A virtual look at Epstein-Barr virus infection: simulation mechanism.

Epstein-Barr virus (EBV) is an important human pathogen that establishes a life-long persistent infection and for which no precise animal model exists. In this paper, we describe in detail an agent-based model and computer simulation of EBV infection. Agents representing EBV and sets of B and T lymphocytes move and interact on a three-dimensional grid approximating Waldeyer's ring, together with abstract compartments for lymph and blood. The simulation allows us to explore the development and resolution of virtual infections in a manner not possible in actual human experiments. Specifically, we identify parameters capable of inducing clearance, persistent infection, or death.

Epstein-Barr virus transactivates the human endogenous retrovirus HERV-K18 that encodes a superantigen.

Superantigens (SAgs) are proteins produced by pathogenic microbes to elicit potent, antigen-independent T cell responses that are believed to enhance the microbes' pathogenicity. Here we show that the human lymphotropic herpesvirus Epstein-Barr virus (EBV) transcriptionally activates the env gene of an endogenous retrovirus, HERV-K18, that possesses SAg activity. SAg activity was demonstrated by MHC class II dependent preferential activation of TCRVB13 T cells in response to murine B cells transfected with the HERV-K18 env gene. This is a unique demonstration of a pathogen inducing a host-encoded Sag and accounts for the previously described EBV associated Sag activity. The T cell activation elicited by the Sag could play a central role in EBV infection and associated diseases.

Epstein-Barr virus: exploiting the immune system.

In vitro, Epstein-Barr virus (EBV) will infect any resting B cell, driving it out of the resting state to become an activated proliferating lymphoblast. Paradoxically, EBV persists in vivo in a quiescent state in resting memory B cells that circulate in the peripheral blood. How does the virus get there, and with such specificity for the memory compartment? An explanation comes from the idea that two genes encoded by the virus--LMP1 and LMP2A--allow EBV to exploit the normal pathways of B-cell differentiation so that the EBV-infected B blast can become a resting memory cell.

Cells expressing the Epstein-Barr virus growth program are present in and restricted to the naive B-cell subset of healthy tonsils.

In this paper we demonstrate, for the first time, that Epstein-Barr virus (EBV)-infected cells expressing the lymphoblastoid growth program are present in healthy carriers of the virus. Previously we observed that latently infected naive B cells are present in tonsils only when viral replication is detected, suggesting that these may represent newly infected B cells. We have tested this idea by performing a reverse transcription-PCR analysis for the expression of latent genes (EBNA2 and the EBNA3s) that are characteristically expressed only by newly infected cells expressing the growth latency program. EBNA2 expression is regularly detected in purified naive (IgD(+)) tonsillar B cells (13 of 16 tonsils tested) but was never found in the IgD(-) population (0 of 16). More detailed analysis revealed that the mRNAs for the latent genes EBNA1 (3 of 3 tonsils tested), EBNA3a (3 of 5), EBNA3b (3 of 5), EBNA3c (3 of 5), LMP1 (6 of 6), and LMP2 (5 of 6) were also present in the IgD(+) population, but the EBNA1Q-K transcript, characteristic of nonlymphoblastoid forms of latency, was never detected (0 of 6). Finally, we demonstrate that the latently infected naive (IgD(+)) cells express CD80 (B7.1), a marker characteristically expressed on activated naive lymphoblasts but absent from resting naive B cells. The infected naive (IgD(+)) population in the tonsil therefore has the viral and cellular phenotype of a B-cell directly infected with EBV-an activated lymphoblast expressing the growth program.

Tonsillar memory B cells, latently infected with Epstein-Barr virus, express the restricted pattern of latent genes previously found only in Epstein-Barr virus-associated tumors.

Epstein-Barr virus (EBV) establishes a life-long persistent infection in most of the human population. In the peripheral blood, EBV is restricted to memory B cells that are resting and express limited genetic information. We have proposed that these memory cells are the site of long-term persistent infection. We now show that memory cells in the tonsil express the genes for EBV nuclear antigen 1 (EBNA1) (from the Qp promoter), latent membrane protein 1 (LMP1), and LMP2a but do not express EBNA2 or the EBNA3s. This pattern of latent gene expression has only been seen previously in EBV-associated tumors such as nasopharyngeal carcinoma, Hodgkin's disease (HD), and T/NK lymphomas. Normal circulating memory B cells frequently reenter secondary lymphoid tissue, where they receive signals essential for their survival. Specifically they require signals from antigen-specific T helper cells and from antigen itself. LMP1 and LMP2 are known to be able to generate these signals in a ligand-independent fashion. We suggest, therefore, that the transcription pattern we have found in latently infected, tonsillar, memory B cells is used because it allows for the expression of LMP1, LMP2a, and EBNA1 in the absence of the immunogenic and growth-promoting EBNA2 and EBNA3 molecules. LMP1 and LMP2a are produced to provide the surrogate rescue and survival signals needed to allow latently infected memory cells to persist, and EBNA1 is produced to allow replication of the viral episome.

EBV persistence involves strict selection of latently infected B cells.

EBV is found preferentially in IgD- B cells in the peripheral blood. This has led to the proposal that the recirculating memory B cell pool is the site of long-lived persistent infection. In this paper we have used CD27, a newly identified specific marker for memory B cells, to test this hypothesis. We show that EBV is tightly restricted in its expression. Less than 1 in 1000 of the infected cells in the peripheral blood are naive (IgD+, CD27-) and <1 in 250 are IgD+ memory cells. Furthermore, EBV was undetectable in the self-renewing peripheral CD5+ or B1 cells, a subset that has not been through a germinal center. No such restriction was observed in tonsillar B cells. Therefore, the virus has access to a range of B cell subsets in the lymph nodes but is tightly restricted to a specific long-lived compartment of B cells, the IgD-, CD27+, and CD5- memory B cells, in the periphery. We suggest that access to this compartment is essential to allow the growth-promoting latent genes to be switched off to create a site of persistent infection that is neither pathogenic nor a target for immunosurveillance.

A model for persistent infection with Epstein-Barr virus: the stealth virus of human B cells.

Most adult humans are infected benignly and for life with the herpesvirus Epstein-Barr virus. EBV has been a focus of research because of its status as a candidate tumor virus for a number of lymphomas and carcinomas. In vitro EBV has the ability to establish a latent infection in proliferating B lymphoblasts. This is the only system available for studying human herpesvirus latency in culture and has been extremely useful for elucidating how EBV promotes cellular growth. However, to understand how EBV survives in the healthy host and what goes awry, leading to disease, it is essential to know how EBV establishes and maintains a persistent infection in vivo. Early studies on the mechanism of EBV persistence produced inconclusive and often contradictory results because the techniques available were crude and insensitive. Recent advances in PCR technology and the application of sophisticated cell fractionation techniques have now provided new insights into the behavior of the virus. Most dramatically it has been shown that EBV in vivo does not establish latency in a proliferating lymphoblast, but in a resting memory B cell. The contrasting behaviors of being able to establish a latent infection in proliferating B blasts and resting memory B cells can be resolved in terms of a model where EBV performs its complete life cycle in B lymphocytes. The virus achieves this not by disrupting normal B cell biology but by using it.

Epstein-barr virus-infected resting memory B cells, not proliferating lymphoblasts, accumulate in the peripheral blood of immunosuppressed patients.

When Epstein-Barr virus (EBV) infects B cells in vitro, the result is a proliferating lymphoblast that expresses at least nine latent proteins. It is generally believed that these cells are rigorously controlled in vivo by cytotoxic T cells. Consistent with this, the latently infected cells in the peripheral blood of healthy carriers are not lymphoblasts. Rather, they are resting memory B cells that are probably not subject to direct immunosurveillance by cytotoxic T lymphocytes (CTLs). When patients become immunosuppressed, the viral load increases in the peripheral blood. The expansion of proliferating lymphoblasts due to the suppressed CTL response is believed to account for this increase and is considered to be a major risk factor for posttransplant lymphoproliferative disease (PTLD) and AIDS-associated B cell lymphoma. Here we show that there is an increase in the numbers of latently infected cells in the peripheral blood of immunosuppressed patients. However, the cells are not proliferating lymphoblasts. They are all latently infected, resting, memory B cells-the same population of infected cells found in the blood of healthy carriers. These results are discussed in the context of a model for EBV persistence that explains why PTLD is usually limited to the lymph nodes.

EBV persistence in memory B cells in vivo.

Epstein-Barr virus establishes latency in vitro by activating human B cells to become proliferating blasts, but in vivo it is benign. In the peripheral blood, the virus resides latently in resting B cells that we now show are restricted to the sIgD memory subset. However, in tonsils the virus shows no such restriction. We propose that EBV indiscriminately infects B cells in mucosal lymphoid tissue and that these cells differentiate to become resting memory B cells that then enter the circulation. Activation to the blastoid stage of latency is an essential intermediate step in this process. Thus, EBV may persist by exploiting the mechanisms that produce and maintain long-term B cell memory.

CD48 binds to heparan sulfate on the surface of epithelial cells.

CD48 is a member of the immunoglobulin superfamily whose cell surface expression is strikingly up-regulated on the surface of Epstein-Barr virus-infected B cells. To date, no ligand for human CD48 has been characterized. In this study, we show that human recombinant CD48 binds to the glycosaminoglycan heparan sulfate on the surface of human epithelial cells. We have produced a monoclonal antibody (615) against epithelial cell surfaces that blocks this binding and show that it too recognizes heparan sulfate. The specific epitope on heparan sulfate that is recognized by the antibody and is involved in binding is also expressed in vivo on the basolateral surfaces of mucosal epithelium and lamina propria.

A ligand for human CD48 on epithelial cells.

CD48 is a member of the Ig superfamily with a high degree of sequence homology to CD58 (LFA-3). In rodents, CD48 is the ligand for CD2 whereas in humans, CD58 is the ligand for CD2. Despite intensive efforts, no ligand for human CD48 has been convincingly demonstrated. We now show that a ligand for human CD48 is present on epithelial cells. The ligand was detected based on the ability of epithelial cells to bind both a decameric, soluble CD48 IgM fusion protein and monomeric CD48 immobilized on plastic dishes. mAbs raised to the ligand completely block binding of CD48 to all epithelial cells tested. We further show that the cell surface proteoglycan CD44 plays an auxiliary role in the binding of epithelial cells to CD48 and that this interaction involves the glycosaminoglycan binding site of CD44. No interaction of human CD48 with CD2 was detected. This is the first clear demonstration that human CD48 can function as an adhesion molecule and suggests a role for CD48 in lymphocyte epithelial cell interactions.

Localization of the major NF-kappaB-activating site and the sole TRAF3 binding site of LMP-1 defines two distinct signaling motifs.

The TRAF3 molecule interacts with the cytoplasmic carboxyl terminus (COOH terminus) of the Epstein-Barr virus-encoded oncogene LMP-1. NF-kappaB activation is a downstream signaling event of tumor necrosis factor receptor-associated factor (TRAF) molecules in other signaling systems (CD40 for example) and is an event caused by LMP-1 expression. One region capable of TRAF3 interaction in LMP-1 is the membrane-proximal 45 amino acids (188-242) of the COOH terminus. We show that this region contains the only site for binding of TRAF3 in the 200-amino acid COOH terminus of LMP-1. The site also binds TRAF2 and TRAF5, but not TRAF6. TRAF3 binds to critical residues localized between amino acids 196 and 212 (HHDDSLPHPQQATDDSG), including the PXQX(T/S) motif, that share limited identity to the CD40 receptor TRAF binding site (TAAPVQETL). Mutation of critical residues in the TRAF3 binding site of LMP-1 that prevents binding of TRAF2, TRAF3, and TRAF5 does not affect NF-kappaB-activating potential. Deletion mapping localized the major NF-kappaB activating region of LMP-1 to critical residues in the distal 4 amino acids of the COOH terminus (383-386). Therefore, TRAF3 binding and NF-kappaB activation occur through two separate motifs at opposite ends of the LMP-1 COOH-terminal sequence.

Identification of the site of Epstein-Barr virus persistence in vivo as a resting B cell.

Epstein-Barr (EBV) is a powerful immortalizing virus for human B lymphocytes in vitro and is associated with several human neoplasias in vivo. Previously, we have shown that the majority of EBV-infected cells in the peripheral blood of healthy, persistently infected individuals do not express the activated phenotype, e.g., high levels of cell surface CD23 and CD80 (B7), characteristically expressed on in vitro-immortalized cells. Here, we show that > or = 90% of the CD23-, virus-infected cells in the peripheral blood are in G0 and therefore resting. The remaining cells may be G1 arrested, but we were unable to detect a significant number of cells traversing the S-G2-M stages of the cell cycle. The mRNA for LMP2A, but not EBNA1 originating from Qp, was readily detected in this population, and these cells appear competent in the processing and presentation of antigen by class I major histocompatibility complex. We propose that these resting B cells are the site of long-term latent persistence for EBV. We further propose that the persistence of the virus in a resting B7- B cell provides an important mechanism to escape immunosurveillance. The demonstration that EBV can persist latently in a resting B cell means that the immortalizing functions of EBV can be down regulated in a normal B cell. This conclusion has important implications for understanding and controlling EBV-associated neoplasia.

An Epstein-Barr virus-associated superantigen.

More than 90% of adults are latently infected with Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, a self-limiting lymphoproliferative disease characterized by extensive T cell activation. Reactivation of this herpesvirus during immunosuppression is often associated with oncogenesis. These considerations led us to analyze the early events that occur after exposure of the immune system to EBV. Strong major histocompatibility complex (MHC) class II-dependent but not MHC-restricted, T cell proliferation was observed in vitro in response to autologous, lytically infected EBV-transformed B cells. By measuring the appearance of the early activation marker CD69 on individual T cell V beta subsets, we could demonstrate selective activation of human V beta 13- T cells. This was confirmed with murine T cell hybridomas expressing various human BV genes. While EBV- Burkitt's lymphoma cells were nonstimulatory, they induced V beta-restricted T cell activation after EBV infection. EBV specific activation was also demonstrated in cord blood cells, excluding a recall-antigen response. Thus, all of the characteristics of a superantigen-stimulated response are seen, indicating that induction of the EBV lytic cycle is associated with the expression of a superantigen in B cells. A model is presented proposing a role for the superantigen in infection, latency, and oncogenesis.

Is EBV persistence in vivo a model for B cell homeostasis?

We have measured the absolute numbers of EBV-infected B cells in the peripheral blood of healthy persistently infected individuals. Single measurements on a panel of 15 healthy individuals demonstrate that the frequency varies over a wide range from 1-50 per 10(6) B cells. Repeat measurements over 1-3.5 years on several individuals whose frequencies varied over a 10-fold range showed that the variation does not represent the fluctuation in the frequency that can occur within an individual; rather, the frequencies are specific to the individual. The frequency within an individual measured over time is stable and contributes less than 10% to the variance seen in the whole population. These measurements suggest that the level of EBV-infected B cells is tightly regulated and we propose that the same homeostatic mechanisms that regulate the levels of normal B cells also regulate B cells latently infected with EBV.

The Kaposi sarcoma-associated herpesvirus (KSHV) is present as an intact latent genome in KS tissue but replicates in the peripheral blood mononuclear cells of KS patients.

Short DNA sequences have been identified, originally in association with Kaposi's sarcoma (KS) biopsies, that are highly homologous to oncogenic, lymphotropic herpesviruses. Recently a virus, Kaposi sarcoma associated herpesvirus (KSHV) or human herpesvirus-8 (HHV-8), bearing these sequences has been identified in a cell line derived from a body cavity-based lymphoma. In this report, we show that the same sequences are present in KS biopsies as DNA molecules of a form and size characteristic of latent herpesviruses-large, covalently closed, circular episomes. The genomes migrate with an apparent size larger than the herpesvirus Epstein-Barr virus (172 kb). This form of the viral genome was found in four of four biopsies and three of five peripheral blood samples from KS patients. Linear forms of the viral genome, characteristic of viral replication, were not detected in the biopsies, but were present in the peripheral blood of three out of five patients. The sequences for KSHV/HHV-8 were also detected in the blood of four of five allograft patients and three of five healthy donors without KS suggesting that the virus is widespread throughout the human population.

Detection of the latent form of Epstein-Barr virus DNA in the peripheral blood of healthy individuals.

Epstein-Barr virus infects resting B cells in vitro and activates them to continuously proliferating lymphoblasts. Activation is essential for the virus to convert its linear genome to the covalently closed circular episomal form in which it persists in proliferating cells. However, in vivo, Epstein-Barr virus persists in resting B cells. We found that in these cells also the virus is present as an episome, suggesting that the cells must, at some time, have been activated and then returned to a resting state. This is the first direct demonstration, for any herpesvirus, of this form of the viral genome in normal persistently infected tissue. Since no linear viral DNA was detected, we estimate that fewer than 1 in 40 cells replicates the virus in the peripheral blood of healthy donors.

Epstein-Barr virus and the B cell: that's all it takes.

Recent experiments demonstrate that a much broader range of B cells harbor Epstein-Barr virus (EBV) in vivo than was previously expected from in vitro studies. In this review it is argued that EBV persists in vivo by integrating its biology with that of the normal B cells within which it resides, and that the B cell provides all the environments necessary for EBV to maintain its life cycle.