Epstein-Barr virus (EBV) receptors, complement receptors, and EBV infectibility of different lymphocyte fractions of human peripheral blood. II. Epstein-Barr virus studies.

In B-cell fractions isolated from human peripheral blood, the frequency of surface immunoglobulin-positive and of complement receptor-positive cells showed a good correlation with the frequency of EBV-binding cells, as detected by membrane fluorescence or by a quantitative bioassay for infectious virus in the absorbed supernatant fluid. There was a close relationship between all three parameters mentioned, the frequency of EBNA-positive cells 2 or 3 days after the infection, and the stimulation of cellular DNA synthesis. So-called O-cell fractions remaining after the removal of nylon adherent and E-rosetting cells contained a certain frequency of complement receptor-positive cells and absorbed EBV to a limited extent, but did not respond to EBV infection with EBNA induction or stimulation of DNA synthesis. None of the T-cell fractions absorbed EBV to a detectable extent. This includes the T_ea+ fraction that contained a certain proportion of complement receptor-positive cells. It is concluded that the previously demonstrated relationship between EBV receptors and complement receptors on B-lymphoblastoid lines also holds for peripheral B lymphocytes. In these cells, virus absorption is followed by an intracellular infectious process, signaled by the appearance of EBNA and cellular DNA synthesis. O cells carry complement receptors and absorb EBV to a certain extent, but do not respond with EBNA synthesis or DNA stimulation, presumably due to intracellular restrictions. T cells do not bind EBV, and the complement receptors present on some cells of the T_ea+ fraction do not function as EBV receptors.     

Relationships between complement activation, complement binding, and EBV absorption by human hematopoietic cell lines.

Complement consumption (C.C.) and C3 deposition on the cell membrane, visualized by membrane fluorescence (CMF), were compared in a collection of established human lymphoid lines. C.C. was independent of the presence of C3 receptors. A positive CMF reaction was seen only in lines that expressed C3 receptors, however. Trypsin treatment abolished CMF and EAC rosetting but had virtually no influence on C.C. EBV absorptive capacity correlated with both C3-receptor expression, as measured by EAC rosetting, and CMF but not with C.C. This is in line with our previous finding on the association of EBV and C3 receptors.     

Epstein-Barr virus (EBV) and human disease: facts, opinions and problems

The human herpesvirus, Epstein-Barr virus (EBV), has classically been associated with two pathologies with frequencies approaching 100%. One of these, Burkitt's lymphoma (BL), is of B-cell origin and the other, nasopharyngeal carcinoma (NPC), is a tumour of poorly differentiated epithelial cells. More recently, EBV had been identified with frequencies from a few percent to 100% (in one case) with a variety of other malignancies. These include Hodgkin's disease (HD; where in the west, the frequency of association is about 50%), sino-nasal T-cell lymphomas, lymphoepitheliomas, some sarcomas and breast cancers, other cancers from the head and neck, and lymphomas arising in patients with immune dysfunctions. Since EBV is ubiquitous, with the vast majority of the world's population having met and seroconverted to the virus, the diversity of tumours with which it has now been associated represents a substantial health burden. In a recent IARC monograph, EBV was classified as a group 1 carcinogen. Here, the data on BL and NPC, as they relate to geographical restrictions, viral strain variation, co-factors in disease, and genetic components are reexamined. We raise the question whether in their origins, these tumours genuinely reflect distinct and independent events, as deemed at present, or may represent a response by different cell types to common extracellular factors. For example, a situation in Kenya apparently existed in the past, where both BL and NPC were observed in ethnic Africans with roughly equal frequencies; more recently, in Kenya, EBV has been identified in nearly 100% of the tumours in children with HD. We also consider tumours where the viral association is reportedly of low frequency, and offer explanations for these data, including the possibility of loss of the viral genome once malignancy has been initiated. If this phenomenon occurs as a frequent secondary event, EBV could be an even greater health risk than presently believed.     

Epstein-Barr virus (EBV) and human hematopoietic cell lines: a review.

Two facts need to be pointed out to help explain why the history of Epstein-Barr virus (EBV) research has been inseparable from that of the studies with human hematopoietic cell lines of neoplastic and non-neoplastic origin. One is that Burkitt lymphoma (BL) cell lines, EBV-positive or-negative, can be established in culture quite easily. Thus, the BL cell lines which Epstein established were indeed some of the first hematopoietic as well as virus-carrying cell lines of human neoplastic origin. The other is that EBV-positive B-cell lymphoblastoid cell lines (B-LCL) of normal origin can be grown from samples of sero-positive individuals. B-LCL were often mistakenly regarded as being of neoplastic origin, but are almost always of normal cell origin. Very rarely, however, B-LCL with the same clonal markers as those of neoplastic cells have also been obtained. While the development of B-LCL has been referred to as the in vitro viral immortalization of human B cells and as a phenomenon representing the potential oncogenicity of EBV, the phenotypic and genotypic differences between B-LCL and EBV-carrying BL cells are obvious, indicating that the development of B-LCL per se does not prove the oncogenic activity of EBV. Two EBV-derived antigens, EBNA2 and latent-infection membrane protein (LMP), which are strongly expressed by B-LCL but not by BL cells, have recently been detected in EBV-positive proliferative B cells in patients with organ transplants, suggesting that the proliferating of B-LCL-like cells may take place as an initial step of the multi-step in vivo oncogenesis of EBV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Survival and PHA-stimulation of gamma-irradiated human peripheral blood T lymphocyte subpopulations

Human peripheral blood T lymphocyte subpopulations were identified and isolated on the basis of their ability to bind IgG (T-G), IgM (T-M), or neither immunoglobulin class (T-null). Lymphocytes were exposed to 0, 0.5, 1.0, 2.5 or 5.0 Gy of 60Co gamma-rays either as a T-cell suspension or as separated T cell subsets. Survival curves, determined 5 days after irradiation, revealed that each subset has radiosensitive and radioresistant portions, and that the T-G cell is the most sensitive subset. Mitotic indices of 48-h cultures showed that the response of unirradiated T lymphocytes to PHA varied greatly among the subsets, the highest indices being obtained for the T-M and the lowest for the T-G cells. With the possible exception of the T-G cells, the subsets are relatively resistant to mitotic effects of gamma-rays. T-G cells suppress the PHA-induced mitotic response of the other T lymphocyte subsets, and this suppressor effect is radiosensitive, being abolished by 1.0 Gy. It is concluded that lymphocytes exposed to greater than or equal to 1 Gy of gamma-rays will have very few dividing B lymphocytes or T-G cells. This together with radiation-induced loss of T-G suppressor action means that the predominant lymphocyte types in mitosis after greater than or equal to 1 Gy are the radioresistant T-M and T-null cells.     

Migration inhibitory factor (MIF) and proliferative responses by human lymphocyte subpopulations separated by sheep erythrocyte rosette formation.

Human peripheral blood lymphocytes were separated by a combination of rosette formation with sheep erythrocytes and differential density centrifugation into subpopulations of rosette positive (T-enriched) cells and rosette negative (T depleted) cells. These were then tested in vitro for the production of macrophage migration inhibitory factor (MIF) and for incorporation of ³H-thymidine in response to specific antigens. Both T enriched and T depleted cell populations produced MIF but only T enriched cells exhibited significant antigen-induced ³H-thymidine incorporation. These findings using a T cell surface marker as the basis for cell separation, a technique which should not alter the B cell surface, confirm an earlier report in which human cells were separated on the basis of surface immunoglobulin, a B cell marker.     

Lytic,nontransforming Epstein-Barr virus (EBV) from a patient with chronic active EBV infection.

A new wild-type isolate of Epstein-Barr virus (EBV) was identified in follow-up studies of a case of chronic active EBV infection in an 8-year-old girl who had high titres of antibody to viral capsid antigen and early antigen (EA) (greater than 20 480 and 2560 respectively), persistent splenomegaly and abnormal immunologic features. More than 10 throat washings from this patient failed to transform cord blood lymphocytes (CBL), but at least 7 were able to induce EA in Raji cells. Supernatants from cultures of the lymphoblastoid cell line obtained by in-vitro infection of this patient''s leukocytes with the B95-8 strain of EBV revealed a herpesvirus particle when examined by electron microscopy. The same supernatants were unable to transform CBL but could induce EA in Raji cells upon superinfection. In 30 or more trials the patient''s lymphocytes never transformed spontaneously but did become positive for EBV nuclear antigen and EA in the first week of culture at least twice. Parallel studies performed on the father of the patient yielded similar results. This, then, is the first report documenting lytic activity associated with a wild-type EBV isolate.     

Identification of an epitope in the major envelope protein of Epstein-Barr virus that mediates viral binding to the B lymphocyte EBV receptor (CR2)

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.     

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV, HHV-8)

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV or human herpesvirus 8, HHV-8) are members of gamma-herpes virus family. Both viruses infect to B cells and cause malignancies such as lymphoma. Since EBV and HHV-8 are so-called 'oncovirus', their oncogenecities have been focused in the researches on EBV and KSHV for a long time. EBV was discovered in 1964, whereas KSHV was identified in 1994. However, KSHV was analyzed rapidly in these fifteen years. One of the recent progresses in the research on EBV and KSHV is that virus-encoded small RNAs were identified in their genomes and characterized. EBV is the first human virus in whose genome microRNA was identified. The oncogenecity of EBV and KSHV remains unclear. Here, I discuss the pathogenesis by EBV and KSHV with special reference to recent progress in this field.     

Hexose transport in Epstein-Barr virus (EBV) negative lymphoma lines and their EBV converted, virus genome carrying sublines

Increased hexose uptake is a marker for viral transformation, as has been shown in non-human fibroblasts transformed by oncogenic viruses. If this phenomenon is a general expression of viral induced transformation it should also apply on different oncogenic virus-cell systems. Recently two human EBV-negative lymphoma lines were converted to a stable EBV-positive state by infection with EBV. According to their biochemical and biological properties they enable us to study events associated with EBV-transformation. We analysed the uptake of (3H) glucosamine and (3H) 2-deoxy-D-glucose into BJAB and Ramos and their EBV-converted sublines and found a clear increase of the rate of uptake of both sugars in the EBV-positive sublines. Control experiments confirmed that the increased uptake was due to alterations on the level of the hexose membrane carriers and not due to increased metabolism. The observation of increased hexose uptake in the only presented available virus transformed human cell system is a strong argument for the general importance of this transformation-associated membrane change.     

The latent membrane protein 1 of Epstein-Barr virus (EBV) primes EBV latency cells for type I interferon production

Epstein-Barr virus (EBV) latency has been associated with a variety of human cancers. Latent membrane protein 1 (LMP-1) is one of the key viral proteins required for transformation of primary B cells in vitro and establishment of EBV latency. We have previously shown that LMP-1 induces the expression of several interferon (IFN)-stimulated genes and has antiviral effect (Zhang, J., Das, S. C., Kotalik, C., Pattnaik, A. K., and Zhang, L. (2004) J. Biol. Chem. 279, 46335-46342). In this report, a novel mechanism related to the antiviral effect of LMP-1 is identified. We show that EBV type III latency cells, in which LMP-1 is expressed, are primed to produce robust levels of endogenous IFNs upon infection of Sendai virus. The priming action is due to the expression of LMP-1 but not EBV nuclear antigen 2 (EBNA-2). The signaling events from the C-terminal activator regions of LMP-1 are essential to prime cells for high IFN production. LMP-1-mediated activation of NF-kappaB is apparently necessary and sufficient for LMP-1-mediated priming effect in DG75 cells, a human B cell line. IFN regulatory factor 7 (IRF-7) that can be activated by LMP-1 is also implicated in the priming action. Taken together, these data strongly suggest that LMP-1 may prime EBV latency cells for IFN production and that the antiviral property of LMP-1 may be an intrinsic part of EBV latency program, which may assist the establishment and/or maintenance of viral latency.     

Amplification of Epstein-Barr virus (EBV) DNA by superinfection with a strain of EBV derived from nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) from a nasopharyngeal carcinoma (NPC) hybrid cell line (NPC-KT) lacking defective viral DNA molecules superinfected Raji cells and induced EBV early antigens (EA), as did virus from P3HR-1 cells, which contained defective molecules. The EBV polypeptides induced by NPC-KT appeared to be identical to those induced by P3HR-1 virus. The ability of NPC-KT virus to induce EA was enhanced more than 10-fold by treatment of superinfected cells with dimethyl sulfoxide; however, dimethyl sulfoxide treatment did not enhance superinfection by P3HR-1 virus. After infection, DNA synthesis of both the superinfecting NPC-KT virus and the resident Raji viral genome was induced. In addition to amplified Raji EBV episomal DNA, a fused terminal fragment of NPC-KT viral DNA was detected. The detection of fused terminal DNA fragments suggests that the superinfecting virion DNA either circularizes or polymerizes after superinfection and is possibly amplified through circular or concatenated replicative intermediates.     

Epstein-Barr virus (EBV)-negative B-lymphoma cell lines for clonal isolation and replication of EBV recombinants.

Previous experiments have demonstrated that positive selection markers recombined into the Epstein-Barr virus (EBV) genome enable the isolation of transforming or nontransforming mutant EBV recombinants in EBV-negative B-lymphoma (BL) cell lines (A. Marchini, J. I. Cohen, and E. Kieff, J. Virol. 66:3214-3219, 1992; F. Wang, A. Marchini, and E. Kieff, J. Virol. 65:1701-1709, 1991). However, virus has been recovered from a BL cell clone (BL41) infected with an EBV recombinant in only one instance (Wang et al., J. Virol. 65:1701-1709, 1991). We now compare the utility of four EBV-negative BL lines, BJAB, BL30, BL41, and Loukes, for isolating EBV recombinants and supporting their subsequent replication. Transforming or nontransforming EBV recombinants carrying a simian virus 40 promoter-hygromycin phosphotransferase (HYG) cassette were cloned by selecting newly infected BL cells for HYG expression. Most of the infected BL clones contained EBV episomes, and EBV gene expression was largely restricted to EBNA-1. Although the BJAB cell line was a particularly good host for isolating EBV recombinants (Marchini et al., J. Virol. 66:3214-3219, 1992), it was largely nonpermissive for virus replication, even in response to heterologous expression of the BZLF1 immediate-early transactivator. In contrast, approximately 50% of infected BL41, BL30, or Loukes cell clones responded to lytic cycle induction. Frequently, a substantial fraction of infected cells expressed the late lytic infection viral protein, gp350/220, and released infectious virus. Since BL cells do not depend on EBV for growth, transforming and nontransforming EBV recombinants were isolated and passaged.     

The mixed lymphocyte reaction (MLR) stimulatory capacity of human lymphocyte subpopulations.

Using various cell separation techniques and combinations of these, suspensions were obtained highly enriched or depleted with respect to their content of E-rosette-forming T cells, Ig-bearing B cells, Fc-receptor-bearing cells, or monocytes. These purified populations were tested for their capacity to stimulate allogeneic cells in a mixed lymphocyte reaction (MLR). It could be demonstrated that the Ig-bearing B cells provide the strongest stimulus in the MLR.     

Detection of Epstein-Barr virus (EBV) DNA sequences using in situ hybridization.

In situ hybridization was used to detect Epstein-Barr virus (EBV) DNA sequences under conditions where the virus DNA is replicating spontaneously and where it is induced to do so following superinfection. The in situ reaction itself is influenced by several parameters, analogous to conventional nucleic acid hybridization, consideration of which should help to optimize the designing of in situ hybridization reactions in general. Both EBV complementary RNA (cRNA) and EBV DNA synthesized in vitro can efficiently detect the virus DNA sequences in situ. The findings presented here can therefore be utilized in both the study of EBV-cell interactions and, more generally, in studies using in situ hybridization as a general approach.