Identification of Protein Kinase Inhibitors with a Selective Negative Effect on the Viability of Epstein-Barr Virus Infected B Cell Lines

Epstein-Barr virus (EBV) is a human herpesvirus, which is causally associated with the development of several B lymphocytic malignancies that include Burkitt''s lymphomas, Hodgkin''s disease, AIDS and posttransplant associated lymphomas. The transforming activity of EBV is orchestrated by several latent viral proteins that mimic and modulate cellular growth promoting and antiapoptotic signaling pathways, which involve among others the activity of protein kinases. In an effort to identify small molecule inhibitors of the growth of EBV-transformed B lymphocytes a library of 254 kinase inhibitors was screened. This effort identified two tyrosine kinase inhibitors and two MEK inhibitors that compromised preferentially the viability of EBV-infected human B lymphocytes. Our findings highlight the possible dependence of EBV-infected B lymphocytes on specific kinase-regulated pathways underlining the potential for the development of small molecule-based therapeutics that could target selectively EBV-associated human B lymphocyte malignancies.     

Differential interleukin-2 and interferon-gamma production by human lymphocyte cultures exceptionally resistant to Epstein-Barr virus immortalization

Epstein-Barr virus (EBV) readily immortalizes human peripheral blood lymphocytes (PBL) in vitro. However, during the past several years, we found that PBL from two exceptional EBV-seropositive healthy adult individuals were refractory to immortalization by EBV. We report here a study aimed at learning about the immunobiological features which differentiate these EBV-resistant (R) PBL from others which are susceptible (S) to EBV immortalization. Results of this investigation indicate that: (a) Following EBV infection, R-PBL produced significantly higher amounts of interferon gamma (IFN-gamma) than S-PBL. There were however no differences in regard to interferon alpha production between these two types (R and S) of EBV-infected cultures. (b) R-PBL had a maximal interleukin-2 (IL-2) production by S-PBL occurred at least 48 hr later, i.e., at Day 7. (c) The percentage of non-B cells expressing the IL-2 receptor was also higher in EBV-infected R-PBL than S-PBL. (d) In contrast, expression of IL-2 receptors after EBV infection was higher on B cells from S-PBL than on B cells from R-PBL. Interestingly, no differences were noted in regard to IL-2 receptor expression between R-PBL and S-PBL treated with mitogens (i.e., phytohemagglutinin and pokeweed mitogen). (e) Finally, using anti-IL-2 and anti-IFN-gamma antibodies in EBV-infected R-PBL cultures, we were able to obtain EBV-induced immortalization of these cultures. Taken together, these results suggest that an early IL-2 synthesis and high IFN-gamma production by EBV-infected PBL play an important role against lymphocyte immortalization by EBV.     

Epstein-Barr virus recombinants from BC-1 and BC-2 can immortalize human primary B lymphocytes with different levels of efficiency and in the absence of coinfection by Kaposi's sarcoma-associated herpesvirus

Epstein-Barr virus (EBV) and Kaposi's sarcoma-associated herpesvirus (KSHV) are human gammaherpesviruses associated with numerous malignancies. Primary effusion lymphoma or body cavity-based lymphoma is a distinct clinicopathological entity that, in the majority of cases, manifests coinfection with KSHV and EBV. In previous analyses, we have characterized the EBV in the BC-1 and BC-2 cell lines as potential intertypic recombinants of the EBV types 1 and 2. In order to examine the infectious and transforming capacities of KSHV and the intertypic EBV recombinants from the BC-1 and BC-2 cell lines, viral replication was induced in these cell lines and fresh human primary B lymphocytes were infected with progeny virus. The transformed clones were analyzed by PCR and Western blotting. All analyzed clones were infected with the intertypic progeny EBV but had no detectable signal for progeny KSHV. Additionally, primary B lymphocytes incubated with viral supernatant containing KSHV alone showed an unsustained initial proliferation, but prolonged growth or immortalization of these cells in vitro was not observed. We also show that the EBV recombinants from BC-1 were less efficient than the EBV recombinants from BC-2 in the ability to maintain the transformed phenotype of the infected human B lymphocytes. From these findings, we conclude that the BC-1 and BC-2 intertypic EBV recombinants can immortalize human primary B lymphocytes, albeit at different levels of efficiency. However, the KSHV induced from BC-1 and BC-2 alone cannot transform primary B cells, nor can it coinfect EBV-positive B lymphocytes under our experimental conditions with B lymphocytes from EBV-seropositive individuals. These results are distinct from those in one previous report and suggest a possible requirement for other factors to establish coinfection with both viral agents.     

Epstein-Barr virus (EBV)-infected monocytes facilitate dissemination of EBV within the oral mucosal epithelium

Epstein-Barr virus (EBV) causes hairy leukoplakia (HL), a benign lesion of oral epithelium that occurs primarily in the setting of human immunodeficiency virus (HIV)-associated immunodeficiency. However, the mechanisms of EBV infection of oral epithelium are poorly understood. Analysis of HL tissues shows a small number of EBV-positive intraepithelial macrophages and dendritic/Langerhans cells. To investigate a role for these cells in spreading EBV to epithelial cells, we used tongue and buccal explants infected ex vivo with EBV. We showed that EBV first infects submucosal CD14(+) monocytes, which then migrate into the epithelium and spread virus to oral epithelial cells, initiating productive viral infection within the terminally differentiated spinosum and granulosum layers. Incubation of EBV-infected monocytes and oral explants with antibodies to CCR2 receptor and monocyte chemotactic protein 1 prevented entry of monocytes into the epithelium and inhibited EBV infection of keratinocytes. B lymphocytes played little part in the spread of EBV to keratinocytes in our explant model. However, cocultivation of EBV-infected B lymphocytes with uninfected monocytes in vitro showed that EBV may spread from B lymphocytes to monocytes. Circulating EBV-positive monocytes were detected in most HIV-infected individuals, consistent with a model in which EBV may be spread from B lymphocytes to monocytes, which then enter the epithelium and initiate productive viral infection of keratinocytes.     

The B subunit of Escherichia coli heat-labile enterotoxin enhances CD8+ cytotoxic-T-lymphocyte killing of Epstein-Barr virus-infected cell lines

Epstein-Barr virus (EBV) is associated with a number of important human cancers, including nasopharyngeal carcinoma, gastric carcinoma, and Hodgkin's lymphoma. These tumors express a viral nuclear antigen, EBV nuclear antigen 1 (EBNA1), which cannot be presented to T cells in a major histocompatibility complex class I context, and the viral latent membrane proteins (LMPs). Although the LMPs are expressed in these tumors, no effective immune response is made. We report here that exposure to the cholera-like enterotoxin B subunit (EtxB) in EBV-infected lymphoblastoid cell lines (LCLs) enhances their susceptibility to killing by LMP-specific CD8(+) cytotoxic T lymphocytes (CTLs) in a HLA class I-restricted manner. CTL killing of LCLs is dramatically increased through both transporter-associated protein-dependent and -independent epitopes after EtxB treatment. The use of mutant B subunits revealed that the enhanced susceptibility of LCLs to CTL killing is dependent on the B subunit's interaction with GM(1) but not its signaling properties. These important findings could underpin the development of novel approaches to treating EBV-associated malignancies and may offer a general approach to increasing the presentation of other tumor and viral antigens.     

Epstein-Barr virus-encoded small RNAs (EBERs) do not modulate interferon effects in infected lymphocytes.

The recent derivation of otherwise isogenic Epstein-Barr virus (EBV) recombinants carrying or lacking the EBV small RNA (EBER) genes enabled us to test whether EBERs are similar to adenovirus VA RNAs in modulating interferon (IFN) effects on virus infection. EBER-positive and -negative EBV recombinants did not differ in their sensitivity to alpha interferon (IFN-alpha)- or IFN-gamma-mediated inhibition of lymphocyte growth transformation. In addition, EBERs did not decrease the inhibitory effects of IFN on vesicular stomatitis virus replication in EBV-transformed lymphocytes. EBER deletion also did not render EBV-transformed B lymphocytes susceptible to an IFN effect on cell proliferation or EBV replication.     

An Epstein-Barr virus isolated from a lymphoblastoid cell line has a 16-kilobase-pair deletion which includes gp350 and the Epstein-Barr virus nuclear antigen 3A

Epstein-Barr virus (EBV) is associated with human cancers, including nasopharyngeal carcinoma, Burkitt's lymphoma, gastric carcinoma and, somewhat controversially, breast carcinoma. EBV infects and efficiently transforms human primary B lymphocytes in vitro. A number of EBV-encoded genes are critical for EBV-mediated transformation of human B lymphocytes. In this study we show that an EBV-infected lymphoblastoid cell line obtained from the spontaneous outgrowth of B cells from a leukemia patient contains a deletion, which involves a region of approximately 16 kbp. This deletion encodes major EBV genes involved in both infection and transformation of human primary B lymphocytes and includes the glycoprotein gp350, the entire open reading frame of EBNA3A, and the amino-terminal region of EBNA3B. A fusion protein created by this deletion, which lies between the BMRF1 early antigen and the EBNA3B latent antigen, is truncated immediately downstream of the junction 21 amino acids into the region of the EBNA3B sequence, which is out of frame with respect to the EBNA3B protein sequence, and indicates that EBNA3B is not expressed. The fusion is from EBV coordinate 80299 within the BMRF1 sequence to coordinate 90998 in the EBNA3B sequence. Additionally, we have shown that there is no detectable induction in viral replication observed when SNU-265 is treated with phorbol esters, and no transformants were detected when supernatant is used to infect primary B lymphocytes after 8 weeks in culture. Therefore, we have identified an EBV genome with a major deletion in critical genes involved in mediating EBV infection and the transformation of human primary B lymphocytes that is incompetent for replication of this naturally occurring EBV isolate.     

An ATM/Chk2-mediated DNA damage-responsive signaling pathway suppresses Epstein-Barr virus transformation of primary human B cells

Epstein-Barr virus (EBV), an oncogenic herpesvirus that causes human malignancies, infects and immortalizes primary human B cells in?vitro into indefinitely proliferating lymphoblastoid cell lines, which represent a model for EBV-induced tumorigenesis. The immortalization efficiency is very low, suggesting that an innate tumor suppressor mechanism is operative. We identify the DNA damage response (DDR) as?a major component of the underlying tumor suppressor mechanism. EBV-induced DDR activation was not due to lytic viral replication, nor did the DDR marks colocalize with latent episomes. Rather, a transient period of EBV-induced hyperproliferation correlated with DDR activation. Inhibition of the DDR kinases ATM and Chk2 markedly increased transformation efficiency of primary B cells. Further, the viral latent oncoprotein EBNA3C was required to attenuate the EBV-induced DDR. We propose that heightened oncogenic activity in early cell divisions activates a growth-suppressive DDR that is attenuated by viral latency products to induce cell immortalization.     

Primary immune responses by cord blood CD4(+) T cells and NK cells inhibit Epstein-Barr virus B-cell transformation in vitro

Epstein-Barr virus (EBV) transformation of B cells from fetal cord blood in vitro varies depending on the individual sample. When a single preparation of EBV was simultaneously used to transform fetal cord blood samples from six different individuals, the virus transformation titer varied from less than zero to 10(5.9). We show that this variation in EBV transformation is associated with a marked primary immune response in cord blood samples predominately involving CD4(+) T cells and CD16(+) CD56(+) NK cells. After virus challenge both CD4(+) T cells and NK cells in fetal cord blood cultures expressed the lymphocyte activation marker CD69. The cytotoxic response against autologous EBV-infected lymphoblastoid cell line (LCL) targets correlated with the number of CD16(+) CD69(+) cells and was inversely correlated with the virus transformation titer. Although NK activity was detected in fresh cord blood and increased following activation by the virus, killing of autologous LCLs was detected only following activation by exposure to the virus. Both activated CD4(+) T cells and CD16(+) NK cells were independently able to kill autologous LCLs. Both interleukin-2 and gamma interferon were produced by CD4(+) T cells after virus challenge. The titer of EBV was lower when purified B cells were used than when whole cord blood was used. Addition of monocytes restored the virus titer, while addition of resting T cells or EBV-activated CD4(+) T-cell blasts reduced the virus titer. We conclude that there are primary NK-cell and Th1-type CD4(+) T-cell responses to EBV in fetal cord blood that limit the expansion of EBV-infected cells and in some cases eliminate virus infection in vitro.     

Immune surveillance and therapy of lymphomas driven by Epstein-Barr virus protein LMP1 in a mouse model

B cells infected by Epstein-Barr virus (EBV), a transforming virus endemic in humans, are rapidly cleared by the immune system, but some cells harboring the virus persist for life. Under conditions of immunosuppression, EBV can spread from these cells and cause life-threatening pathologies. We have generated mice expressing the transforming EBV latent membrane protein 1 (LMP1), mimicking a constitutively active CD40 coreceptor, specifically in B cells. Like human EBV-infected cells, LMP1+ B cells were efficiently eliminated by T cells, and breaking immune surveillance resulted in rapid, fatal lymphoproliferation and lymphomagenesis. The lymphoma cells expressed ligands for a natural killer (NK) cell receptor, NKG2D, and could be targeted by an NKG2D-Fc fusion protein. These experiments indicate a central role for LMP1 in the surveillance and transformation of EBV-infected B cells in vivo, establish a preclinical model for B cell lymphomagenesis in immunosuppressed patients, and validate a new therapeutic approach.     

Transmembrane peptides used to investigate the homo-oligomeric interface and binding hotspot of latent membrane protein 1

Epstein-Barr virus (EBV), a human γ-herpesvirus, establishes lifelong infection by targeting the adaptive immune system of the host through memory B cells. Although normally benign, EBV contributes to lymphoid malignancies and lymphoproliferative syndromes in immunocompromised individuals. The viral oncoprotein latent membrane protein 1 (LMP-1) is essential for B lymphocyte immortalization by EBV. The constitutive signaling activity of LMP-1 is dependent on homo-oligomerization of its six-spanning hydrophobic transmembrane domain (TMD). However, the mechanism driving LMP-1 intermolecular interaction is poorly understood. Here, we show that the fifth transmembrane helix (TM5) of LMP-1 strongly self-associates, forming a homotrimeric complex mediated by a polar residue embedded in the membrane, D150. Replacement of this aspartic acid residue with alanine disrupts TM5 self-association in detergent micelles and bacterial cell membranes. A full-length LMP-1 variant harboring the D150A substitution is deficient in NFκB activation, supporting the key role of the fifth transmembrane helix in constitutive activation of signaling by this oncoprotein.     

Epstein-Barr virus with the latent infection nuclear antigen 3B completely deleted is still competent for B-cell growth transformation in vitro

The Epstein-Barr virus (EBV) nuclear antigen 3B (EBNA-3B) is considered nonessential for EBV-mediated B-cell growth transformation in vitro based on three virus isolates with EBNA-3B mutations. Two of these isolates could potentially express truncated EBNA-3B products, and, similarly, we now show that the third isolate, IB4, has a point mutation and in-frame deletion of 263 amino acids. In order to test whether a virus with EBNA-3B completely deleted can immortalize B-cell growth, we first cloned the EBV genome as a bacterial artificial chromosome (BAC) and showed that the BAC-derived virus was B-cell immortalization competent. Deletion of the entire EBNA-3B open reading frame from the EBV BAC had no adverse impact on growth of EBV-immortalized B cells, providing formal proof that EBNA-3B is not essential for EBV-mediated B-cell growth transformation in vitro.     

The tyrosine kinase lck is critically involved in the growth transformation of human B lymphocytes.

Epstein-Barr virus (EBV) exposure of human B lymphocytes induces rapid, Ca(2+)-dependent tyrosine phosphorylation of two cytosolic proteins, one likely the CD21 EBV receptor and another unknown species of 55-60 kDa. We now identify the latter protein as the tyrosine kinase lck (p56lck). In T cells many activation events reduce the high constitutive p56lck expression levels typical for that lineage, and they induce the appearance of a 60-kDa lck species. We now demonstrate that in B cells exposed to EBV the at best low constitutive p56lck expression levels are rapidly and transiently up-regulated without generation of 60-kDa lck. lck-specific antisense oligonucleotides block p56lck induction and prevent subsequent B cell activation and immortalization whereas B cell activation by nononcogenic agents was unaffected. We propose that p56lck superinduction is a transformation prerequisite which signals entry into the oncogenic growth transformation process.     

Inhibition of polyamine synthesis in human B lymphocytes during primary infection with Epstein-Barr virus (EBV) blocks cellular DNA synthesis but not the expression of EBV-encoded nuclear antigens (EBNA)

Inhibition of polyamine synthesis by 2-difluoromethylornithine (DFMO) treatment had no apparent effect on the initial manifestation of Epstein-Barr virus (EBV) infection in human B lymphocytes, because the expression of EBV-encoded nuclear antigens (EBNA) occurred normally. However, many subsequent steps in the transformation process were inhibited by DFMO treatment. These include cellular DNA synthesis and immunoglobulin (IgM, IgG and IgA) synthesis and secretion. Consequently, DFMO treatment blocked the progression of the transformation process of EBV-infected B lymphocytes. EBV-carrying marmoset B lymphocytes (B95-8 cells) were also blocked in their DNA synthesis when treated with DFMO. At variance with other DNA synthesis inhibitors, which induce virus production very effectively in B95-8 cells, DFMO caused no increase in the number of cells expressing the early antigens associated with the lytic cycle.     

An efficient method for the rapid establishment of Epstein-Barr virus immortalization of human B lymphocytes

Abstract. Several methods have been developed for the immortalization of B lymphocytes by Epstein-Barr virus (EBV). We developed an efficient method which reduces the time from culture initiation to immortalization and cryopreservation. Two infections of EBV to lymphocytes, and the use of phorbol ester-induced EBV stock significantly improved immortalization efficiency and reduced the time between initiation and immortalization and cryopreservation. The resulting cell bank was used to produce DNA for genetic studies focusing on the genes involved in immune and autistic disorders.