[show abstract][hide abstract] ABSTRACT: Epstein-Barr Virus (EBV) is an oncogenic γ-herpesvirus that capably establishes both latent and lytic modes of infection in host cells and causes malignant diseases in humans. Nuclear antigen 2 (EBNA2)-mediated transcription of both cellular and viral genes is essential for the establishment and maintenance of the EBV latency program in B lymphocytes. Here, we employed a protein affinity pull-down and LC-MS/MS analysis to identify nucleophosmin (NPM1) as one of the cellular proteins bound to EBNA2. Additionally, the specific domains that are responsible for protein-protein interactions were characterized as EBNA2 residues 300 to 360 and the oligomerization domain (OD) of NPM1. As in c-MYC, dramatic NPM1 expression was induced in EBV positively infected B cells after three days of viral infection, and both EBNA2 and EBNALP were implicated in the transactivation of the NPM1 promoter. Depletion of NPM1 with the lentivirus-expressed short-hairpin RNAs (shRNAs) effectively abrogated EBNA2-dependent transcription and transformation outgrowth of lymphoblastoid cells. Notably, the ATP-bound state of NPM1 was required to induce assembly of a protein complex containing EBNA2, RBP-Jκ, and NPM1 by stabilizing the interaction of EBNA2 with RBP-Jκ. In a NPM1-knockdown cell line, we demonstrated that an EBNA2-mediated transcription defect was fully restored by the ectopic expression of NPM1. Our findings highlight the essential role of NPM1 in chaperoning EBNA2 onto the latency-associated membrane protein 1 (LMP1) promoters, which is coordinated with the subsequent activation of transcriptional cascades through RBP-Jκ during EBV infection. These data advance our understanding of EBV pathology and further imply that NPM1 can be exploited as a therapeutic target for EBV-associated diseases.
[show abstract][hide abstract] ABSTRACT: Latent Epstein-Barr virus (EBV) infection is associated with human B cell lymphomas and certain carcinomas. EBV episome persistence, replication, and gene expression are dependent on EBV-encoded nuclear antigen 1 (EBNA1)'s DNA binding domain (DBD)/dimerization domain (DD)-mediated sequence-specific DNA binding activity. Homodimerization of EBNA1 is essential for EBNA1 DNA binding and transactivation. In this study, we characterized a novel small molecule EBNA1 inhibitor EiK1, screened from the previous high throughput screening (HTS). The EiK1 compound specifically inhibited the EBNA1-dependent, OriP-enhanced transcription, but not EBNA1-independent transcription. A Surface Plasmon Resonance Biacore assay revealed that EiK1 associates with EBNA1 amino acid 459-607 DBD/DD. Consistent with the SPR data, in vitro gel shift assays showed that EiK1 suppressed the activity of EBNA1 binding to the cognate familial repeats (FR) sequence, but not control RBP-Jκ binding to the Jκ site. Subsequently, a cross-linker-mediated in vitro multimerization assay and EBNA1 homodimerization-dependent yeast two-hybrid assay showed that EiK1 significantly inhibited EBNA1 dimerization. In an attempt to identify more highly specific peptide inhibitors, small peptides encompassing the EBNA1 DBD/DD were screened for inhibition of EBNA1 DBD-mediated DNA binding function. The small peptide P85, covering EBNA1 a.a. 560-574, significantly blocked EBNA1 DNA binding activity in vitro, prevented dimerization in vitro and in vivo, associated with EBNA1 in vitro, and repressed EBNA1-dependent transcription in vivo. Collectively, this study describes two novel inhibitors of EBNA1 dimerization. This study demonstrates that EBNA1 homodimerization can be effectively targeted by a small molecule or peptide.
Biochemical and Biophysical Research Communications 06/2012; 424(2):251-6. · 2.41 Impact Factor
[show abstract][hide abstract] ABSTRACT: Downregulation of the CD99 antigen on the surface of Hodgkin's lymphoma (HL) cells via EBV LMP1-mediated NF-κB suppression of Sp1 transcriptional activity is known to be associated with the appearance of pathogenic Reed-Sternberg cells. Here, we show that in addition, EBV LMP1 heterologous NF-κB activators such as CD30 and CD40 repress the CD99 promoter, which contains multiple Sp1-binding sites but no NF-κB binding sites. In addition, NF-κB-inducing kinase (NIK) repressed the CD99 promoter while NIK kinase mutants and JNK inhibitory protein failed to do so. Of the NF-κB subunits, NF-κB2 (p52) alone or in combination with other Rel subunits consistently inhibited the CD99, while NF-κB1 (p50) showed a marginal repressive effect. Furthermore, while transfection of LMP1 repressed the CD99 promoter in wild-type or NF-κB1 deficient MEFs, the same repression was not observed in NF-κB2 (p52)-deficient MEFs, indicating that NF-κB2 (p52) is required for LMP1-mediated repression of the CD99 promoter. Consistently, basal activity of the CD99 promoter was significantly higher in IKKα(-/-) and IKKβ(-/-) MEFs, but not in IKKΓ(-/-) MEFs compared to the wild-type control MEFs. Sp1-binding sites were directly used in the repression, because a synthetic Sp1 reporter with 10 Sp1-binding sites from the CD99 promoter was repressed by LMP1 or p52 transfection. These data indicate that LMP1-mediated NF-κB2 exhibits the major inhibitory role in the transcription at the CD99 promoter.
Molecules and Cells 10/2011; 32(6):555-60. · 2.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Latent Epstein-Barr virus (EBV) infection causes human lymphomas and carcinomas. EBV usually persists as an episome in malignant cells. EBV episome persistence, replication, and gene expression are dependent on EBNA1 binding to multiple cognate sites in oriP. To search for inhibitors of EBNA1- and oriP-dependent episome maintenance or transcription, a library of 40,550 small molecules was screened for compounds that inhibit EBNA1- and oriP-dependent transcription and do not inhibit EBNA1- and oriP-independent transcription. This screening identified roscovitine, a selective inhibitor of cyclin-dependent kinase 1 (CDK1), CDK2, CDK5, and CDK7. Based on motif predictions of EBNA1 serine 393 as a CDK phosphorylation site and (486)RALL(489) and (580)KDLVM(584) as potential cyclin binding domains, we hypothesized that cyclin binding to EBNA1 may enable CDK1, -2, -5, or -7 to phosphorylate serine 393. We found that Escherichia coli-expressed EBNA1 amino acids 387 to 641 were phosphorylated in vitro by CDK1-, -2-, -5-, and -7/cyclin complexes and serine 393 phosphorylation was roscovitine inhibited. Further, S393A mutation abrogated phosphorylation. S393A mutant EBNA1 was deficient in supporting EBNA1- and oriP-dependent transcription and episome persistence, and roscovitine had little further effect on the diminished S393A mutant EBNA1-mediated transcription or episome persistence. Immunoprecipitated FLAG-EBNA1 was phosphorylated in vitro, and roscovitine inhibited this phosphorylation. Moreover, roscovitine decreased nuclear EBNA1 and often increased cytoplasmic EBNA1, whereas S393A mutant EBNA1 was localized equally in the nucleus and cytoplasm and was unaffected by roscovitine treatment. These data indicate that roscovitine effects are serine 393 specific and that serine 393 is important in EBNA1- and oriPCp-dependent transcription and episome persistence.
Journal of Virology 03/2011; 85(6):2859-68. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: To test whether transgenic Epstein-Barr virus nuclear antigen 1 (EBNA1) expression in C57BL/6 mouse lymphocytes causes lymphoma, EBNA1 expressed in three FVB lineages at two or three times the level of latent infection was crossed up to six successive times into C57BL/6J mice. After five or six crosses, 14/36, (38%) EBNA1 transgenic mice, 11/31 (36%) littermate EBNA1-negative controls, and 9/25 (36%) inbred C57BL/6J mice housed in the same facility had lymphoma. These data indicate that EBNA1 does not significantly increase lymphoma prevalence in C57BL/6J mice.
Journal of Virology 05/2008; 82(8):4180-3. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: The lymphoma-inducing potential of Ig heavy-chain enhancer- and promoter-regulated Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) was evaluated in three transgenic FVB mouse lineages. EBNA1 was expressed at a higher level in transgenic B220(+) splenocytes than in EBV-infected lymphoblastoid cell lines. EBNA1 was also expressed in B220(-) transgenic splenocytes and thymocytes. Before killing and assessments at 18-26 months, EBNA1-transgenic mice did not differ from control mice in mortality. At 18-26 months EBNA1-transgenic mice did not differ from littermate control in ultimate body weight, in spleen size or weight, in lymph node, kidney, liver, or spleen histology, in splenocyte fractions positive for cluster of differentiation (CD)3epsilon, CD4, CD8, CD62L, B220, CD5, IgM, IgD, MHC class II, CD11b, or CD25, or in serum IgM, IgG, or total Ig levels. Lymphomas were not found in spleens or other organs of 18- to 26-month-old EBNA1-transgenic (n=86) or control (n=45) FVB mice. EBNA1-transgenic lineages had a higher pulmonary adenoma prevalence than did littermate controls (39% versus 7%). However, the adenoma prevalence was not higher in EBNA1-transgenic mice than has been described for FVB mice, and EBNA1 was not expressed in normal pulmonary epithelia or adenomas.
Proceedings of the National Academy of Sciences 02/2005; 102(3):820-5. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: Epstein-Barr virus (EBV) latent infection membrane protein 1 (LMP1)-induced NF-kappaB activation is important for infected cell survival. LMP1 activates NF-kappaB, in part, by engaging tumor necrosis factor (TNF) receptor-associated factors (TRAFs), which also mediate NF-kappaB activation from LTbetaR and CD40. LTbetaR and CD40 activation of p100/NF-kappaB2 is now known to be NIK/IKKalpha-dependent and IKKbeta/IKKgamma independent. In the experiments described here, we found that EBV LMP1 induced p100/NF-kappaB2 processing in human lymphoblasts and HEK293 cells. LMP1-induced p100 processing was NIK/IKKalpha dependent and IKKbeta/IKKgamma independent. Furthermore, the LMP1 TRAF-binding site was required for p100 processing and p52 nuclear localization, whereas the LMP1 death domain-binding site was not. Moreover, the LMP1 TRAF-binding site preferentially caused RelB nuclear accumulation. In murine embryo fibroblasts (MEFs), IKKbeta was essential for LMP1 up-regulation of macrophage inflammatory protein (MIP)-2, TNFalpha, I-TAC, ELC, MIG, and CXCR4 RNAs. Interestingly, in IKKalpha knockout MEFs, LMP1 hyperinduced MIP-2, TNFalpha, and I-TAC expression, consistent with a role for IKKalpha in down-modulating canonical IKKbeta activation or its effects. In contrast, LMP1 failed to up-regulate CXCR4 and MIG RNA in IKKalpha knockout MEFs, indicating a dependence on noncanonical IKKalpha activation. Furthermore, LMP1 up-regulation of MIP-2 RNA in MEFs was both IKKbeta- and IKKgamma-dependent, whereas LMP1 upregulation of MIG and I-TAC RNA was fully IKKgamma independent. Thus, LMP1 induces typical canonical IKKbeta/IKKgamma-dependent, atypical canonical IKKbeta-dependent/IKKgamma-independent, and noncanonical NIK/IKKalpha-dependent NF-kappaB activations; NIK/IKKalpha-dependent NF-kappaB activation is principally mediated by the LMP1 TRAF-binding site.
Proceedings of the National Academy of Sciences 02/2004; 101(1):141-6. · 9.74 Impact Factor