Experimental infection of NOD/SCID mice reconstituted with human CD34+ cells with Epstein-Barr virus

Epstein-Barr virus (EBV)-induced lymphoproliferative disease is an important complication in the context of immune deficiency. Impaired T-cell immunity allows the outgrowth of transformed cells with the subsequent production of predominantly B-cell lymphomas. Currently there is no in vivo model that can adequately recapitulate EBV infection and its association with B-cell lymphomas. NOD/SCID mice engrafted with human CD34(+) cells and reconstituted mainly with human B lymphocytes may serve as a useful xenograft model to study EBV infection and pathogenesis. We therefore infected reconstituted mice with EBV. High levels of viral DNA were detected in the peripheral blood of all infected mice. All infected mice lost weight and showed decreased activity levels. Infected mice presented large visible tumors in multiple organs, most prominently in the spleen. These tumors stained positive for human CD79a, CD20, CD30, and EBV-encoded RNAs and were light chain restricted. Their characterization is consistent with that of large cell immunoblastic lymphoma. In addition, tumor cells expressed EBNA1, LMP1, and LMP2a mRNAs, which is consistent with a type II latency program. EBV(+) lymphoblastoid cell lines expressing human CD45, CD19, CD21, CD23, CD5, and CD30 were readily established from the bone marrow and spleens of infected animals. Finally, we also demonstrate that infection with an enhanced green fluorescent protein (EGFP)-tagged virus can be monitored by the detection of infected EGFP(+) cells and EGFP(+) tumors. These data demonstrate that NOD/SCID mice that are reconstituted with human CD34(+) cells are susceptible to infection by EBV and accurately recapitulate important aspects of EBV pathogenesis.     

Epstein-Barr virus lytic infection is required for efficient production of the angiogenesis factor vascular endothelial growth factor in lymphoblastoid cell lines

Although Epstein-Barr virus (EBV)-associated malignancies are primarily composed of cells with one of the latent forms of EBV infection, a small subset of tumor cells containing the lytic form of infection is often observed. Whether the rare lytically infected tumor cells contribute to the growth of the latently infected tumor cells is unclear. Here we have investigated whether the lytically infected subset of early-passage lymphoblastoid cell lines (LCLs) could potentially contribute to tumor growth through the production of angiogenesis factors. We demonstrate that supernatants from early-passage LCLs infected with BZLF1-deleted virus (Z-KO LCLs) are highly impaired in promoting endothelial cell tube formation in vitro compared to wild-type (WT) LCL supernatants. Furthermore, expression of the BZLF1 gene product in trans in Z-KO LCLs restored angiogenic capacity. The supernatants of Z-KO LCLs, as well as supernatants from LCLs derived with a BRLF1-deleted virus (R-KO LCLs), contained much less vascular endothelial growth factor (VEGF) in comparison to WT LCLs. BZLF1 gene expression in Z-KO LCLs restored the VEGF level in the supernatant. However, the cellular level of VEGF mRNA was similar in Z-KO, R-KO, and WT LCLs, suggesting that lytic infection may enhance VEGF translation or secretion. Interestingly, a portion of the vasculature in LCL tumors in SCID mice was derived from the human LCLs. These results suggest that lytically infected cells may contribute to the growth of EBV-associated malignancies by enhancing angiogenesis. In addition, as VEGF is a pleiotropic factor with effects other than angiogenesis, lytically induced VEGF secretion may potentially contribute to viral pathogenesis.     

Use of adenovirus vectors expressing Epstein-Barr virus (EBV) immediate-early protein BZLF1 or BRLF1 to treat EBV-positive tumors

The Epstein-Barr virus (EBV) genome is present in a variety of tumor types, including virtually all undifferentiated nasopharyngeal carcinomas (NPC) and a portion of gastric carcinomas. The uniform presence of the EBV genome in certain tumors (versus only a very small number of normal B cells) suggests that novel therapies which specifically target EBV-positive cells for destruction might be effective for treating such tumors. Although the great majority of EBV-positive tumor cells are infected with one of the latent forms of EBV infection, expression of either viral immediate-early protein (BZLF1 or BRLF1) is sufficient to convert the virus to the lytic form of infection. Induction of the lytic form of EBV infection could potentially result in death of the tumor cell. Here we have examined the efficacy of adenovirus vectors expressing the BZLF1 or BRLF1 proteins for treatment of EBV-positive epithelial tumors. The BZLF1 and BRLF1 vectors induced preferential killing of EBV-positive, versus EBV-negative, gastric carcinoma cells in vitro. Infection of C18 NPC tumors (grown in nude mice) with either the BZLF1 or BRLF1 vector, but not a control adenovirus vector, induced expression of early lytic EBV genes in tumor cells. Injection of C18 tumors with the BZLF1 or BRLF1 adenovirus vector, but not the control vector, also significantly inhibited growth of the tumors in nude mice. The addition of ganciclovir did not significantly affect the antitumor effect of the BZLF1 and BRLF1 adenovirus vectors. These results suggest a potential cancer therapy against EBV-related tumors.     

An Epstein-Barr Virus (EBV) mutant with enhanced BZLF1 expression causes lymphomas with abortive lytic EBV infection in a humanized mouse model

Immunosuppressed patients are at risk for developing Epstein-Barr Virus (EBV)-positive lymphomas that express the major EBV oncoprotein, LMP1. Although increasing evidence suggests that a small number of lytically infected cells may promote EBV-positive lymphomas, the impact of enhanced lytic gene expression on the ability of EBV to induce lymphomas is unclear. Here we have used immune-deficient mice, engrafted with human fetal hematopoietic stem cells and thymus and liver tissue, to compare lymphoma formation following infection with wild-type (WT) EBV versus infection with a "superlytic" (SL) mutant with enhanced BZLF1 (Z) expression. The same proportions (2/6) of the WT and SL virus-infected animals developed B-cell lymphomas by day 60 postinfection; the remainder of the animals had persistent tumor-free viral latency. In contrast, all WT and SL virus-infected animals treated with the OKT3 anti-CD3 antibody (which inhibits T-cell function) developed lymphomas by day 29. Lymphomas in OKT3-treated animals (in contrast to lymphomas in the untreated animals) contained many LMP1-expressing cells. The SL virus-infected lymphomas in both OKT3-treated and untreated animals contained many more Z-expressing cells (up to 30%) than the WT virus-infected lymphomas, but did not express late viral proteins and thus had an abortive lytic form of EBV infection. LMP1 and BMRF1 (an early lytic viral protein) were never coexpressed in the same cell, suggesting that LMP1 expression is incompatible with lytic viral reactivation. These results show that the SL mutant induces an "abortive" lytic infection in humanized mice that is compatible with continued cell growth and at least partially resistant to T-cell killing.     

Functional analysis of the CD4(+) T-cell response to Epstein-Barr virus: T-cell-mediated activation of resting B cells and induction of viral BZLF1 expression

In contrast to the major role played by Epstein-Barr virus (EBV)-specific CD8(+) cytotoxic T-cell responses in immunosurveillance, recent studies have offered the apparently paradoxical suggestion that development of EBV-driven human B-cell lymphoproliferative disorders and tumors in SCID/hu mice is dependent on the presence of T cells, in particular CD4(+) T cells. This study presents a functional analysis of the CD4(+) T-cell response to EBV and shows that while CD4(+) T cells may be cytotoxic, they also express Th2 cytokines and CD40 ligand (gp39) and possess B-cell helper function. We show that EBV-specific CD4(+) T cells can provide non-HLA-restricted help for activation of resting B cells via a gp39-CD40-dependent pathway and are able to induce expression of BZLF1, a viral lytic cycle transactivator in latently infected resting B cells, ultimately resulting in rapid outgrowth of transformed B-cell colonies. These results support the proposal that CD4(+) T cells may play a key role in reactivation of latent EBV infection and may thus contribute to the pathogenesis of EBV-driven lymphoproliferative disorders.     

EBV and systemic lupus erythematosus: a new perspective

We have proposed that EBV uses mature B cell biology to access memory B cells as a site of persistent infection. A central feature of this model is that EBV adapts its gene expression profile to the state of the B cell it resides in and that the level of infection is stable over time. This led us to question whether changes in the behavior or regulation of mature B cells would alter the state of EBV persistence. To investigate this, we studied the impact of systemic lupus erythematosus (SLE), a disease characterized by immune dysfunction, on EBV infection. We show that patients with SLE have abnormally high frequencies of EBV-infected cells in their blood, and this is associated with the occurrence of SLE disease flares. Although patients with SLE have frequencies of infected cells comparable to those seen in immunosuppressed patients, in SLE the effect was independent of immunosuppressive therapy. Aberrant expression of viral lytic (BZLF1) and latency (latency membrane proteins 1 and 2a) genes was also detected in the blood of SLE patients. We conclude that the abnormal regulation of EBV infection in SLE patients reflects the sensitivity of the virus to perturbation of the immune system.     

The ZIIR element of the Epstein-Barr virus BZLF1 promoter plays a central role in establishment and maintenance of viral latency

The Epstein-Barr virus (EBV) BZLF1 gene encodes the immediate-early (IE) protein Zta, which plays a central role in regulating the switch between viral latency and lytic replication. A silencing element, ZIIR, is located between the ZID and ZII positive regulatory elements in the BZLF1 promoter Zp. We report here the phenotypes of variants of EBV strain B95.8 containing base substitution mutations in this ZIIR element. HEK293 cells infected with ZIIR mutant (ZIIRmt) virus produced at least 20-fold more viral IE Zta and Rta and early (E) EAD protein than did cells infected with the parental wild-type (WT) virus, leading to viral DNA replication and production of infectious virus. However, ZIIR mutant virus was 1/10 as efficient as WT virus in establishing proliferating B-cell clones following infection of human primary blood B cells. The ZIIRmt-infected lymphoblastoid cell lines (LCLs) that did grow out exhibited a phenotype similar to the one observed in 293 cells, including marked overproduction of IE and E gene products relative to WT-infected LCLs and lytic replication of the viral genome. Incubation of the ZIIRmt-infected LCLs with the chemical inducer 12-O-tetradecanoyl-phorbol-13-acetate (TPA) led to much greater activation of Zp than did the same treatment of WT- or ZVmt-infected LCLs. Furthermore, a protein kinase C (PKC) inhibitor, bis-indolylmaleimide, eliminated this activation by TPA. Thus, we conclude that ZIIR is a potent silencing element of Zp; it plays a key role in establishment and maintenance of EBV latency by inhibiting activation of Zp through the PKC signal transduction pathway.     

Cytolytic CD4(+)-T-cell clones reactive to EBNA1 inhibit Epstein-Barr virus-induced B-cell proliferation

In the absence of immune surveillance, Epstein-Barr virus (EBV)-infected B cells generate neoplasms in vivo and transformed cell lines in vitro. In an in vitro system which modeled the first steps of in vivo immune control over posttransplant lymphoproliferative disease and lymphomas, our investigators previously demonstrated that memory CD4(+) T cells reactive to EBV were necessary and sufficient to prevent proliferation of B cells newly infected by EBV (S. Nikiforow et al., J. Virol. 75:3740-3752, 2001). Here, we show that three CD4(+)-T-cell clones reactive to the latent EBV antigen EBNA1 also prevent the proliferation of newly infected B cells from major histocompatibility complex (MHC) class II-matched donors, a crucial first step in the transformation process. EBNA1-reactive T-cell clones recognized B cells as early as 4 days after EBV infection through an HLA-DR-restricted interaction. They secreted Th1-type and Th2-type cytokines and lysed EBV-transformed established lymphoblastoid cell lines via a Fas/Fas ligand-dependent mechanism. Once specifically activated, they also caused bystander regression and bystander killing of non-MHC-matched EBV-infected B cells. Since EBNA1 is recognized by CD4(+) T cells from nearly all EBV-seropositive individuals and evades detection by CD8(+) T cells, EBNA1-reactive CD4(+) T cells may control de novo expansion of B cells following EBV infection in vivo. Thus, EBNA1-reactive CD4(+)-T-cell clones may find use as adoptive immunotherapy against EBV-related lymphoproliferative disease and many other EBV-associated tumors.     

Persistent infection drives the development of CD8+ T cells specific for late lytic infection antigens in lymphocryptovirus-infected macaques and Epstein-Barr virus-infected humans

We examined the CD8(+) T cell repertoire against lytic infection antigens in rhesus macaques persistently infected with the Epstein-Barr virus (EBV)-related lymphocryptovirus (rhLCV). CD8(+) T cells specific for late (L) antigens were detected at rates comparable to those for early antigens and were associated with increasing duration of infection. L antigen-specific CD8(+) T cells were also readily detected in adult, EBV-positive humans. Thus, viral major histocompatibility complex class I (MHCI) immune evasion genes expressed during lytic LCV infection do not prevent L-specific CD8(+) T cell development over time during persistent infection.     

Inhibition of polo-like kinase 1 (PLK1) facilitates reactivation of gamma-herpesviruses and their elimination

Both Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV) establish the persistent, life-long infection primarily at the latent status, and associate with certain types of tumors, such as B cell lymphomas, especially in immuno-compromised individuals including people living with HIV (PLWH). Lytic reactivation of these viruses can be employed to kill tumor cells harboring latently infected viral episomes through the viral cytopathic effects and the subsequent antiviral immune responses. In this study, we identified that polo-like kinase 1 (PLK1) is induced by KSHV de novo infection as well as lytic switch from KSHV latency. We further demonstrated that PLK1 depletion or inhibition facilitates KSHV reactivation and promotes cell death of KSHV-infected lymphoma cells. Mechanistically, PLK1 regulates Myc that is critical to both maintenance of KSHV latency and support of cell survival, and preferentially affects the level of H3K27me3 inactive mark both globally and at certain loci of KSHV viral episomes. Furthremore, we recognized that PLK1 inhibition synergizes with STAT3 inhibition to efficiently induce KSHV reactivation. We also confirmed that PLK1 depletion or inhibition yields the similar effect on EBV lytic reactivation and cell death of EBV-infected lymphoma cells. Lastly, we noticed that PLK1 in B cells is elevated in the context of HIV infection and caused by HIV Nef protein to favor KSHV/EBV latency.     

Impaired transporter associated with antigen processing-dependent peptide transport during productive EBV infection

Human herpesviruses, including EBV, persist for life in infected individuals. During the lytic replicative cycle that is required for the production of infectious virus and transmission to another host, many viral Ags are expressed. Especially at this stage, immune evasion strategies are likely to be advantageous to avoid elimination of virus-producing cells. However, little is known about immune escape during productive EBV infection because no fully permissive infection model is available. In this study, we have developed a novel strategy to isolate populations of cells in an EBV lytic cycle based on the expression of a reporter gene under the control of an EBV early lytic cycle promoter. Thus, induction of the viral lytic cycle in transfected EBV(+) B lymphoma cells resulted in concomitant reporter expression, allowing us, for the first time, to isolate highly purified cell populations in lytic cycle for biochemical and functional studies. Compared with latently infected B cells, cells supporting EBV lytic cycle displayed down-regulation of surface HLA class I, class II, and CD20, whereas expression levels of other surface markers remained unaffected. Moreover, during lytic cycle peptide transport into the endoplasmic reticulum, was reduced to <30% of levels found in latent infection. Because steady-state levels of TAP proteins were unaffected, these results point toward EBV-induced interference with TAP function as a specific mechanism contributing to the reduced levels of cell surface HLA class I. Our data implicate that EBV lytic cycle genes encode functions to evade T cell recognition, thereby creating a window for the generation of viral progeny.     

Epstein-Barr virus BZLF1 gene, a switch from latency to lytic infection, is expressed as an immediate-early gene after primary infection of B lymphocytes

We demonstrate here that the Epstein-Barr virus (EBV) BZLF1 gene, a switch from latent infection to lytic infection, is expressed as early as 1.5 h after EBV infection in Burkitt's lymphoma-derived, EBV-negative Akata and Daudi cells and primary B lymphocytes. Since BZLF1 mRNA is expressed even when the cells are infected with EBV in the presence of anisomycin, an inhibitor of protein synthesis, its expression does not require prerequisite protein synthesis, indicating that BZLF1 is expressed as an immediate-early gene following primary EBV infection of B lymphocytes.