The Epstein-Barr virus (EBV) small RNA EBER1 binds and relocalizes ribosomal protein L22 in EBV-infected human B lymphocytes.

Epstein-Barr virus (EBV), an oncogenic herpesvirus, encodes two small RNAs (EBERs) that are expressed at high levels during latent transformation of human B lymphocytes. Here we report that a 15-kDa cellular protein called EAP (for EBER associated protein), previously shown to bind EBER1, is in fact the ribosomal protein L22. Approximately half of the L22 in EBV-positive cells is contained within the EBER1 ribonucleoprotein (RNP) particle, whereas the other half residues in monoribosomes and polysomes. Immunofluorescence with anti-L22 antibodies demonstrates that L22 is localized in the cytoplasm and the nucleoli of uninfected human cells, as expected, whereas EBV-positive lymphocytes also show strong nucleoplasmic staining. In situ hybridization indicates that the EBER RNPs are predominantly nucleoplasmic, suggesting that L22 relocalization correlates with binding to EBER1 in vivo. Since incubation of uninfected cell extracts with excess EBER1 RNA does not remove L22 from preexisting ribosomes, in vivo binding of L22 by EBER1 may precede ribosome assembly. The gene encoding L22 has recently been identified as the target of a chromosomal translocation in certain patients with leukemia, suggesting that L22 levels may be a determinant in cell transformation.     

In situ demonstration of Epstein-Barr virus small RNAs (EBER 1) in acquired immunodeficiency syndrome-related lymphomas: correlation with tumor morphology and primary site

Some acquired immunodeficiency syndrome (AIDS)-related lymphomas (ARLs) are infected with Epstein-Barr virus (EBV), although the frequency and importance of this association is disputed. Using paraffin section RNA in situ hybridization (ISH) with digoxigenin-labeled riboprobes, we screened 16 central nervous system (CNS) non-Hodgkin's lymphomas (NHLs), 101 systemic NHLs, and 11 Hodgkin's disease cases arising in human immunodeficiency virus-seropositive individuals for EBV-encoded small RNA (EBER 1) expression, an EBV gene product transcribed in abundance during latent infection. Tumor cells contained EBV in 85 of 128 ARLs (66%), but infection rates differed with lymphoma type. EBER 1 was expressed in tumor cells in 11 of 11 Hodgkin's disease cases (100%), 15 of 16 CNS NHLs (94%), and 46 of 60 systemic immunoblast- rich/large-cell lymphomas (77%), but in only 12 of 35 Burkitt-type (small noncleaved cell) (34%) and 1 of 6 monomorphic centroblastic (diffuse large noncleaved cell) (17%) lymphomas. In most EBV-positive ARLs, all recognizable viable tumor cells expressed EBER 1. We conclude that (1) EBV infects tumor cells in all AIDS-related Hodgkin's disease cases, in virtually all primary CNS ARLs, and in most systemic immunoblast-rich/large-cell ARLs; (2) only a minority of Burkitt-type and monomorphic centroblastic lymphomas are associated with EBV; and (3) EBER-ISH is ideal for the histopathologic detection of latent EBV in routine tissue specimens.     

In vitro stimulation of lymphocytes of donors seronegative for Epstein-Barr virus by preparations of inactivated Epstein-Barr virus.

Heat-inactivated preparations of Epstein-Barr virus stimulated human lymphocytes as assayed by incorporation of [(3)H]thymidine. The inactivated Epstein-Barr virus stimulated the lymphocytes of all five seropositive donors, 11 of 14 seronegative donors (aged eight to 26 years), and none of 15 neonates. Control antigens prepared from a human lymphoid cell line devoid of the Epstein-Barr virus genome did not stimulate the lymphocytes of seronegative donors. Fetal calf serum at the concentration used for suspension of Epstein-Barr virus did not stimulate or only minimally stimulated the lymphocytes of seronegative donors. The reactivity of the histocompatibility antigens found on human lymphocytes was abolished by procedures used for inactivation of the virus.     

GB virus C replicates in primary T and B lymphocytes

GB virus C (GBV-C) infection is common in humans and may persist for decades, although most infected persons clear the virus and subsequently develop antibodies to the envelope glycoprotein. GBV-C replicates in peripheral blood mononuclear cells (PBMCs) and CD4(+) T lymphocytes in vitro, and depletion of CD4(+) T lymphocytes has been proposed as the reason for clearance of GBV-C among persons positive for human immunodeficiency virus. We identified GBV-C RNA in purified CD4(+) and CD8(+) T lymphocytes and CD19(+) B lymphocytes removed ex vivo from infected donors and found that GBV-C replicated in vitro in these PBMC subsets, suggesting that GBV-C is a panlymphotropic virus.     

Epstein-Barr virus infection in vitro can rescue germinal center B cells with inactivated immunoglobulin genes

Immunoglobulin genotyping of Epstein-Barr virus (EBV)-positive posttransplantation lymphoproliferative disease has suggested that such lesions often arise from atypical post-germinal center B cells, in some cases carrying functionally inactivated immunoglobulin genes. To investigate whether EBV can rescue cells that are failed products of the somatic hypermutation process occurring in germinal centers (GCs), we isolated GC cells from tonsillar cell suspensions and exposed them to EBV in vitro. Screening more than 100 EBV-transformed cell lines of GC origin identified 6 lines lacking surface immunoglobulin, a phenotype never seen among lines derived from circulating naive or memory B cells. Furthermore, 3 of the 6 surface immunoglobulin-negative GC lines carried inactivating mutations in the immunoglobulin H (IgH) variable gene sequence. The ability of EBV to rescue aberrant products of the germinal center reaction in vitro strengthens the probability that a parallel activity contributes to EBV's lymphomagenic potential in vivo.     

Expression of Epstein-Barr virus (EBV) DNA and cloned DNA fragments in human lymphocytes following Sendai virus envelope-mediated gene transfer.

Purified EBV DNA and cloned DNA fragments were trapped in Sendai virus (SV) envelopes during envelope reconstitution. The DNA-loaded reconstituted envelopes (RSVE/DNA) served as gene-transfer vehicles using the capability of RSVE to fuse with normal and tumor cells. The efficiency of RSVE-mediated EBV DNA transfer into lymphoid tumor cells and fresh human lymphocytes was 5-10% of the enveloped 3H-labeled EcoRI fragment B of EBV DNA. Purified intracellular EBV (B95-8 strain) DNA induced EBV nuclear antigen (EBNA) in 0.2-1% of human lymphocytes, transiently stimulated cellular DNA synthesis, but did not fully transform cells. Cloned Sal I F1 fragment [approximately equal to 9 kilobase pairs (kbp)] and a smaller BamHI K (5.2 kbp) fragment from the same region of B95-8 EBV DNA induced EBNA in 2-4% of human lymphocytes but did not stimulate DNA synthesis nor transform cells. Cloned BamHI D1 fragment (approximately equal to 9 kbp) from AG-876 virus DNA, or a combination of cloned BamHI X and H fragments (approximately equal to 2 and 7 kbp, respectively) from the similar region of B95-8 virus DNA, significantly stimulated lymphocyte DNA synthesis, but EBNA could not be detected and transformation was not achieved. Early antigen and viral capsid antigen were not observed with any of the fragments tested. Our results suggest that the induction of EBNA and stimulation of lymphocyte proliferation are not controlled by the same region of EBV DNA.     

Chromosome site for Epstein-Barr virus DNA in a Burkitt tumor cell line and in lymphocytes growth-transformed in vitro.

The Epstein-Barr virus (EBV) genome stably persists in latently infected Burkitt tumor cells and growth-transformed B lymphocytes. These cells usually contain multiple copies of episomal viral DNA. Cytological hybridization of recombinant viral DNA fragments to metaphase chromosomes of two latently infected cell lines demonstrates that viral DNA localizes to both chromatids of one homologue of chromosome 1 in Namalwa, a Burkitt tumor cell line, and to both chromatids of one homologue of chromosome 4 in IB4, a cell line with transformed growth properties in vitro. The site of chromosome association remains stable in a clone of IB4 cells. Probes from five separate regions of the EBV genome hybridize to the same chromosome regions. A previously undescribed achromatic site is identified within the region of EBV chromosome cytological hybridization. These observations suggest that most or all of the EBV genome is integrated into the chromosomal DNA of Namalwa and IB4 cells. Although the chromosomal sites of EBV DNA association are among those regions with homology to the EBV IR3 repeated DNA sequence, EBV IR3 did not mediate recombination between EBV and chromosomal DNA.     

The 3;21 translocation in myelodysplasia results in a fusion transcript between the AML1 gene and the gene for EAP, a highly conserved protein associated with the Epstein-Barr virus small RNA EBER 1.

In the 8;21 translocation, the AML1 gene, located at chromosome band 21q22, is translocated to chromosome 8 (q22), where it is fused to the ETO gene and transcribed as a chimeric gene. AML1 is the human homolog of the recently cloned mouse gene pebp2 alpha B, homologous to the DNA binding alpha subunit of the polyoma enhancer factor pebp2. AML1 is also involved in a translocation with chromosome 3 that is seen in patients with therapy-related acute myeloid leukemia and myelodysplastic syndrome and in chronic myelogenous leukemia in blast crisis. We have isolated a fusion cDNA clone from a t(3;21) library derived from a patient with therapy-related myelodysplastic syndrome; this clone contains sequences from AML1 and from EAP, which we have now localized to band 3q26. EAP has previously been characterized as a highly expressed small nuclear protein of 128 residues (EBER 1) associated with Epstein-Barr virus small RNA. The fusion clone contains the DNA binding 5' part of AML1 that is fused to ETO in the t(8;21) and, in addition, at least one other exon. The translocation replaces the last nine codons of AML1 with the last 96 codons of EAP. The fusion does not maintain the correct reading frame of EAP and may not lead to a functional chimeric protein.     

Abnormal responses of rheumatoid arthritis lymphocytes to Epstein-Barr virus infection in vitro: evidence for multiple defects.

Blood lymphocytes from 53 patients with rheumatoid arthritis (RA) and 44 controls were cultured with the polyclonal B cell activator Epstein-Barr virus (EBV). Culture supernatants were removed at weekly intervals and the amount of IgM secreted by the lymphocytes measured by an enzyme-linked immunosorbent assay (ELISA). Three major differences in the pattern of EBV-induced IgM synthesis by RA versus control lymphocytes were observed. Lymphocytes from RA patients, in general, produced less IgM after one week in culture than controls. In contrast, they increased their IgM secretion significantly by the end of the second week, whereas control lymphocyte cultures showed little change in IgM secretion at this time. Control lymphocytes from EBV seropositive individuals produced undetectable amounts of IgM after five weeks in culture. However, lymphocytes from 40% of the RA patients, even though they were EBV seropositive, secreted greater than 2000 ng/ml (microgram/l) IgM after five weeks. The data are discussed in terms of defective B and T cell responses to EBV in lymphocytes from patients with RA.     

Persistently high Epstein-Barr virus (EBV) loads in peripheral blood lymphocytes from patients with chronic active EBV infection

Chronic active Epstein-Barr virus infection (CAEBV) is a severe illness with unusual EBV activation that persists for years, and its pathogenesis is largely unknown. After the creation of an accurate and reproducible polymerase chain reaction system to quantify EBV DNA, virus loads in peripheral blood lymphocytes (PBL) were determined in 54 children: 15 with CAEBV, 16 with infectious mononucleosis (IM), and 23 healthy children. Children with CAEBV and those with IM had high virus loads. Lower loads were detected in 47% of seropositive healthy donors. There were two distinct differences between children with CAEBV and those with IM: The former had greater viral replication (10(3)-10(7) copies/2.5x10(5) PBL) than those with IM, and viral replication declined in children with IM whereas active replication persisted for years in subjects with CAEBV. Persisting high virus loads are a possible diagnostic criterion for CAEBV. EBV loads may enable classification and prognosis of EBV infections.     

Effect of adenine arabinoside on Epstein-Barr virus in vitro.

The effect of adenine arabinoside (ara-A) on Epstein-Barr virus (EBV) in cultures of lymphoid cells was examined with use of an EBV-producing cell line, P3HR-1, and a nonproducing cell line, Raji. The presence of EBV-associated viral capsid antigen (VCA) and early antigen (EA) was detected by indirect immunofluorescence. Ara-A inhibited the expression of VCA in P3HR-1 cells at concentrations fivefold below those that inhibited cell multiplication; there was no concomitant accumulation of EA. Ara-A did not inhibit superinfection of Raji cells with EBV and did not induce EA until high concentrations of the compound were reached. The inhibition of the expression of VCA but not EA is consistent with a postulated inhibition by ara-A of viral-directed synthesis of DNA. At low concentrations, ara-A may exert a more specific antiviral effect than that reported for idoxuridine and cytosine arabinoside in this system.     

Safety of allogeneic Epstein-Barr virus (EBV)-specific cytotoxic T lymphocytes for patients with refractory EBV-related lymphoma

Epstein-Barr virus (EBV) causes lymphomas in immunocompromised individuals such as recipients of stem cell or organ transplants and patients with acquired immunodeficiency syndrome (AIDS). EBV has also been detected in the Reed-Sternberg cells of approximately 50% of all cases of Hodgkin's disease (HD). The purpose of this study was to examine the safety, and the clinical and immunological effects of infusing allogeneic EBV-specific cytotoxic T lymphocytes (CTL) for patients with refractory EBV-positive malignancies. In this pilot study, we have treated four patients with EBV-related lymphoma using allogeneic EBV-specific CTL. Two patients received EBV-specific CTL derived from partially human leucocyte antigen (HLA)-matched donors and the other two from HLA-matched siblings. No complications were observed as a result of the CTL infusions and all patients showed increased levels of EBV-specific CTL precursors (CTLp) post infusion. Of the two organ transplant patients, one had refractory disease and has sustained a complete remission following the T-cell infusions. The second has also been disease free since T-cell infusions, although the efficacy cannot be definitively attributed to CTL therapy because this patient received local radiation therapy prior to immunotherapy. A patient with AIDS-related, EBV-positive lymphoma had disease progression following CTL infusions. One HD patient received HLA 4/6 matched T cells from an unrelated donor and showed a decrease in the size of affected lymph nodes and resolution of B-symptoms post infusion. In conclusion, adoptive immunotherapy with allogeneic EBV-specific CTL is safe and may have efficacy in patients with high-risk or refractory EBV-related tumours.     

Biological aspects of Epstein-Barr virus (EBV)-infected lymphocytes in chronic active EBV infection and associated malignancies

Most primary Epstein-Barr virus (EBV) infections are clinically inapparent, but occasionally EBV infection can cause acute infectious mononucleosis. EBV has been linked to a variety of hematologic and non-hematologic malignancies. Chronic active EBV (CAEBV) infection designates a recently identified EBV-associated syndrome characterized by a variety of serious hematological disorders, including malignant lymphoma. EBV was found to infect circulating T- and/or NK-cells in patients with CAEBV infection. These EBV-infected T- and/or NK-cells express EBNA-1, LMP-1, and LMP-2A, a type II form of EBV latency, which is also observed in nasopharyngeal carcinoma (NPC), Hodgkin's disease (HD), and peripheral T-cell lymphoma. CAEBV infections may thus represent a subset of EBV-associated T- and/or NK-cell lymphoproliferative disorders.     

Deficient control of in vitro Epstein-Barr virus infection in patients with ankylosing spondylitis.

Infectious Epstein-Barr (EB) virus obtained from the B95-8 marmoset cell line was used to infect mononuclear cells from healthy controls and patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS), and outgrowth of B cells into lymphoblastoid cell lines was assessed by visual microscopy and uptake of tritiated thymidine over a 28 day period. When undiluted virus was used lymphocytes from both patients with AS and RA and from normal controls outgrew into lymphoblastoid cell lines (LCLs) by day 28 of culture. At dilutions of 1/10, 1/20, and 1/40, however, the control cells showed regression of proliferation at approximately day 14 of culture, whereas the cells from patients with AS and RA continued to proliferate and outgrew into LCLs (transformation scores of cells from patients with AS compared with controls at day 28 p less than 0.05 in all cases; thymidine uptake at a 1/40 dilution at day 28, patients with AS compared with controls p less than 0.01). Hence these results suggest that there is a defect in the cellular response to EB virus induced B cell proliferation in patients with AS similar to that seen with cells from RA donors.