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Here we isolated the Fab clones 1-A11, 6-C6, 10-B2, and 15-H10 and confirmed their recognition of LMP1 expressed on the surface of EBV+ B95.8 cells. Recognition of LMP1 on the cell membrane by clone 15-H10 was verified through immunofluorescent assay, while other clones failed the recognition. One of the possible reasons could be the stringent washing in this assay that ruptured the low affinity Fab-LMP1 binding. EBV infection is related with multiple diseases, and conventional approaches used in laboratory diagnostic tests for EBV infection and pathogenesis have their limitations [(generate false positive results or lack the ability to localize the expression of EBV within target cells) ( Young and Rickinson 2004 )]. The LMP1-specific Fab clones reported here need further evaluation in both affinity and specificity in testing EBV+ patient samples, and hopefully it could have potential applications in EBV diagnostics and directly targeting EBV-related tumors in adoptive T cell therapy.

High plasma lactate is associated with poor prognosis of many malignancies, but its role in virally mediated cancer progression and underlying molecular mechanisms are unclear. Epstein-Barr virus (EBV), the first human oncogenic virus, causes several cancers, including B-cell lymphoma. Here, we report that lactate dehydrogenase A (LDH-A) expression and lactate production are elevated in EBV-immortalized B lymphoblastic cells, and lactic acid (LA; acidic lactate) at low concentration triggers EBV-infected B-cell adhesion, morphological changes, and proliferation in vitro and in vivo. Moreover, LA-induced responses of EBV-infected B cells uniquely occurs in viral latency type III, and it is dramatically associated with the inhibition of global viral microRNAs, particularly the miR-BHRF1 cluster, and the high expression of SMAD3, JUN, and COL1A genes. The introduction of miR-BHRF1-1 blocks the LA-induced effects of EBV-infected B cells. Thus, this may be a novel mechanism to explain EBV-immortalized B lymphoblastic cell malignancy in an LA microenvironment.

Latent Epstein-Barr virus (EBV) infection is strongly associated with several malignancies, including B-cell lymphomas and epithelial tumors. EBNA1 is a key antigen expressed in all EBV-associated tumors during latency that is required for maintenance of the EBV episome DNA and the regulation of viral gene transcription. However, the mechanism utilized by EBV to maintain latent infection at the levels of posttranslational regulation remains largely unclear. Here, we report that EBNA1 contains two SUMO-interacting motifs (SIM2 and SIM3), and mutation of SIM2, but not SIM3, dramatically disrupts the EBNA1 dimerization, while SIM3 contributes to the polySUMO2 modification of EBNA1 at lysine 477 in vitro. Proteomic and immunoprecipitation analyses further reveal that the SIM3 motif is required for the EBNA1-mediated inhibitory effects on SUMO2-modified STUB1, SUMO2-mediated degradation of USP7, and SUMO1-modified KAP1. Deletion of the EBNASIM motif leads to functional loss of both EBNA1-mediated viral episome maintenance and lytic gene silencing. Importantly, hypoxic stress induces the SUMO2 modification of EBNA1, and in turn the dissociation of EBNA1 with STUB1, KAP1 and USP7 to increase the SUMO1 modification of both STUB1 and KAP1 for reactivation of lytic replication. Therefore, the EBNA1SIM motif plays an essential role in EBV latency and is a potential therapeutic target against EBV-associated cancers., Author summary The Small Ubiquitin-related modifier (SUMO) modification of proteins is a reversible post-translational regulation involved in control of gene transcription, among other functions. Epstein-Barr virus (EBV) infects most people worldwide and contributes to the development of several types of cancers due to its ability to induce cell proliferation and survival. EBNA1 is expressed in all forms of EBV-associated tumors. In this study, we found that EBNA1 contains a SUMO-interacting motif (SIM) named EBNA1SIM, which is required for EBNA1 to exert inhibitory effects on a SUMO2-modified complex (SC2) including STUB1, KAP1 and USP7. Disruption of EBNA1SIM leads to loss of both EBNA1-mediated viral episome maintenance and lytic gene silencing. Importantly, hypoxia-mediated reactivation of viral lytic replication induces the EBNA1 dissociation from STUB1 in the SC2 complex. This discovery not only opens a new insight on the interplay between host and virus, but it also provides a therapeutic target specific against EBV-associated cancers.

High plasma lactate is associated with poor prognosis of many malignancies, but its role in virally mediated cancer progression and underlying molecular mechanisms are unclear. Epstein-Barr virus (EBV), the first human oncogenic virus, causes several cancers, including B-cell lymphoma. Here, we report that lactate dehydrogenase A (LDH-A) expression and lactate production are elevated in EBV-immortalized B lymphoblastic cells, and lactic acid (LA; acidic lactate) at low concentration triggers EBV-infected B-cell adhesion, morphological changes, and proliferation in vitro and in vivo . Moreover, LA-induced responses of EBV-infected B cells uniquely occurs in viral latency type III, and it is dramatically associated with the inhibition of global viral microRNAs, particularly the miR-BHRF1 cluster, and the high expression of SMAD3 , JUN , and COL1A genes. The introduction of miR-BHRF1-1 blocks the LA-induced effects of EBV-infected B cells. Thus, this may be a novel mechanism to explain EBV-immortalized B lymphoblastic cell malignancy in an LA microenvironment. IMPORTANCE The tumor microenvironment is complicated, and lactate, which is created by cell metabolism, contributes to an acidic microenvironment that facilitates cancer progression. However, how LA operates in virus-associated cancers is unclear. Thus, we studied how EBV (the first tumor virus identified in humans; it is associated with many cancers) upregulates the expression of LDH-A and lactate production in B lymphoma cells. Elevated LA induces adhesion and the growth of EBV-infected B cells by inhibiting viral microRNA transcription. Thus, we offer a novel understanding of how EBV utilizes an acidic microenvironment to promote cancer development.

Epstein–Barr virus (EBV) is a ubiquitous human herpesvirus associated with a large number of lymphoid and epithelial malignancies. As a successful pathogen it has co-evolved with its human host for millions of years. EBV has the unique ability to establish life-long latent infection in primary human B lymphocytes. During latent infection, a small subset of viral proteins is expressed. These proteins are essential for maintenance of the EBV genome as well as the deregulation of various signaling pathways that facilitate the proliferation and survival of the infected cells. Epstein–Barr nuclear antigen (EBNA)3C is one of the latent proteins shown to be essential for transformation of primary human B lymphocytes in vitro. EBNA3C primarily functions as a transcriptional regulator by interacting with a number of well known cellular and viral transcriptional factors. We have recently identified several binding partners for EBNA3C including proteins that regulate cell cycle and chromatin remodeling. We are actively engaged in discerning the biological significance of these interactions. This review summarizes our current understanding of how EBNA3C usurps cellular pathways that promote B-cell transformation.

Denis Burkitt identified the existence of a highly aggressive lymphoproliferative disorder, Burkitt’s lymphoma (BL), which was highly prevalent in young children in the equatorial belt of Africa. Burrkitt’s lymphoma can also be seen in children as well as immunocompromised individuals in other areas with endemic malaria. Burkitt’s lymphoma was the first human tumour to be causally associated with Epstein–Barr virus (EBV). Intense studies over the past almost five decades have demonstrated a major role for EBV in the pathogenesis of this unique neoplasm. However, the precise contribution of EBV to the pathogenesis of BL has not been fully determined. In this chapter, we will discuss different aspects of EBV-mediated B-cell transformation that contributes to the development of BL. Additionally, we will also summarize the critical roles of the EBV latency proteins in oncogenesis and their contribution to cellular proliferation, deregulation of cell signaling and inhibition of the apoptotic process. This review will provide clues to the probable strategies for therapeutic intervention of EBV-associated Burkitt’s lymphoma progression which is dependent on the interplay between EBV-encoded latent antigens and their interactions with host cellular proteins and their environment.

The Epstein-Barr nuclear antigen 3C (EBNA3C), one of the essential latent antigens for Epstein-Barr virus (EBV)-induced immortalization of primary human B lymphocytes in vitro, has been implicated in regulating cell proliferation and anti-apoptosis via interaction with several cellular and viral factors. Gemin3 (also named DDX20 or DP103) is a member of DEAD RNA helicase family which exhibits diverse cellular functions including DNA transcription, recombination and repair, and RNA metabolism. Gemin3 was initially identified as a binding partner to EBNA2 and EBNA3C. However, the mechanism by which EBNA3C regulates Gemin3 function remains unclear. Here, we report that EBNA3C directly interacts with Gemin3 through its C-terminal domains. This interaction results in increased stability of Gemin3 and its accumulation in both B lymphoma cells and EBV transformed lymphoblastoid cell lines (LCLs). Moreover, EBNA3C promotes formation of a complex with p53 and Gemin3 which blocks the DNA-binding affinity of p53. Small hairpin RNA based knockdown of Gemin3 in B lymphoma or LCL cells remarkably attenuates the ability of EBNA3C to inhibit the transcription activity of p53 on its downstream genes p21 and Bax, as well as apoptosis. These findings provide the first evidence that Gemin3 may be a common target of oncogenic viruses for driving cell proliferation and anti-apoptotic activities., Author Summary Gemin3 (DDX20 or DP103) is a member of the DEAD-box family of RNA helicases involved in various cellular processes including DNA transcription and RNA processing. The Epstein-Barr virus (EBV) encoded nuclear antigen 3C (EBNA3C) is essential for EBV-induced immortalization of primary human B-lymphocytes in vitro. In this study, we discovered that Gemin3 directly binds to the tumor suppressor p53, and acts as a negative regulator blocking p53 functions. Importantly, EBNA3C induces Gemin3 accumulation and enhances the formation of the complex of Gemin3 and p53 in EBV- transformed primary human B lymphocytes. Remarkably, inhibition of Gemin3 production leads to cell death of B lymphoma cells, particularly EBNA3C positive cells. This is the first evidence which shows that Gemin3 directly impairs p53 function in EBV positive cells, and that Gemin3 could be a potential target for EBV-associated cancer therapy.