Localization of the Major NF-κB-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-κB 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-κB-activating potential. Deletion mapping localized the major NF-κB 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-κB activation occur through two separate motifs at opposite ends of the LMP-1 COOH-terminal sequence

The Expression Pattern of Epstein-Barr Virus Latent Genes In Vivo Is Dependent upon the Differentiation Stage of the Infected B Cell

AbstractEpstein-Barr virus–infected B cells in vivo demonstrate three distinct patterns of latent gene expression, depending on the differentiation stage of the cell. Tonsillar naive B cells express the EBNA2-dependent lymphoblastoid phenotype, characteristic of direct infection. Germinal center centroblasts and centrocytes as well as tonsillar memory B cells express a more restricted pattern of latent genes (EBNA1(Q-K)+, LMP1+, LMP2+, EBNA2−) that has only been seen previously in EBV-positive tumors. Peripheral memory cells express an even more restricted pattern where no latent genes are expressed, with the possible exception of LMP2. These results are consistent with a model where EBV uses the normal biology of B lymphocytes to gain access to and persist within the long-lived memory B cell compartment

Epstein-Barr Virus Transactivates the Human Endogenous Retrovirus HERV-K18 that Encodes a Superantigen

AbstractSuperantigens (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

A multifactorial role for P. falciparum malaria in endemic Burkitt\u27s lymphoma pathogenesis

Endemic Burkitt\u27s lymphoma (eBL) arises from the germinal center (GC). It is a common tumor of young children in tropical Africa and its occurrence is closely linked geographically with the incidence of P. falciparum malaria. This association was noted more than 50 years ago. Since then we have learned that eBL contains the oncogenic herpes virus Epstein-Barr virus (EBV) and a defining translocation that activates the c-myc oncogene. However the link to malaria has never been explained. Here we provide evidence for a mechanism arising in the GC to explain this association. Accumulated evidence suggests that eBL arises in the GC when deregulated expression of AID (Activation-induced cytidine deaminase) causes a c-myc translocation in a cell latently infected with Epstein-Barr virus (EBV). Here we show that P. falciparum targets GC B cells via multiple pathways to increase the risk of eBL. 1. It causes deregulated expression of AID, thereby increasing the risk of a c-myc translocation. 2. It increases the number of B cells transiting the GC. 3. It dramatically increases the frequency of these cells that are infected with EBV and therefore protected from c-myc induced apoptosis. We propose that these activities combine synergistically to dramatically increase the incidence of eBL in individuals infected with malaria

A Novel Persistence Associated EBV miRNA Expression Profile Is Disrupted in Neoplasia

We have performed the first extensive profiling of Epstein-Barr virus (EBV) miRNAs on in vivo derived normal and neoplastic infected tissues. We describe a unique pattern of viral miRNA expression by normal infected cells in vivo expressing restricted viral latency programs (germinal center: Latency II and memory B: Latency I/0). This includes the complete absence of 15 of the 34 miRNAs profiled. These consist of 12 BART miRNAs (including approximately half of Cluster 2) and 3 of the 4 BHRF1 miRNAs. All but 2 of these absent miRNAs become expressed during EBV driven growth (Latency III). Furthermore, EBV driven growth is accompanied by a 5–10 fold down regulation in the level of the BART miRNAs expressed in germinal center and memory B cells. Therefore, Latency III also expresses a unique pattern of viral miRNAs. We refer to the miRNAs that are specifically expressed in EBV driven growth as the Latency III associated miRNAs. In EBV associated tumors that employ Latency I or II (Burkitt's lymphoma, Hodgkin's disease, nasopharyngeal carcinoma and gastric carcinoma), the Latency III associated BART but not BHRF1 miRNAs are up regulated. Thus BART miRNA expression is deregulated in the EBV associated tumors. This is the first demonstration that Latency III specific genes (the Latency III associated BARTs) can be expressed in these tumors. The EBV associated tumors demonstrate very similar patterns of miRNA expression yet were readily distinguished when the expression data were analyzed either by heat-map/clustering or principal component analysis. Systematic analysis revealed that the information distinguishing the tumor types was redundant and distributed across all the miRNAs. This resembles “secret sharing” algorithms where information can be distributed among a large number of recipients in such a way that any combination of a small number of recipients is able to understand the message. Biologically, this may be a consequence of functional redundancy between the miRNAs

The Dynamics of EBV Shedding Implicate a Central Role for Epithelial Cells in Amplifying Viral Output

To develop more detailed models of EBV persistence we have studied the dynamics of virus shedding in healthy carriers. We demonstrate that EBV shedding into saliva is continuous and rapid such that the virus level is replaced in ≤2 minutes, the average time that a normal individual swallows. Thus, the mouth is not a reservoir of virus but a conduit through which a continuous flow stream of virus passes in saliva. Consequently, virus is being shed at a much higher rate than previously thought, a level too high to be accounted for by replication in B cells in Waldeyer's ring alone. Virus shedding is relatively stable over short periods (hours-days) but varies through 3.5 to 5.5 logs over longer periods, a degree of variation that also cannot be accounted for solely by replication in B cells. This variation means, contrary to what is generally believed, that the definition of high and low shedder is not so much a function of variation between individuals but within individuals over time. The dynamics of shedding describe a process governing virus production that is occurring independently ≤3 times at any moment. This process grows exponentially and is then randomly terminated. We propose that these dynamics are best explained by a model where single B cells sporadically release virus that infects anywhere from 1 to 5 epithelial cells. This infection spreads at a constant exponential rate and is terminated randomly, resulting in infected plaques of epithelial cells ranging in size from 1 to 105 cells. At any one time there are a very small number (≤3) of plaques. We suggest that the final size of these plaques is a function of the rate of infectious spread within the lymphoepithelium which may be governed by the structural complexity of the tissue but is ultimately limited by the immune response

A Virtual Look at Epstein–Barr Virus Infection: Biological Interpretations

The possibility of using computer simulation and mathematical modeling to gain insight into biological and other complex systems is receiving increased attention. However, it is as yet unclear to what extent these techniques will provide useful biological insights or even what the best approach is. Epstein–Barr virus (EBV) provides a good candidate to address these issues. It persistently infects most humans and is associated with several important diseases. In addition, a detailed biological model has been developed that provides an intricate understanding of EBV infection in the naturally infected human host and accounts for most of the virus' diverse and peculiar properties. We have developed an agent-based computer model/simulation (PathSim, Pathogen Simulation) of this biological model. The simulation is performed on a virtual grid that represents the anatomy of the tonsils of the nasopharyngeal cavity (Waldeyer ring) and the peripheral circulation—the sites of EBV infection and persistence. The simulation is presented via a user friendly visual interface and reproduces quantitative and qualitative aspects of acute and persistent EBV infection. The simulation also had predictive power in validation experiments involving certain aspects of viral infection dynamics. Moreover, it allows us to identify switch points in the infection process that direct the disease course towards the end points of persistence, clearance, or death. Lastly, we were able to identify parameter sets that reproduced aspects of EBV-associated diseases. These investigations indicate that such simulations, combined with laboratory and clinical studies and animal models, will provide a powerful approach to investigating and controlling EBV infection, including the design of targeted anti-viral therapies

The Effect of Epstein-Barr Virus Latent Membrane Protein 2 Expression on the Kinetics of Early B Cell Infection

Infection of human B cells with wild-type Epstein-Barr virus (EBV) in vitro leads to activation and proliferation that result in efficient production of lymphoblastoid cell lines (LCLs). Latent Membrane Protein 2 (LMP2) is expressed early after infection and previous research has suggested a possible role in this process. Therefore, we generated recombinant EBV with knockouts of either or both protein isoforms, LMP2A and LMP2B (Δ2A, Δ2B, Δ2A/Δ2B) to study the effect of LMP2 in early B cell infection. Infection of B cells with Δ2A and Δ2A/Δ2B viruses led to a marked decrease in activation and proliferation relative to wild-type (wt) viruses, and resulted in higher percentages of apoptotic B cells. Δ2B virus infection showed activation levels comparable to wt, but fewer numbers of proliferating B cells. Early B cell infection with wt, Δ2A and Δ2B viruses did not result in changes in latent gene expression, with the exception of elevated LMP2B transcript in Δ2A virus infection. Infection with Δ2A and Δ2B viruses did not affect viral latency, determined by changes in LMP1/Zebra expression following BCR stimulation. However, BCR stimulation of Δ2A/Δ2B cells resulted in decreased LMP1 expression, which suggests loss of stability in viral latency. Long-term outgrowth assays revealed that LMP2A, but not LMP2B, is critical for efficient long-term growth of B cells in vitro. The lowest levels of activation, proliferation, and LCL formation were observed when both isoforms were deleted. These results suggest that LMP2A appears to be critical for efficient activation, proliferation and survival of EBV-infected B cells at early times after infection, which impacts the efficient long-term growth of B cells in culture. In contrast, LMP2B did not appear to play a significant role in these processes, and long-term growth of infected B cells was not affected by the absence of this protein. © 2013 Wasil et al