Human plasma accelerates immortalization of B lymphocytes by Epstein-Barr virus

Abstract.   Objective : Serum and plasma contain species-specific factors that modulate cell population growth and function, and that are required for proliferation of most cell cultures. Foetal calf serum (FCS) is the most common source of these growth factors. We studied the effect of human plasma (HP) on the immortalization process of B lymphocytes by Epstein–Barr virus (EBV) was studied. Materials and methods : The effect of HP as compared to FCS was done through assessment of cell proliferation. Results : It was found that HP (autologous and non-autologous plasma) is more effective than FCS in generating lymphoblastoid cell lines, regardless of EBV status of the donors: 65% of HP-supplemented cultures developed into lymphoblastoid cell lines by 7–14 culture days, as compared to 16% of cultures with FCS. In addition, 6% of HP-supplemented cultures did not achieve becoming lymphoblastoid cell lines by day 35 in comparison to 94% of cultures with FCS. The higher proliferative effect of HP was not altered by heat inactivation or filtration. HP maintained its proliferative activity at 4 °C over 8 months, thus indicating that HP contains a stable growth factor(s), which accelerates B-lymphocyte immortalization. Conclusion : The results support other studies that recommend the use of autologous plasma for tissue culture, mainly in the case of autologous transplantation. Furthermore, the use of HP allows preparation of lymphoblastoid cell lines from a small amount of peripheral blood in a shorter period of time and with a higher rate of success.     

Pathogenesis of Epstein-Barr virus (EBV)-carrying lymphomas

The EBV carrier state is almost general in men. The virus induces B lymphocyte proliferation in vitro, but this is counteracted in vivo by the immune response. Therefore, EBV-induced malignancies occur only when the immune response is impaired, e.g. in transplant recipients. The versatility of the viral gene expression strategy secures the consistent maintainance of the virus in healthy individuals. The viral proteins required for transformation render the cell immunogenic. Expression of the transforming genes leads to rejection, but these genes are not required for the maintenance of the viral genome. EBV is an important contributor for malignant transformation, even when it does not directly induce cell proliferation. Several mechanisms have been unravelled in EBV-associated tumors whereby the virus may modify the cellular phenotype and may influence the interaction of tumor cells with their microenvironment. The virus carrier state can lead to the evasion of apoptosis and can intensify the response to growth promoting signals, too.     

Interaction of Epstein-Barr virus (EBV) with human B-lymphocytes

Epstein-Barr virus, EBV, and humans have a common history that reaches back to our primate ancestors. The virus co-evolved with man and has established a largely harmless and highly complex co-existence. It is carried as silent infection by almost all human adults. A serendipitous discovery established that it is the causative agent of infectious mononucleosis.Still, EBV became known first in 1964, in a rare, geographically prevalent malignant lymphoma of B-cell origin, Burkitt lymphoma BL. Its association with a malignancy prompted intensive studies and its capacity to immortalize B-lymphocytes in vitro was soon demonstrated. Consequently EBV was classified therefore as a potentially tumorigenic virus. Despite of this property however, the virus carrier state itself does not lead to malignancies because the transformed cells are recognized by the immune response. Consequently the EBV induced proliferation of EBV carrying B-lymphocytes is manifested only under immunosuppressive conditions.The expression of EBV encoded genes is regulated by the cell phenotype. The virus genome can be found in malignancies originating from cell types other than the B-lymphocyte. Even in the EBV infected B-cell, the direct transforming capacity is restricted to a defined window of differentiation. A complex interaction between virally encoded proteins and B-cell specific cellular proteins constitute the proliferation inducing program.In this short review we touch upon aspects which are the subject of our present work.We describe the mechanisms of some of the functional interactions between EBV encoded and cellular proteins that determine the phenotype of latently infected B-cells.The growth promoting EBV encoded genes are not expressed in the virus carrying BL cells. Still, EBV seems to contribute to the etiology of this tumor by modifying events that influence cell survival and proliferation. We describe a possible growth promoting mechanism in the genesis of Burkitt lymphoma that depends on the presence of EBV.     

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.     

Epstein-Barr virus (EBV) antigenic determinants on subunits of the EBV determined nuclear antigen (EBNA)

In order to characterize the substructure of the Epstein-Barr virus determined nuclear antigen (EBNA) which is considered to have a molecular weight of 180 K in its native form, we have examined the antigenic specificity of the polypeptides obtained after denaturation of this molecule. Two procedures were employed; treatment by sodium dodecyl sulfate (SDS) and heat followed by gel electrophoresis, or denaturation by guanidine hydrochloride followed by gel filtration, which allowed us to detect a specific antigenic activity in the 50 K region, following dialysis. The denatured molecules could be reassociated into larger molecules (50 to 180 K) which retain the property of binding to fixed nuclei, as does native EBNA. These results indicate that EBNA has a polymeric structure and that 50 K subunits carry the antigenic determinants.     

Epstein-Barr virus (EBV) Rta-mediated EBV and Kaposi's sarcoma-associated herpesvirus lytic reactivations in 293 cells

Epstein-Barr virus (EBV) Rta belongs to a lytic switch gene family that is evolutionarily conserved in all gamma-herpesviruses. Emerging evidence indicates that cell cycle arrest is a common means by which herpesviral immediate-early protein hijacks the host cell to advance the virus's lytic cycle progression. To examine the role of Rta in cell cycle regulation, we recently established a doxycycline (Dox)-inducible Rta system in 293 cells. In this cell background, inducible Rta modulated the levels of signature G1 arrest proteins, followed by induction of the cellular senescence marker, SA-β-Gal. To delineate the relationship between Rta-induced cell growth arrest and EBV reactivation, recombinant viral genomes were transferred into Rta-inducible 293 cells. Somewhat unexpectedly, we found that Dox-inducible Rta reactivated both EBV and Kaposi's sarcoma-associated herpesvirus (KSHV), to similar efficacy. As a consequence, the Rta-mediated EBV and KSHV lytic replication systems, designated as EREV8 and ERKV, respectively, were homogenous, robust, and concurrent with cell death likely due to permissive lytic replication. In addition, the expression kinetics of EBV lytic genes in Dox-treated EREV8 cells was similar to that of their KSHV counterparts in Dox-induced ERKV cells, suggesting that a common pathway is used to disrupt viral latency in both cell systems. When the time course was compared, cell cycle arrest was achieved between 6 and 48 h, EBV or KSHV reactivation was initiated abruptly at 48 h, and the cellular senescence marker was not detected until 120 h after Dox treatment. These results lead us to hypothesize that in 293 cells, Rta-induced G1 cell cycle arrest could provide (1) an ideal environment for virus reactivation if EBV or KSHV coexists and (2) a preparatory milieu for cell senescence if no viral genome is available. The latter is hypothetical in a transient-lytic situation.     

A Ca2+/calmodulin-dependent protein kinase, CaM kinase-Gr, expressed after transformation of primary human B lymphocytes by Epstein-Barr virus (EBV) is induced by the EBV oncogene LMP1.

CaM kinase-Gr is a multifunctional Ca2+/calmodulin-dependent protein kinase which is enriched in neurons and T lymphocytes. The kinase is absent from primary human B lymphocytes but is expressed in Epstein-Barr virus (EBV)-transformed B-lymphoblastoid cell lines, suggesting that expression of the kinase can be upregulated by an EBV gene product(s). We investigated the basis of CaM kinase-Gr expression in EBV-transformed cells and the mechanisms that regulate its activity therein by using an EBV-negative Burkitt lymphoma cell line, BJAB, and BJAB cells converted to expression of individual EBV proteins by single-gene transfer. CaM kinase-Gr expression was upregulated in BJAB cells by EBV latent-infection membrane protein 1 (LMP1) but not by LMP2A or by nuclear proteins EBNA1, EBNA2, EBNA3A, and EBNA3C. In LMP1-converted BJAB cells, the kinase was functional and was dramatically activated upon cross-linking of surface immunoglobulin M. Overlapping cDNA clones that encode human CaM kinase-Gr were sequenced, revealing 81% amino acid identity between the rat and human proteins. Transfection of BJAB cells with an expression construct for the human enzyme resulted in a functional kinase which was shown by epitope tagging to localize primarily to cytoplasmic and perinuclear structures. Induction of CaM kinase-Gr expression by LMP1 provides the first example of a Ca2+/calmodulin-dependent protein kinase upregulated by a viral protein. In view of the key role played by LMP1 in B-lymphocyte immortalization by EBV, these findings implicate CaM kinase-Gr as a potential mediator of B-lymphocyte growth transformation.     

Human peripheral blood monocytes release a 30,000 dalton factor (30 KD MF) that stimulates immunoglobulin production by activated B cells

Supernatants (SN) of well-washed adherent human monocytes, obtained from T cell-depleted peripheral blood mononuclear cells, contain a 30,000 dalton protein (30 KD MF) that increases immunoglobulin (Ig) synthesis by EBV-activated B cells two- to fourfold. This factor is released spontaneously during the first 20 hr after monocytes are placed in culture. SN containing 30 KD MF are inactive in the thymocyte co-stimulator assay, under conditions that will detect as little as 0.5 U of purified IL 1. The addition of autologous T cells to isolated adherent monocytes, previously depleted of T cells, suppresses the release or activity of this B cell stimulator in a dose-dependent manner. In addition, 30 KD MF stimulates a two- to fourfold increase in IgA production by cells of an EBV-transformed B cell line (JB/FF line) without increasing incorporation of [3H]thymidine. In contrast, stimulation of this B cell line with up to 10 U of purified IL 1 increases IgA synthesis by less than 50%, and addition of up to 100 U of recombinant IL 2 causes no change whatsoever in IgA production. However, co-stimulation with 30 KD MF and recombinant IL 2 or recombinant gamma-interferon induces more Ig production than is caused by the monocyte factor alone. These observations suggest that the monocyte, in addition to acting as an antigen-presenting cell and source of IL 1, facilitates B cell differentiation by producing a factor which acts both independently and in synergy with cytokines produced by T cells to stimulate Ig production by B lymphocytes.     

Two families of sequences in the small RNA-encoding region of Epstein-Barr virus (EBV) correlate with EBV types A and B

DNA sequence analysis was carried out on the 1-kilobase SacI-EcoRI region of the EcoRI J fragment of four strains of Epstein-Barr virus (EBV) (MABA, P3HR-1, FF41, and NPC-5), and the sequences were compared with the prototype sequence from strain B95-8. Ten single-base changes which grouped the strains into two families (1 and 2) were found. Restriction endonuclease polymorphisms predicted from the sequences were used to classify the EBV DNA from a further 26 EBV-positive cell lines into these two families. The EBNA-2 types (A or B) of the strains were found to correlate with the J region type; EBNA-2 type A DNA regularly contained J region sequence type 1, while EBNA-2 type B DNA generally carried J region sequence type 2. These data are consistent with the notion of there being two distinct families of EBV with discrete, conserved differences in DNA sequence.     

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) 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.     

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.     

Human cytotoxic T lymphocytes (CTL) against Epstein-Barr virus (EBV) infected cells: EBV specificity and involvement of major histocompatibility complex determinants in the lysis exerted by anti-EBV CTL toward HLA-compatible and allogeneic target cells

Human peripheral blood lymphocytes (PBL), from anti-Epstein-Barr virus (EBV)-seropositive donors, were stimulated by EBV and were shown to be cytotoxic toward autologous, HLA-compatible, and fully allogeneic EBV-transformed target cells. The lysis was not due to natural killer (NK) cells since the target cells used were resistant to lysis by fresh PBL and by virus-stimulated PBL-depleted of AET-SRBC-rosetting T cells (the latter being still fully cytotoxic on K562 NK-susceptible target cells). Conversely only E-rosette-purified (T) lymphocytes killed EBV-transformed HLA-compatible and allogeneic target cells. Moreover, anti-MHC antibodies inhibited the cytotoxicity exerted by EBV-induced cytotoxic T lymphocytes (CTL) on both autologous and allogeneic target cells. Finally the lysis was EBV specific since PHA blasts were not killed and since only EBV-transformed cells could compete for lysis with the EBV-positive target cells. Efficient competition was achieved by EBV-transformed cells autologous or allogeneic to the targets, even when effector and target cells were fully allogeneic. All together, the data suggest that human anti-EBV CTL may recognize nonpolymorphic HLA determinants on the target cells in association with the virus-induced antigens.     

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.