Retinoic acid and steroids inhibit Epstein-Barr virus-induced nuclear antigen, DNA synthesis and lymphocyte transformation

Retinoic acid, dexamethasone and prednisolone were evaluated for their effects on Epstein-Barr virus (EBV)-induced nuclear antigen (EBNA), DNA synthesis and transformation of human thymus-independent, B lymphocytes. It was found that continuous treatment of target cells with these agents completely inhibited EBV-induced transformation events. However, discontinuous treatment of the virus-infected cultures with these agents resulted in the recovery of DNA synthesis, and the appearance of EBNA and transformation. When the cells were treated with these agents 8 days after the virus infection, the inhibitors had no effect. These results show that only continuous treatment with these agents inhibited EBV-mediated transformation; these inhibitors had no effect once the EBNA and EBV-induced DNA synthesis were initiated in target lymphocytes.     

Abortive expression of the Epstein-Barr virus (EBV) cycle in a variety of EBV DNA-containing cell lines, as reflected by nucleic acid hybridization in situ.

A variety of Epstein-Barr virus (EBV) DNA-containing cell lines have been tested for the expression of the EBV-associated antigens EBNA (nuclear antigen), EA (early antigen), and VCA (viral capsid antigen), and for the presence of cells containing disproportionate amounts of EBV DNA. The antigen tests utilized immunofluorescence and 125I-labelled antibodies combined with autoradiography. EBV-DNA was detected by in situ hybridization with 3H-labelled EBV RNA complementary to P3HR-1 EBV DNA (P-EBVcRNA). The P-EBVcRNA has been shown to represent the majority of the P3HR-1 EBV DNA sequences. It was concluded that EBV DNA-containing cell lines can be divided into those that express only EBNA, those that express EBNA and EA and those that express EBNA, EA and VCA and also contain cells that undergo disproportionate EBV DNA synthesis. Consequently, in some cell lines there is an abortive expression of the EBV cycle in that some cells spontaneously express EA but fail to continue further to viral DNA synthesis. A similar pattern can be found after experimental induction of the EBV cycle, suggesting that related mechanisms govern the spontaneous expression of the EBV cycle and the extent of its inducibility.     

Early events in transformation of human cord leukobytes by Epstain-Barr virus: induction of DNA synthesis, mitosis and the virus-associated nuclear antigen synthesis

The present investigations were undertaken to establish the early events in EBV-induced transformation of human cord lymphocytes with particular reference to the induction of DNA synthesis, mitosis and viral synthesis. When cord leukocytes were exposed to a 3 h pulse of²H-TdR at 3 h intervals following EBV infection and examined by autoradiography, blastoid cells with labelled nuclei appeared 12-24 h after virus exposure, reaching a maximum of 7.5% in 15 h. When the infected leukocytes were reincubated after each pulse for an additional 20-40 h with colchicine added 10 h prior to termination of incubation, 18% of the positive cells were in mitosis. The cells were examined by immunofluorescence for the synthesis of EBV-associated antigens. Early antigen (EA)—and viral capsid antigen (VCA)—were not detected, but EBV-associated nuclear antigen (EBNA) synthesis became evident as early as 2 days after infection. The frequency of EBNA-positive cells, mostly blastoid cells, increased with continued incubation, and at 10 days the majority of the cells were positive. These findings strongly suggest that the human cord leukocytes are non-permissive for replication of EBV, and that the virus can induce in them early DNA synthesis followed by high-frequency mitosis and cell proliferation, in close association of the viral genome with the host-cell genome as shown by the synthesis of EBV-specific “footprint”.     

Heterogeneity of Epstein-Barr virus derived from P3HR-1 cells.

Infection of cells of the EBV-free human B-lymphoma lines BJAB and Ramos resulted in conversion of these cells to EBV-genome carriers expressing EBNA. EBV isolates from P3HR-1 cells induced a heterogeneous EBNA pattern: both a faintly granular pattern and brilliant EBNA-expression were observed. The two types of EBNA-expressing cells could be separated upon cloning. Brilliantly EBNA-expressing cells always segregated varying percentages of EBNA-negative cells. An EBNA-negative subclone derived from these cells was devoid of detectable EBV DNA. Nucleic acid hybridization experiments failed to reveal a correlation between the intensity of EBNA expression and the number of EBV genome equivalents per cell. EBV genome-containing cells had an average of 14-fold more cells showing EA synthesis after superinfection by P3HR-1 virus, when compared with EBNA-negative cells infected under identical conditions. Studies on the kinetics of EA induction in EBNA-positive and EBNA-negative cells indicate that complementation is required for the induction of EA after superinfection.     

Early activation of the proto-oncogene c-fgr during Epstein-Barr virus immortalization.

In an attempt to clarify the chronological relationships between Epstein-Barr virus (EBV) infection, B cell immortalization and c-fgr activation, we evaluated for the presence of EBV-determined nuclear antigen (EBNA), cellular DNA synthesis and expression of c-fgr-specific RNA following infection of human peripheral blood lymphocytes with B95-8 EBV. High expression of c-fgr was observed prior to EBNA detection and cellular DNA synthesis in EBV-infected cells. These results suggest that activation of c-fgr is an essential event during the early phase of EBV immortalization.     

Lymphoblastoid transformation and kinetics of appearance of viral nuclear antigen (EBNA) in cord-blood lymphocytes infected by Epstein-Barr Virus (EBV).

Human cord-blood lymphocytes were infected with B95.8 Epstein-Barr virus (EBV) before and after separation into B- and T-cell populations. Lymphoblastoid cells exhibiting B-cell characteristics appeared after 2 to 3 days of culture in the total population and in the separated B-cell subpopulation but not in the T-cell subpopulation. EBV nuclear antigen (EBNA) was detected concurrently with the appearance of lymphoblastoid cells. The proportion of EBNA-positive cells corresponded to that of lymphoblastoid cells, and reached 50% after 4 days. EBNA was present only in cells with B-cell markers. These observations indicate that only B-cells are susceptible to EBV infection, that the transformation occurs within a few days and that EBNA is a valid early marker for susceptibility to EBV transformation.     

Persistence of Epstein-Barr viral nuclear antigen (EBNA) in cells entering the EB viral cycle.

It was shown by double immunofluorescence studies that Epstein-Barr viral nuclear antigen (EBNA) was preserved in EBV-infected cells after they had entered the productive viral cycle, as signalled by the appearance of the early antigen (EA)complex. A nuclear component of the EA comples could be clearly distinguished from EBNA with regard to antigenic specificity.     

Transient expression of the Epstein-Barr virus LMP1 gene in human primary B cells induces cellular activation and DNA synthesis.

The Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) and Epstein-Barr virus nuclear antigen 2 (EBNA2) are expressed in EBV-immortalized human B cells. It has previously been shown that transfection of the LMP1 and EBNA2 genes into Burkitt's lymphoma cell lines results in the up-regulation of CD23, CD21, ICAM-1 and LFA-1 cell-surface proteins. In the present study, the effects of transient expression of the LMP1 and EBNA2 genes were studied in normal primary human B cells pretreated with UV-inactivated EBV particles. To identify and purify cells which express the transfected DNA we used a gene encoding a surface molecule, CD2, as a co-transfection marker. We show that transient expression of the LMP1 gene, from heterologous promoters, is sufficient to induce cellular enlargement and up-regulation of surface expression of the activation markers CD23, CD21, ICAM-1 and LFA-1 in primary B cells. Most importantly, we show that transient expression of the LMP1 gene is sufficient to induce DNA synthesis in human primary B cells. Transient EBNA2 expression enhanced the effect of transient LMP1 expression on CD21 and CD23 cell-surface expression but, under our experimental conditions, inhibited the induction of DNA synthesis by LMP1. We conclude that activation of primary B cells with inactivated EBV particles, followed by transient expression of only two viral genes, EBNA2 and LMP1, is sufficient to reconstitute some of the early events of B-cell immortalization by EBV.     

Induction of Epstein-Barr virus-associated nuclear antigen during in vitro transformation of human lymphoid cells.

Human lymphoid cells isolated from the peripheral blood of adults, from cord blood, and from fetal liver, spleen, bone marrow, and thymus were cultivated with or without a cell-free preparation of Epstein-Barr virus (EBV) with demonstrated transforming activity. The cultures were examined for the EBV-associated nuclear antigen (EBNA) and for transfromation into permanent lymphoblastoid cell lines (LCL). EBNA, seen only in cultures that had received exogenous EBV, was detected between days 1 and 6 after addition of EBV, most frequently on day 3. EBNA-positive cells had a lymphoblastoid appearance. Transformation into established LCL became apparent between days 12 and 19. The addition of pokeweed mitogen to cultures containing EBV enhanced the development of EBNA, whereas phytohemagglutinin or concanavalin A had no such effect. Neither EBNA nor transfomration was observed in lymphoid cells from fetal thymus. In fetal spleen, bone marrow, and liver cells, EBV regularly induced EBNA and LCL transformation.     

EBV is necessary for proliferation of dually infected primary effusion lymphoma cells

Epstein Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are found together in approximately 80% of primary effusion lymphomas (PEL), but their contribution to these cancers is unclear. We found that dominant-negative derivatives of EBNA1 inhibited EBV-positive PEL cells from forming colonies. Those rare PEL cells that proliferated after expression of the dominant-negative derivatives usually expressed these derivatives at low or undetectable levels and continued to maintain their EBV genomes. Those proliferating cells expressing higher levels of the derivatives expressed mutant derivatives that could not bind DNA. These findings indicate that EBV is required to sustain proliferation, as measured by colony formation of dually infected PEL cells. The dominant-negative derivatives of EBNA1 had no effect on the colony-forming ability of five EBV-negative, KSHV-negative hematopoietic cell lines. Surprisingly, they did inhibit the colony-forming ability of EBV-negative, KSHV-positive PEL cells. The small fraction of cells that continued to proliferate expressed only mutants of the EBNA1 derivatives that could no longer bind DNA. These findings indicate that the site-specific DNA-binding activity of EBNA1 or its derivatives when expressed efficiently in EBV-negative, KSHV-positive PEL cells inhibits their colony formation possibly through their binding to the KSHV genome.     

Epstein-Barr virus nuclear antigen 2 disrupts mitotic checkpoint and causes chromosomal instability

The Epstein-Barr virus nuclear antigen 2 (EBNA2) plays a keyrole in transformation of B-lymphocytes mediated by Epstein-Barrvirus (EBV) and can induce tumor formation in transgenic mice.However, the precise mechanism underlying EBNA2-mediated tumorigenesisremains elusive. Here, we report that EBNA2 can compromise mitoticspindle checkpoint (MSC) induced by the spindle inhibitor nocodazoleand cause chromosomal instability (CIN) in HEp-2, U2-OS andBJAB cells. When EBNA2-expressing cells were treated with nocodazole,they exited mitosis prematurely and initiated another roundof DNA synthesis. Nucleolocalization of EBNA2 was essentialfor EBNA2 to compromise MSC and to cause CIN. The metaphasechromosome spread data indicated that the EBNA2-expressing U2-OScells showed a more heterogenous chromosome number distributionthan the vector-transfected and parental cells. The median chromosomenumber for EBNA2-expressing, vector-transfected and parentalU2-OS cells is 75, 65 and 64, respectively. EBNA2 was shownto be able to downregulate mitotic arrest deficient 2 (MAD2)2- to 3-fold and upregulate polo-like kinase 1 (PLK1) 2-fold.The dysregulation of MAD2 and PLK1 may lead to activation ofanaphase promoting complex/ cyclosome and premature degradationof securin. Indeed, we found that when MSC was induced by nocodazole,securin was prematurely degraded in EBNA2-expressing cells.Finally, we show that EBNA2 could induce micronuclei and multinucleiformation in HEp-2 and U2-OS cells. Together, these studiesreveal a new function of EBNA2 in cell-cycle regulation andmay shed light on the role of EBNA2 in EBV-mediated tumorigenesis. Abbreviations: APC/C, anaphase promoting complex/ cyclosome; BrdU, bromodeoxyuridine; CIN, chromosomal instability; EBV, Epstein-Barr virus; EBNA2, Epstein-Barr virus nuclear antigen 2; ER, estrogen receptor; FACS, fluorescence-activated cell sorting; MAD2, mitotic arrest deficient 2; MSC, mitotic spindle checkpoint; 4-OHT, 4-hydroxytamoxifen; PLK1, polo-like kinase 1 Received June 1, 2008; revised December 5, 2008; accepted December 20, 2008.     

EBV infection of mitogen-stimulated human B lymphocytes

B lymphocytes from human blood were treated with the mitogen Protein A (Staph, aureus), which induces DNA synthesis in, as well as differeniation of, the B lymphocytes. The cells were subsequently exposed to Epstein-Barr virus (EBV). EBV-infected and mitogen-stimulated cells were detected simultaneously, by using a combination of immunofluorescence (for EBNA) and autoradiography (for DNA-synthesis). In the initial phase (1-2 days) after addition of the mitogen, stimulated cells were as susceptible to infection as resting cells. Thereafter, their susceptibility decreased. This suggests that, although initiation of DNA-synthesis does not seem to limit sensitivity to the viral infection, differentiation of the B cells might do so. The intensity and pattern of EBNA-staining in prestimulated cells different from that of the controls.     

EBV-transformed lymphoblastoid cell lines down-regulate ebna in parallel with secretory differentiation

Four monoclonal and one polyclonal lymphoblastoid cell line (LCL) were studied with regard to cytoplasmic immunoglobulin (clg) expression, presence of the Epstein-Barr virus (EBV)-determined nuclear antigen (EBNA) and DNA synthesis. Each line was found to consist of two subpopulations, with only minimal overlap. Proliferating, EBNA-positive, clg-negative cells formed the majority. The minority were EBNA-negative, contained abundant clg and were largely non-proliferating. This suggests the continuous occurrence of a maturation process within each LCL. The concomitant downregulation of EBNA raises the interesting question whether continued synthesis of the nuclear antigen is incompatible with differentiation for epigenetic reasons, or, alternatively, whether differentiation takes place when the viral genomes are suppressed or lost.     

Differential inducibility of Epstein-Barr virus in cloned, non-producer Raji cells.

Cells of the human lymphoblastoid non-producer line Raji were cloned in soft agar. Individual colonies were isolated and analyzed for their inducibility of the Epstein-Barr virus-associated early antigen (EA). The induction of EA by the tumor promoter TPA varied among the different cell clones. Clones with very high and very low inducibility of the resident Epstein-Barr virus genome were further analyzed. Constant differences in the inducibility of EA were observed after activation by tumor promoters, 5-iododeoxyuridine or antibodies to human IgM. Induction of EA synthesis by superinfection with Epstein-Barr virus from the P3HR-1 line, however, did not vary among the clones tested. No differences in expression of the Epstein-Barr virus-associated nuclear antigen (EBNA) were noted in cells of clones with high or low susceptibility to EA induction. DNA reassociation kinetics demonstrated that Raji cells with high susceptibility to EA induction contained a significantly higher number of Epstein-Barr virus genome equivalents per cell than cells with low susceptibility. Treatment of Raji cells with the tumor promoter TPA did not change the ratio of Epstein-Barr virus-specific DNA to cellular DNA.     

Cellular DNA synthesis in productive infection with adenoviruses

The effect of adenoviruses on cellular DNA synthesis in productive infection of hamster kidney cells and human embryonic lung cells was studied. (1) Treatment with 5-fluorouracil (FU) strongly reduced incorporation of ³H-dT into DNA of uninfected hamster kidney cultures. Under such conditions, the amount of incorporation of ³H-dT into DNA in FU-treated Ad5-infected cultures was about one-third of that observed in untreated ones. Analysis by DNA-DNA hybridization revealed that the major proportion of newly synthetized ³H-DNA was cellular between 16 to 32 h after infection in FU-treated infected cultures. Synthesis of viral DNA and infectious virus was slightly delayed and reduced in FU-treated cells. (2) With the aim of keeping the specific activities of ³H-dT derivatives in acid-soluble pools in cells as constant as possible, long-term labelling of DNA with ³H-dT after infection was carried out, in the presence of aminopterin, hypoxanthine and glycine to prevent endogenous biosynthesis of thymidylic acid. Incorporation of 3H-dT into cellular DNA was much greater in infected than in uninfected cultures after labelling for 24 h. These results strongly suggest that cellular DNA synthesis was induced in productive infection of hamster kidney cells by Ad5. (3) Infection of monolayer cultures of human embryo lung (HEL) cells with adenovirus types 2, 3, 4, 5 and 12 (Ad2, Ad3, Ad4, Ad5 and Ad12) resulted in productive infection with delayed cytopathic effect (CPE). ³H-dT uptake into DNA was stimulated in contact-inhibited HEL cells by infection. Apparent increase in incorporation of ³H-dT into cellular DNA was detectable for a long period in infected cultures, suggesting that induction of cellular DNA synthesis took place by infection. Thus the time of appearance of CPE seems to be correlated with the period during which induction of cellular DNA synthesis is detectable.     

V-val突变型EB病毒核抗原1的功能研究

背景与目的:EB病毒核抗原1(Epstein-Barr viral nuclear antigen 1,EBNA 1)对于维持EB病毒的潜伏感染有重要作用。V-val变异型的EBNA1与鼻咽癌有密切的相关性。本研究旨在比较原型和V-val变异型的EBNA1基因在上皮细胞中的功能的差异。方法:用PCR方法扩增出原型和V-val变异型EBNA1基因的全长并克隆到pGFP载体上,转染HEK293细胞,检测两种亚型的EBNA1蛋白的表达对细胞生物学性状的影响。以含有EB病毒增强子FR序列的荧光素酶载体作为报告基因,比较两种亚型的EBNA1基因对质粒的转录激活能力。结果:原型和V-val变异型EBNA1基因的表达对HEK293细胞的生长速度没有明显影响,但表达原型EBNA1的细胞的克隆形成能力明显低于V-val亚型。在裸鼠致瘤实验中,接种表达原型和V-val变异型EBNA1细胞的实验组均未见肿瘤形成。但是在瞬时转染实验中,表达V-val变异型EBNA1基因的HEK293细胞的荧光素酶活性明显高于原型EBNA1基因。结论:原型和变异型EBNA1均未发现有明显的转化细胞的能力,但是V-val变异型EBNA1蛋白与原型相比,其转录激活的功能明显增强。     

Interaction of the B95-8 and P3HR-1 substrains of Epstein-Barr virus (EBV) with peripheral human lymphocytes.

Two substrains of the Epstein-Barr virus derived from the B95-8 and P3HR-1 cell lines were studied for their interaction with human peripheral lymphocytes. It has been previously shown that B95-8 virus has and P3HR-1 virus lacks lymphocyte-transforming ("immortalizing") properties. DNA stimulation induced by B95-8 virus showed a good correlation with the number of surface Ig-positive cells. P3HR-1 virus added before B95-8 virus completely abolished the stimulation of DNA synthesis. It also prevented EBNA induction by B95-8 virus. P3HR-1 virus added after B95-8 virus diminished DNA stimulation by the latter in a time-dependent fashion. P3HR-1 virus did not inhibit DNA stimulation by phytohaemagglutinin but was inhibitory if added before a B cell mitogen (Staphylococcus Aureus). The origin of P3HR-1 virus and its relationship to the transformation process are discussed.     

Association of Epstein-Barr virus with nasopharyngeal carcinoma in Alaskan native patients: serum antibodies and tissue EBNA and DNA

Biopsy specimens from Alaskan Native patients with nasopharyngeal carcinoma (NPC) and from other patients seen on the otolaryngology service were tested for Epstein-Barr virus-specific DNA and nuclear antigen (EBNA). Serum samples from both groups were tested for various EBV-related antibodies. EBV DNA and EBNA results were in agreement in 29 of 31 tissue specimens tested by the two methods. Ten of 11 biopsies containing NPC cells were positive for EBV DNA. Two NPC patients had biopsies that showed only atypical epithelium but were also positive for EBV DNA or EBNA. The other tissue specimens were negative except for biopsies from two patients: one with a parotid gland lymphoepithelial lesion; another with undifferentiated carcinoma of salivary gland origin.