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.     

Interleukin-1beta expression in human gastric carcinoma with Epstein-Barr virus infection

The KT tumor is a transplantable strain of a human Epstein-Barr virus (EBV)-associated gastric carcinoma (EBVaGC), established in severe combined immunodeficiency (SCID) mice, with which the cytokine expression of EBVaGC can be investigated without interference from the infiltrating lymphocytes. As a part of a high-density oligonucleotide array (GeneChip) analysis of EBVaGC, the interleukin-1beta (IL-1beta) gene was the only cytokine gene that showed markedly higher expression in the KT tumor cells than in two tumor strains of EBV-negative GC. The results were confirmed by Northern blotting, Western blotting, and enzyme-linked immunosorbent assay. Furthermore, we demonstrated a positive signal for IL-1beta mRNA in the carcinoma cells of a surgically resected EBVaGC, but not in EBV-negative GC, by in situ hybridization. In vitro, IL-1beta increased the cell growth of a GC cell line, TMK1. Thus, IL-1beta may act as an autocrine growth factor in EBVaGC.     

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.     

Structural characterization of the human B lymphocyte-restricted differentiation antigen CD22. Comparison with CD21 (complement receptor type 2/Epstein-Barr virus receptor)

CD22 and CD21 are glycoproteins primarily expressed on normal and neoplastic human B cells. The surface expression of these two molecules parallel each other during normal B cell differentiation, and the reported relative mobilities for CD22 and CD21 are 130/140 kDa and 140 kDa, respectively. Herein we present a detailed analysis of the biosynthesis and structure of CD22 and also compare it directly to CD21. Electrophoresis under reducing and nonreducing conditions suggested that CD22 and CD21 may have similarities in intra-chain disulfide bond formation. Biosynthesis and processing of CD22 and CD21 were very similar with respect to kinetics and post-translational modification, and both could be phosphorylated. However, endoglycosidase digestion (using N-glycanase and endoglycosidase H) and peptide mapping (using V8 protease and N-chlorosuccinimide) strongly suggested that CD22 and CD21 are distinct gene products.     

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.     

A model for persistent infection with Epstein-Barr virus: the stealth virus of human B cells.

Most adult humans are infected benignly and for life with the herpesvirus Epstein-Barr virus. EBV has been a focus of research because of its status as a candidate tumor virus for a number of lymphomas and carcinomas. In vitro EBV has the ability to establish a latent infection in proliferating B lymphoblasts. This is the only system available for studying human herpesvirus latency in culture and has been extremely useful for elucidating how EBV promotes cellular growth. However, to understand how EBV survives in the healthy host and what goes awry, leading to disease, it is essential to know how EBV establishes and maintains a persistent infection in vivo. Early studies on the mechanism of EBV persistence produced inconclusive and often contradictory results because the techniques available were crude and insensitive. Recent advances in PCR technology and the application of sophisticated cell fractionation techniques have now provided new insights into the behavior of the virus. Most dramatically it has been shown that EBV in vivo does not establish latency in a proliferating lymphoblast, but in a resting memory B cell. The contrasting behaviors of being able to establish a latent infection in proliferating B blasts and resting memory B cells can be resolved in terms of a model where EBV performs its complete life cycle in B lymphocytes. The virus achieves this not by disrupting normal B cell biology but by using it.     

Experimental infection of NOD/SCID mice reconstituted with human CD34+ cells with Epstein-Barr virus

Epstein-Barr virus (EBV)-induced lymphoproliferative disease is an important complication in the context of immune deficiency. Impaired T-cell immunity allows the outgrowth of transformed cells with the subsequent production of predominantly B-cell lymphomas. Currently there is no in vivo model that can adequately recapitulate EBV infection and its association with B-cell lymphomas. NOD/SCID mice engrafted with human CD34(+) cells and reconstituted mainly with human B lymphocytes may serve as a useful xenograft model to study EBV infection and pathogenesis. We therefore infected reconstituted mice with EBV. High levels of viral DNA were detected in the peripheral blood of all infected mice. All infected mice lost weight and showed decreased activity levels. Infected mice presented large visible tumors in multiple organs, most prominently in the spleen. These tumors stained positive for human CD79a, CD20, CD30, and EBV-encoded RNAs and were light chain restricted. Their characterization is consistent with that of large cell immunoblastic lymphoma. In addition, tumor cells expressed EBNA1, LMP1, and LMP2a mRNAs, which is consistent with a type II latency program. EBV(+) lymphoblastoid cell lines expressing human CD45, CD19, CD21, CD23, CD5, and CD30 were readily established from the bone marrow and spleens of infected animals. Finally, we also demonstrate that infection with an enhanced green fluorescent protein (EGFP)-tagged virus can be monitored by the detection of infected EGFP(+) cells and EGFP(+) tumors. These data demonstrate that NOD/SCID mice that are reconstituted with human CD34(+) cells are susceptible to infection by EBV and accurately recapitulate important aspects of EBV pathogenesis.     

Epstein-Barr virus in somatic cell hybrids between mouse cells and human nasopharyngeal carcinoma cells.

Somatic cell hybrids between mouse cells and cells derived directly from NPC biopsies were produced in order to study the association of the Epstein-Barr virus (EBV) genome and the expression of Epstein-Barr nuclear antigen (EBNA) with the human chromosome(s). All attempts to correlate the presence of EBV-DNA and the expression of EBNA with the presence of a particular human chromosome(s) showed that the segregation of EBV-DNA or of EBNA and human chromosomes was dysconcordant. The data, therefore, suggest that in the hybrids studied the presence of EBA-DNA is not determined by the presence of a specific human chromosome.     

Meta-analysis of the relationship between Epstein-Barr virus infection and clinicopathological features of patients with gastric carcinoma

Epstein-Barr virus (EBV) infection has been causally associated with occurrence of many malignant neoplasms. EBV-encoded small RNAs (EBERs) have been detected from about 10% of gastric carcinoma tissue cells, suggesting that EBV infection is associated with the development of gastric carcinoma. The present study pooled the data from the papers concerning EBV-related gastric cancers and performed a meta-analysis of 22 research papers. Among these papers, a total of 5475 cases with gastric cancer were enrolled, of whom 411 cases were found EBV-positive, with the EBV-positive rate being 7.5%. Among the EBV-positive gastric cancer cases, the detection rate was 11.1% in males and 3.0% in females. Compared with EBV-negative gastric cancer, EBV-positive gastric cancer had less lymph node metastasis. Based on the histological typing, of the EBV-positive gastric cancers, the diffuse type was 8.1%, and intestinal type was 8.0%. The examined specimen types included stored paraffin blocks and fresh surgically removed specimens, their EBV positive rates were 7.9% and 6.5% respectively. In terms of geographical distribution, the detection rate of EBV-positive gastric cancer was 9.4% in America, 6.1% in Asia and 9.1% in Europe. Meta-analysis showed that EBV infection occurred only in gastric cancer tissue cells and was significantly associated with the patients’ gender, lymph node metastases, and the location where tumor tissue generated and geographical distribution (P0.05), but was not significantly associated with the patients’ histological types of tumor and the types of specimens (P0.05). These results suggested that EBV-positive gastric cancer has distinct clinicopathological features.     

Persistent Epstein-Barr virus infection in a human T-cell line: unique program of latent virus expression.

The growth transforming potential of Epstein-Barr virus (EBV) for Burkitt's lymphoma and nasopharyngeal carcinoma is now extended to other neoplasia, such as Hodgkin's disease, peripheral T-cell tumor and gastric cancer. We have generated an EBV recombinant with a selectable marker at the viral thymidine kinase locus. Recombinant EBV was successfully infected into a human T-cell line, MT-2. Following incubation in the selective medium, drug resistant MT-2 cell clones were isolated and proved to be infected with recombinant EBV. EBV-infected MT-2 cell clones expressed EBNA 1 and LMP 1 and very little of EBNA 2, showing the BamHI F promoter-driven latency II form of infection, which is seen in non-B-cell tumors. This is the first report of in vitro generation of latency II type EBV infection. The present system of persistent EBV infection in T cells should be a good model for investigating the pathogenic role of EBV in non-B-cell tumors.     

Induction of Epstein-Barr virus nuclear antigen and DNA synthesis in a human epithelial cell line after Epstein-Barr virus infection.

The association of Epstein-Barr virus (EBV) with nasopharyngeal carcinoma is supported by the presence of EBV genomes in the epithelial elements of the tumor and by elevated antibody titers to EBV-specific antigens in the patients; the levels of these titers are related to the clinical course of the disease. However, since most laboratory data suggest that EBV is a B-lymphotropic virus, it is unclear how the virus becomes associated with the epithelial elements of the nasopharynx. The purpose of the present work was to find a human model system to study this association. A human epithelial line (U) was found that could be directly infected by EBV, and viral functions, the induction of EBV nuclear antigen and cellular DNA synthesis, were demonstrated. The U line was established in 1957 by the late H. J. Van Kooten (Kok-Doorschodt at the University of Utrecht), and although it is no longer diploid, it exhibits density inhibition. When U cells were infected with EBV, EBV nuclear antigen was expressed in 6 to 16% of the cells, 1 and 2 days after infection with B95-8 virus, but not with the P3HR-1 strain. No evidence for virus replication was obtained; immunofluorescence staining for early antigens and virus capsid antigens gave negative results. Quantitative adsorption experiments for EBV indicated that the adsorption capacity of U cells is significant (60% of Raji cells). The present results also demonstrated that infection with the virus overcomes block(s) in cellular DNA synthesis caused by 5-fluorodeoxyuridine. The induction of DNA synthesis was determined by increased incorporation of [3H]thymidine into the cells. The highest level of isotope incorporation was observed at about 15 h after infection and thereafter decreased. Analysis of the induced DNA indicated that it was of cellular origin.     

The role of virus-specific CD4+ T cells in the control of Epstein-Barr virus infection

Epstein-Barr virus (EBV) establishes lifelong persistent infections in humans and has been implicated in the pathogenesis of several human malignancies. Protective immunity against EBV is mediated by T cells, as indicated by an increased incidence of EBV-associated malignancies in immunocompromised patients, and by the successful treatment of EBV-associated post-transplant lymphoproliferative disease (PTLD) in transplant recipients by the infusion of polyclonal EBV-specific T cell lines. To implement this treatment modality as a conventional therapeutic option, and to extend this protocol to other EBV-associated diseases, generic and more direct approaches for the generation of EBV-specific T cell lines enriched in disease-relevant specificities need to be developed. To this aim, we studied the poorly defined EBV-specific CD4+ T cell response during acute and chronic infection.     

Contributions of the Epstein-Barr virus EBNA1 protein to gastric carcinoma

Approximately 10% of gastric carcinomas (GC) are comprised of cells latently infected with Epstein-Barr virus (EBV); however, the mechanism by which EBV contributes to the development of this malignancy is unclear. We have investigated the cellular effects of the only EBV nuclear protein expressed in GC, EBNA1, focusing on promyelocytic leukemia (PML) nuclear bodies (NBs), which play important roles in apoptosis, p53 activation, and tumor suppression. AGS GC cells infected with EBV were found to contain fewer PML NBs and less PML protein than the parental EBV-negative AGS cells, and these levels were restored by silencing EBNA1. Conversely, EBNA1 expression was sufficient to induce the loss of PML NBs and proteins in AGS cells. Consistent with PML functions, EBNA1 expression decreased p53 activation and apoptosis in response to DNA damage and resulted in increased cell survival. In addition, EBNA1 mutants unable to bind CK2 kinase or ubiquitin-specific protease 7 had decreased ability to induce PML loss and to interfere with p53 activation. PML levels in EBV-positive and EBV-negative GC biopsy specimens were then compared by immunohistochemistry. Consistent with the results in the AGS cells, EBV-positive tumors had significantly lower PML levels than EBV-negative tumors. The results indicate that EBV infection of GC cells leads to loss of PML NBs through the action of EBNA1, resulting in impaired responses to DNA damage and promotion of cell survival. Therefore, PML disruption by EBNA1 is one mechanism by which EBV may contribute to the development of gastric cancer.     

Epstein-Barr virus (EBV) infection in B-cell non-Hodgkin's lymphomas in children: virus latency and its correlation with CD21 and CD23 molecules

Epstein Barr virus (EBV) infection of human B lymphocytes in vitro results in immortalisation of the cells and augmented membranous expression of numerous B-cell activation molecules, including CD23. Other studies demonstrated that only those B lymphocytes which carry the surface CD21 (EBV receptor) become transformation-competent. Inspired by the relatively unclear relations between expression of EBV and those of CD21 and CD23 in in vivo conditions we have decided to define correlations between tissue markers of EBV and of CD21 and CD23 molecules in B-cell non-Hodgkin's lymphomas (NHLs) in children. The studies were performed on an archival tissue material originating from children with B-cell NHLs (n=26) using immunocytochemical techniques, in situ hybridisation, and PCR. Our studies confirmed the latent phase of EBV infection in all of the EBV-positive patients. Viral proteins as well as viral RNAs (EBERs) was found both in the cytoplasm, in cell nuclei and in cell membranes of mainly the transformed lymphocytes B. Expression of the latent proteins (EBNA2 and LMP1) and that of EBERs in B-cell NHLs was significantly higher as compared to children with nonneoplastic lesions. The studies demonstrated reciprocally positive correlations between expressions of CD21 and CD23 in our children, but no correlation could be demonstrated between expression of EBV tissue markers and that of CD21 and/or CD23. Positive correlation was confirmed between expression of EBNA2 and LMP1 as well as between expression of the two proteins and EBERs in B-cell NHLs. Our studies have shown mainly latency III pattern of EBV. We have also demonstrated a novel form of EBV latency with no EBERs expression. The high detectability of EBV-positive cases both in the group of B-cell NHLs (77%), and in the group with non-neoplastic lesions (64%) suggested that only more pronounced tissue expression of EBV markers in B-cell NHLs as compared to the non-neoplastic material may point to a potential role of EBV in pathogenesis of lymphoma in this group of population in our country.