissues of different types from Northern China, a non-NPC endemic area, were tested for the five subtypes by nested-PCR and DNA sequencing. In addition, EBV type 1 and type 2 classification was typed by using standard PCR assays across type-specific regions of the EBNA3C genes. Four EBNA1 subtypes were identified: V-val (68.2%, 75/110), P-thrV (15.5%, 17/110), V-leuV (3.6%, 4/110) and P-ala (10.9%, 12/110). The distribution of the EBNA1 subtypes in the four lymphoma groups was not significantly different (p = 0.075), neither was that of the EBV type 1/type 2 (p = 0.089). Compared with the previous data of gastric carcinoma (GC), nasopharyngeal carcinoma (NPC) and throat washing (TW) from healthy donors, the distribution of EBNA1 subtypes in lymphoma differed significantly (p = 0.016), with a little higher frequency of P-ala subtype. The EBV type distribution between lymphoma and the other three groups was significantly different (p = 0.000, p = 0.000, p = 0.001, respectively). The proportion of type 1 and type 2 mixed infections was higher in lymphoma than that in GC, NPC and TW. In lymphomas, the distribution of EBNA1 subtypes in the three EBV types was not significantly different (p = 0.546). These data suggested that the variation patterns of EBNA1 gene may be geographic-associated rather than tumor-specific and the role of EBNA1 gene variations in tumorigenesis needs more extensive and deep explorations.

SLAM-associated protein modulates CD8 T cell responses primed by antigen-presenting B cells (IRM4P.503)

Abstract Mutations in the SH2D1A gene, that encodes SLAM-associated protein (SAP), cause X-linked lymphoproliferative disease (XLP), a rare congenital immunodeficiency defined by exquisite sensitivity to Epstein-Barr virus (EBV). The inability of boys with XLP to control EBV infection results in fulminant, often fatal, infectious mononucleosis, massive expansions of EBV-infected B cells and malignant B cell lymphomas. However, the precise mechanism of how SAP loss contributes to susceptibility to EBV remains unclear. Given the unique B cell tropism of EBV and the purported role of SAP in regulating lymphocyte/lymphocyte interactions, we hypothesized that SAP expression within CD8 T cells is critical for T cells to mount responses towards antigen-expressing B cells. Wild type and Sh2d1a-/- CD8 T cells responded strongly when stimulated in vitro with cognate antigen-coated whole splenocytes. However, Sh2d1a-/- CD8 T cells exhibited greatly diminished proliferation and effector functions relative to wild type CD8 T cells when cultured with antigen-coated B cells in vitro or challenged with antigen-expressing B cell lymphoma in vivo. These results demonstrate that SAP is required for the priming of CD8 T cells when B cells act as the primary APC. Together, our findings suggest that the susceptibility of XLP patients to EBV infection may at least in part be a consequence of an inability of their naïve CD8 T cells to differentiate into effectors upon encountering virally-infected B cells.

Epstein–Barr Virus Infection Induces Aberrant TLR Activation Pathway and Fibroblast–Myofibroblast Conversion in Scleroderma

Scleroderma (SSc) is a complex and heterogeneous connective tissue disease mainly characterized by autoimmunity, vascular damage, and fibrosis that mostly involve the skin and lungs. Epstein–Barr virus (EBV) is a lymphotropic γ-herpesvirus that has co-evolved with human species, infecting >95% of the adult population worldwide, and has been a leading candidate in triggering several autoimmune diseases. Here we show that EBV establishes infection in the majority of fibroblasts and endothelial cells in the skin of SSc patients, characterized by the expression of the EBV noncoding small RNAs (EBERs) and the increased expression of immediate-early lytic and latency mRNAs and proteins. We report that EBV is able to persistently infect human SSc fibroblasts in vitro, inducing an aberrant innate immune response in infected cells. EBV–Toll-like receptor (TLR) aberrant activation induces the expression of selected IFN-regulatory factors (IRFs), IFN-stimulated genes (ISGs), transforming growth factor-β1 (TGFβ1), and several markers of fibroblast activation, such as smooth muscle actin and Endothelin-1, and all of these genes play a key role in determining the profibrotic phenotype in SSc fibroblasts. These findings imply that EBV infection occurring in mesenchymal, endothelial, and immune cells of SSc patients may underlie the main pathological features of SSc including autoimmunity, vasculopathy, and fibrosis, and provide a unified disease mechanism represented by EBV reactivation.

Epstein‐Barr Virus Infects B and Non‐B Lymphocytes in HIV‐1–Infected Children and Adolescents

Epstein-Barr virus (EBV) is a widespread, persistent herpesvirus that can transform B cells and that is associated with malignant lymphomas. EBV dynamics and specific immunity in human immunodeficiency virus (HIV)-1-infected children are unknown. We found that, in 74% of EBV-seropositive, HIV-1-infected children, EBV DNA loads at the start of highly active antiretroviral therapy (HAART) were comparable with those in acutely EBV-infected, HIV-negative children. EBV DNA load remained elevated in most HIV-1-infected children for months to years of follow-up. Frequencies of interferon-gamma-producing EBV-specific CD8+ T cells were comparable with those in healthy control children, and antibodies to EBV nuclear antigen were detected in 73% of EBV-seropositive children. Detectable EBV DNA load was not correlated with HIV-1 RNA level or with CD4+ T cell count increase after the start of HAART. Because of its resemblance to chronic active EBV, we studied the cellular tropism of EBV in these patients. EBV DNA was found not only in the CD19+ B cell fraction but also--at stable levels--in the CD4+ and CD8+ T cell fractions. Although the reason for the aberrant T cell tropism of EBV remains unclear, these data may provide an explanation for the differential EBV dynamics in the presence of normal serological findings.

Capacity of Epstein-Barr virus to infect monocytes and inhibit their development into dendritic cells is affected by the cell type supporting virus replication.

Epstein-Barr virus (EBV) is a ubiquitous human herpesvirus that is involved in the pathogenesis of a wide spectrum of malignant and non-malignant diseases. Strong evidence implicates T lymphocytes in the control of EBV replication and tumorigenesis, but cellular components of the innate immune system are poorly characterized in terms of their function in the development of EBV-specific immunity or interaction with the virus. This study demonstrates that EBV virions produced in epithelial cells surpass their B cell-derived counterparts in the capacity to enter monocytes and inhibit their development into dendritic cells (DCs). Different ratios of the gp42 and gH glycoproteins in the envelope of virions that were derived from major histocompatibility complex class II-positive or -negative cells accounted primarily for the differences in EBV tropism. EBV is shown to enter both monocytes and DCs, although the cells are susceptible to virus-induced apoptosis only if infected at early stages of DC differentiation. The purified gH/gL heterodimer binds efficiently to monocytes and DCs, but not to B cells, suggesting that high expression levels of a putative binding partner for gH contribute to virus entry. This entry takes place despite very low or undetectable expression of CD21, the canonical EBV receptor. These results indicate that the site of virus replication, either in B cells or epithelial cells, alters EBV tropism for monocytes and DCs. This results in a change in the virus's immunomodulating capacity and may have important implications for the regulation of virus-host interactions during primary and chronic EBV infection.

Epstein-Barr Virus Infection of CR2-Transfected Epithelial Cells Reveals the Presence of MHC Class II on the Virion

Epithelial cell lines transfected with the Epstein-Barr virus (EBV) receptor CR2 are susceptible to infection by EBV. Following infection with certain EBV strains we found that these cells became positive for MHC class II. The class II was confirmed as being of viral and not target cell origin by immunostaining with HLA-specific monoclonal antibodies. Electron microscopic immunogold staining confirmed the presence of MHC class II on the surface of the virion. While some MHC class I was also found on the EB virion, other cell surface molecules were absent. Dual color immunofluorescence and confocal microscopy analysis demonstrated colocalization of class II with EBV-encoded structural proteins (MA and VCA) in infected epithelial cells. However, preincubation of EBV with antibodies against either MHC class I or MHC class II failed to affect either EBV binding or EBV infection. The presence of MHC on the surface of the EB virion may be a consequence of the intracellular pathways through which productive virus exits from the cell and may influence the target cell tropism of EBV.

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.

Epstein-Barr virus (EBV) infection of murine L cells expressing recombinant human EBV/C3d receptor.

The normal host range of Epstein-Barr virus (EBV) is limited to primate B lymphocytes and certain epithelial cells that express the C3d/EBV receptor [complement receptor 2 (CR2, CD21)]. In the present study, expansion of the tissue tropism of EBV has been accomplished by stably transfecting the murine fibroblast L cell line with pMT.CR2. neo.1, a eukaryotic expression vector promoting the transcription of a complementary DNA insert encoding human CR2. High CR2-expressing transfected L cells were selected by fluorescence-activated cell sorting. The recombinant CR2 was shown to have the same molecular weight as wild-type CR2 from Raji cells and to mediate the binding by the transfectants of particles bearing the iC3b and C3d fragments of the third component of complement. All CR2-expressing L cells, but not nontransfected controls, also bound EBV, as assessed by indirect immunofluorescence. After a 60-hr culture, approximately 0.5% of the CR2-expressing cells preincubated with EBV demonstrated immunofluorescent staining of EBV nuclear antigen with serum from a patient with nasopharyngeal carcinoma. No fluorescent staining of cells was seen with monoclonal antibodies to the early antigen complex or to gp350/220, indicating that the infection was predominantly latent. Infected cells cultured for up to 4 weeks remained EBV nuclear antigen-positive. The capacity of recombinant human CR2 to confer on murine L cells susceptibility to stable latent infection by EBV indicates that this receptor is a primary determinant of the tissue tropism of EBV and may facilitate studies of cell-specific factors that regulate the viral growth cycle.

Role of the CD21 antigen in lymphocyte transformation by Epstein-Barr virus.

The human CD21 membrane antigen has been shown to bind Epstein-Barr Virus (EBV) and some binding and functional data imply a role for this interaction in the B cell tropism of EBV. In this report the technique of limit dilution analysis was used to provide evidence that B cell transformation by EBV only occurred when CD21 antigen was present and that all cells expressing CD21 were transformation-competent. The rate of transformation varied, being increased if B cells were activated by an additional B cell mitogen, and being dependent on, although not strictly proportional to, the intensity of CD21 expression. The possibility that EBV might enter by other means was examined by selectively deleting CD21 antigen from the cell surface. This was accompanied by a profound decrease in the transformation frequency. It is concluded that CD21 antigen is necessary for EBV transformation and that CD21 expression is the major limiting factor in these events.

Infectious mononucleosis fifty years after the discovery of the Paul-Bunnell test.

The Paul-Bunnell test, now 50 years old, is still of fundamental importance in the diagnosis of infectious mononucleosis, even though various immunologic methods have been developed in clinical practice to identify constituents of the Epstein-Barr virus (EBV). The demonstration of sheep red cell agglutinins in infectious mononucleosis (Paul-Bunnell) was in fact the first observation of the presence of heterophil antibodies in this viral disease which was later shown to evoke a polyclonal antibody response to a wide spectrum of autoantigens as well. The selective tropism of EBV for B lymphocytes and the reactivity of T lymphocytes to these infected B lymphocytes are the pathophysiological elements characteristic of infectious mononucleosis, and are the reason for this massive antibody response.