Differential methylation of Epstein-Barr virus latency promoters facilitates viral persistence in healthy seropositive individuals

Epstein-Barr virus (EBV) establishes a life-long infection in humans, with distinct viral latency programs predominating during acute and chronic phases of infection. Only a subset of the EBV latency-associated antigens present during the acute phase of EBV infection are expressed in the latently infected memory B cells that serve as the long-term EBV reservoir. Since the EBV immortalization program elicits a potent cellular immune response, downregulation of viral gene expression in the long-term latency reservoir is likely to facilitate evasion of the immune response and persistence of EBV in the immunocompetent host. Tissue culture and tumor models of restricted EBV latency have consistently demonstrated a critical role for methylation of the viral genome in maintaining the restricted pattern of latency-associated gene expression. Here we extend these observations to demonstrate that the EBV genomes in the memory B-cell reservoir are also heavily and discretely methylated. This analysis reveals that methylation of the viral genome is a normal aspect of EBV infection in healthy immunocompetent individuals and is not restricted to the development of EBV-associated tumors. In addition, the pattern of methylation very likely accounts for the observed inhibition of the EBV immortalization program and the establishment and maintenance of a restricted latency program. Thus, EBV appears to be the first example of a parasite that usurps the host cell-directed methylation system to regulate pathogen gene expression and thereby establish a chronic infection.     

Simulating Epstein-Barr virus infection with C-ImmSim

MOTIVATION: Epstein-Barr virus (EBV) infects greater than 90% of humans benignly for life but can be associated with tumors. It is a uniquely human pathogen that is amenable to quantitative analysis; however, there is no applicable animal model. Computer models may provide a virtual environment to perform experiments not possible in human volunteers. RESULTS: We report the application of a relatively simple stochastic cellular automaton (C-ImmSim) to the modeling of EBV infection. Infected B-cell dynamics in the acute and chronic phases of infection correspond well to clinical data including the establishment of a long term persistent infection (up to 10 years) that is absolutely dependent on access of latently infected B cells to the peripheral pool where they are not subject to immunosurveillance. In the absence of this compartment the infection is cleared. AVAILABILITY: The latest version 6 of C-ImmSim is available under the GNU General Public License and is downloadable from www.iac.cnr.it/~filippo/cimmsim.html     

Role of B-cell proliferation in the establishment of gammaherpesvirus latency

Murine gammaherpesvirus 68 (gammaHV68) provides a tractable small animal model with which to study the mechanisms involved in the establishment and maintenance of latency by gammaherpesviruses. Similar to the human gammaherpesvirus Epstein-Barr virus (EBV), gammaHV68 establishes and maintains latency in the memory B-cell compartment following intranasal infection. Here we have sought to determine whether, like EBV infection, gammaHV68 infection in vivo is associated with B-cell proliferation during the establishment of chronic infection. We show that gammaHV68 infection leads to significant splenic B-cell proliferation as late as day 42 postinfection. Notably, gammaHV68 latency was found predominantly in the proliferating B-cell population in the spleen on both days 16 and 42 postinfection. Furthermore, virus reactivation upon ex vivo culture was heavily biased toward the proliferating B-cell population. DNA methyltransferase 1 (Dnmt1) is a critical maintenance methyltransferase which, during DNA replication, maintains the DNA methylation patterns of the cellular genome, a process that is essential for the survival of proliferating cells. To assess whether the establishment of gammaHV68 latency requires B-cell proliferation, we characterized infections of conditional Dnmt1 knockout mice by utilizing a recombinant gammaHV68 that expresses Cre-recombinase (gammaHV68-Cre). In C57BL/6 mice, the gammaHV68-Cre virus exhibited normal acute virus replication in the lungs as well as normal establishment and reactivation from latency. Furthermore, the gammaHV68-Cre virus also replicated normally during the acute phase of infection in the lungs of Dnmt1 conditional mice. However, deletion of the Dnmt1 alleles from gammaHV68-infected cells in vivo led to a severe ablation of viral latency, as assessed on both days 16 and 42 postinfection. Thus, the studies provide direct evidence that the proliferation of latently infected B cells is critical for the establishment of chronic gammaHV68 infection.     

A molecular link between malaria and Epstein-Barr virus reactivation

Although malaria and Epstein-Barr (EBV) infection are recognized cofactors in the genesis of endemic Burkitt lymphoma (BL), their relative contribution is not understood. BL, the most common paediatric cancer in equatorial Africa, is a high-grade B cell lymphoma characterized by c-myc translocation. EBV is a ubiquitous B lymphotropic virus that persists in a latent state after primary infection, and in Africa, most children have sero-converted by 3 y of age. Malaria infection profoundly affects the B cell compartment, inducing polyclonal activation and hyper-gammaglobulinemia. We recently identified the cystein-rich inter-domain region 1alpha (CIDR1alpha) of the Plasmodium falciparum membrane protein 1 as a polyclonal B cell activator that preferentially activates the memory compartment, where EBV is known to persist. Here, we have addressed the mechanisms of interaction between CIDR1alpha and EBV in the context of B cells. We show that CIDR1alpha binds to the EBV-positive B cell line Akata and increases the number of cells switching to the viral lytic cycle as measured by green fluorescent protein (GFP) expression driven by a lytic promoter. The virus production in CIDR1alpha-exposed cultures was directly proportional to the number of GFP-positive Akata cells (lytic EBV) and to the increased expression of the EBV lytic promoter BZLF1. Furthermore, CIDR1alpha stimulated the production of EBV in peripheral blood mononuclear cells derived from healthy donors and children with BL. Our results suggest that P. falciparum antigens such as CIDR1alpha can directly induce EBV reactivation during malaria infection that may increase the risk of BL development for children living in malaria-endemic areas. To our knowledge, this is the first report to show that a microbial protein can drive a latently infected B cell into EBV replication.     

Human CD8+ T cell memory generation in Puumala hantavirus infection occurs after the acute phase and is associated with boosting of EBV-specific CD8+ memory T cells

The induction and maintenance of T cell memory is incompletely understood, especially in humans. We have studied the T cell response and the generation of memory during acute infection by the Puumala virus (PUUV), a hantavirus endemic to Europe. It causes a self-limiting infection with no viral persistence, manifesting as hemorrhagic fever with renal syndrome. HLA tetramer staining of PBMC showed that the CD8(+) T cell response peaked at the onset of the clinical disease and decreased within the next 3 wk. Expression of activation markers on the tetramer-positive T cells was also highest during the acute phase, suggesting that the peak population consisted largely of effector cells. Despite the presence of tetramer-positive T cells expressing cytoplasmic IFN-gamma, PUUV-specific cells producing IFN-gamma in vitro were rare during the acute phase. Their frequency, as well as the expression of IL-7R alpha mRNA and surface protein, increased during a follow-up period of 6 wk and probably reflected the induction of memory T cells. Simultaneously with the PUUV-specific response, we also noted in seven of nine patients an increase in EBV-specific T cells and the transient presence of EBV DNA in three patients, indicative of viral reactivation. Our results show that in a natural human infection CD8(+) memory T cells are rare during the peak response, gradually emerging during the first weeks of convalescence. They also suggest that the boosting of unrelated memory T cells may be a common occurrence in human viral infections, which may have significant implications for the homeostasis of the memory T cell compartment.     

Toll/IL-1 receptor domain-containing adaptor inducing IFN-beta (TRIF)-mediated signaling contributes to innate immune responses in the lung during Escherichia coli pneumonia

Peripheral blood memory B cells latently infected with EBV bear somatic mutations and are typically isotype switched consistent with being classical Ag-selected memory B cells. In this work, we performed a comparative analysis of the expressed Ig genes between large sets of EBV-infected and uninfected peripheral blood B cells, isolated from the same infectious mononucleosis patients, to determine whether differences exist that could reveal the influence of EBV on the production and maintenance of these cells. We observed that EBV(+) cells on average accumulated more somatic hypermutations than EBV(-) cells. In addition, they had more replacement mutations and a higher replacement-silent ratio of mutations in their CDRs. We also found that EBV occupies a skewed niche within the memory compartment, due to its exclusion from the CD27(+)IgD(+)IgM(+) subset, but this skewing does not affect the overall structure of the compartment. These results indicate that EBV impacts the mutation and selection process of infected cells but that once they enter memory they cannot be distinguished from uninfected cells by host homeostasis mechanisms.     

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.     

The replisome pausing factor Timeless is required for episomal maintenance of latent Epstein-Barr virus

The Epstein-Barr virus (EBV) genome is maintained as an extrachromosomal episome during latent infection of B lymphocytes. Episomal maintenance is conferred by the interaction of the EBV-encoded nuclear antigen 1 (EBNA1) with a tandem array of high-affinity binding sites, referred to as the family of repeats (FR), located within the viral origin of plasmid replication (OriP). How this nucleoprotein array confers episomal maintenance is not completely understood. Previous studies have shown that DNA replication forks pause and terminate with high frequency at OriP. We now show that cellular DNA replication fork pausing and protection factors Timeless (Tim) and Tipin (Timeless-interacting protein) accumulate at OriP during S phase of the cell cycle. Depletion of Tim inhibits OriP-dependent DNA replication and causes a complete loss of the closed-circular form of EBV episomes in latently infected B lymphocytes. Tim depletion also led to the accumulation of double-strand breaks at the OriP region. These findings demonstrate that Tim is essential for sustaining the episomal forms of EBV DNA in latently infected cells and suggest that DNA replication fork protection is integrally linked to the mechanism of plasmid maintenance.     

Epstein-Barr virus microRNAs are evolutionarily conserved and differentially expressed

The pathogenic lymphocryptovirus Epstein-Barr virus (EBV) is shown to express at least 17 distinct microRNAs (miRNAs) in latently infected cells. These are arranged in two clusters: 14 miRNAs are located in the introns of the viral BART gene while three are located adjacent to BHRF1. The BART miRNAs are expressed at high levels in latently infected epithelial cells and at lower, albeit detectable, levels in B cells. In contrast to the tissue-specific expression pattern of the BART miRNAs, the BHRF1 miRNAs are found at high levels in B cells undergoing stage III latency but are essentially undetectable in B cells or epithelial cells undergoing stage I or II latency. Induction of lytic EBV replication was found to enhance the expression of many, but not all, of these viral miRNAs. Rhesus lymphocryptovirus, which is separated from EBV by > or =13 million years of evolution, expresses at least 16 distinct miRNAs, seven of which are closely related to EBV miRNAs. Thus, lymphocryptovirus miRNAs are under positive selection and are likely to play important roles in the viral life cycle. Moreover, the differential regulation of EBV miRNA expression implies distinct roles during infection of different human tissues.     

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 provides a new paradigm: a requirement for the immediate inhibition of apoptosis

DNA viruses such as herpesviruses are known to encode homologs of cellular antiapoptotic viral Bcl-2 proteins (vBcl-2s), which protect the virus from apoptosis in its host cell during virus synthesis. Epstein-Barr virus (EBV), a human tumor virus and a prominent member of γ-herpesviruses, infects primary resting B lymphocytes to establish a latent infection and yield proliferating, growth-transformed B cells in vitro. In these cells, 11 viral genes that contribute to cellular transformation are consistently expressed. EBV also encodes two vBcl-2 genes whose roles are unclear. Here we show that the genetic inactivation of both vBcl-2 genes disabled EBV's ability to transform primary resting B lymphocytes. Primary B cells infected with a vBcl-2-negative virus did not enter the cell cycle and died of immediate apoptosis. Apoptosis was abrogated in infected cells in which vBcl-2 genes were maximally expressed within the first 24 h postinfection. During latent infection, however, the expression of vBcl-2 genes became undetectable. Thus, both vBcl-2 homologs are essential for initial cellular transformation but become dispensable once a latent infection is established. Because long-lived, latently infected memory B cells and EBV-associated B-cell lymphomas are derived from EBV-infected proapoptotic germinal center B cells, we conclude that vBcl-2 genes are essential for the initial evasion of apoptosis in cells in vivo in which the virus establishes a latent infection or causes cellular transformation or both.     

Evidence for early infection of nonneoplastic natural killer cells by Epstein-Barr virus

We examined lymph nodes and tonsils from patients with infectious mononucleosis by combined detection of EBV-encoded RNA and a specific marker of natural killer (NK) cells, PEN5. A small number of Epstein-Barr virus (EBV) latently infected nonneoplastic NK cells were detected. Our data demonstrate that NK cells are natural targets of EBV and that infection of these cells is an early event observed during primary EBV infection.     

Immature and transitional B cells are latency reservoirs for a gammaherpesvirus

Gammaherpesviruses, including Kaposi's sarcoma-associated herpesvirus (KSHV; also known as human herpesvirus 8 [HHV-8]), Epstein-Barr virus (EBV), and murine gammaherpesvirus 68 (MHV68; also known as gammaherpesvirus 68 [γHV68] or murine herpesvirus 4 [MuHV-4]), establish lifelong latency in the resting memory B cell compartment. However, little is known about how this reservoir of infected mature B cells is maintained for the life of the host. In the context of a normal immune system, the mature B cell pool is naturally maintained by the renewable populations of developing B cells that arise from hematopoiesis. Thus, recurrent infection of these developing B cell populations could allow the virus continual access to the B cell lineage and, subsequent to differentiation, the memory B cell compartment. To begin to address this hypothesis, we examined whether MHV68 establishes latency in developing B cells during a normal course of infection. In work described here, we demonstrate the presence of viral genome in bone marrow pro-pre-B cells and immature B cells during early latency and immature B cells during long-term latency. Further, we show that transitional B cells in the spleen are latently infected and express the latency-associated nuclear antigen (LANA) throughout chronic infection. Because developing B cells normally exhibit a short life span and a high rate of turnover, these findings suggest a model in which gammaherpesviruses may gain access to the mature B cell compartment by recurrent seeding of developing B cells.