Suppression of Epstein-Barr nuclear antigen 1 (EBNA1) by RNA interference inhibits proliferation of EBV-positive Burkitt’s lymphoma cells

Hong, Mei; Murai, Yoshihiro; Kutsuna, Tomohiko; Takahashi, Hiroyuki; Nomoto, Kazuhiro; Cheng, Chunmei; Ishizawa, Shin; Zhao, Qing‐Li; Ogawa, Ryohei; Harmon, Brian V.; Tsuneyama, Koichi; Takano, Yasuo

doi:10.1007/s00432-005-0036-x

Cited by 59 publications

(52 citation statements)

References 49 publications

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“…The EBNA1 DNA binding domain recognizes 24 cognate sites in the EBV episome OriP element, while the EBNA1 N-terminal arginine-rich elements (amino acids [aa] 61 to 83 and 325 to 376) tether EBV episomes to chromosomes for partitioning to progeny cell nuclei and enhance transcription (6,38,59,61,68,80,81,83). Experiments with dominant negative EBNA1 mutants, EBNA1 antisense oligonucleotides, and EBNA1-specific RNA interference confirm that EBNA1 is essential for EBV episome persistence in dividing cells (36,40,55,63,84). An EBNA1 inhibitor might terminate latent EBV infection and abort EBV effects in nonmalignant and malignant diseases.…”

Section: Latent Epstein-barr Virus (Ebv) Infection Causes Human Lymphmentioning

confidence: 99%

Roscovitine Inhibits EBNA1 Serine 393 Phosphorylation, Nuclear Localization, Transcription, and Episome Maintenance

Kang

Lee

Soni

et al. 2011

J Virol

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Latent Epstein-Barr virus (EBV) infection causes human lymphomas and carcinomas. EBV usually persists asan 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.Epstein-Barr virus (EBV) replicates in oropharyngeal epithelial cells (62,70,85). Subsequent latent infection of mature B lymphocytes can result in infectious mononucleosis and is essential for long-term latency and reactivation and continues to be shed in saliva (21). Latently EBV-infected B cells can emerge as Burkitt's lymphomas (BL) or Hodgkin's disease in otherwise healthy people or as lymphoproliferative disease in people who have T-lymphocyte deficiencies due to HIV infection, transplantation, advanced age, or a genetic predisposition (4,8,25,34,49,62,85). In immunocompetent people, particularly those of southern Chinese descent, latent EBV infection can also cause nasopharyngeal carcinoma, which is the most common EBV malignancy worldwide (25,34). Latent EBV infection is a significant cause of morbidity and mortality.In latent infections, including malignancies, EBV usually persists as a multicopy episome (1,47,56,60). EBV genome integration is unusual (33, 52). EBNA1 is essential for EBV episome persistence in dividing cells and enhances episome transcription (59,61,83). The EBNA1 DNA binding domain recognizes 24 cognate sites in the EBV episome OriP element, while ...

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Section: Latent Epstein-barr Virus (Ebv) Infection Causes Human Lymphmentioning

confidence: 99%

Roscovitine Inhibits EBNA1 Serine 393 Phosphorylation, Nuclear Localization, Transcription, and Episome Maintenance

Kang

Lee

Soni

et al. 2011

J Virol

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“…The protein also acts as a transcriptional regulator of both viral and host promoters and may promote lytic reactivation of the virus (5,6). Furthermore, multiple lines of evidence indicate that EBNA1 contributes to the development of EBV associated tumours through increasing cell proliferation and survival and possibly by inducing oxidative stress (7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17).…”

Section: Introductionmentioning

confidence: 99%

Modelling the structure of full-length Epstein–Barr virus nuclear antigen 1

2014

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Abstract:Epstein-Barr virus (EBV) is a clinically important human virus associated with several cancers and is the etiologic agent of infectious mononucleosis. The viral nuclear antigen-1 (EBNA1) is central to the replication and propagation of the viral genome and likely contributes to tumourigenesis. We have compared EBNA1 homologues from other primate lymphocryptoviruses (LCV) and found that the central glycine/alanine repeat (GAr) domain, as well as predicted cellular protein (USP7 and CK2) binding sites are present in homologues in the Old World primates, but not the marmoset; suggesting that these motifs may have co-evolved. Using the resolved structure of the C-terminal one third of EBNA1 (homodimerisation and DNA binding domain), we have gone on to develop monomeric and dimeric models in silico of the full length protein.The C-terminal domain is predicted to be structurally highly similar between homologues, indicating conserved function. Zinc could be stably incorporated into the model, bonding with two N-terminal cysteines predicted to facilitate multimerisation. The GAr contains secondary structural elements in the models, while the protein binding regions are unstructured, irrespective of the prediction approach used and sequence origin. These intrinsically disordered regions may facilitate the diversity observed in partner interactions. We hypothsise that the structured GAr could mask the disordered regions, thereby protecting the protein from default degradation. In the dimer conformation, the C-terminal tails of each monomer wrap around a proline-rich protruding loop of the partner monomer, providing dimer stability, a feature which could be exploited in therapeutic design. Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporation EBV-B95-8 hu-EBNA1CyEBV-TsBB6 cy-EBNA1 CeHV15 rh-EBNA1CeHV12 ba-EBNA1 CalHV3 ma EBNA1Modelling the structure of full length Epstein-Barr Virus Nuclear Antigen Figure S2. EBNA1 model comparison.The EBV B95-8 EBNA1 sequence was input to I-TASSER, which selected the EBNA1 C-terminal domain crystal structure (1B3T) and several fragment templates comprising: yeast fatty acid synthetase (2PFF), a-L-fucosidase (2Z8X), photosynthetic reaction centre (1C51), type A collagen (1YOF) and dimeric 6-phosphoglucouronate dehydrogenase (2ZYD). The 1B3T template was also used to generate models in MOE. EBNA1 models constructed using I-TASSER and MOE (and the composite of these two generated in Modeller9v8 (shown above), were assessed for structural plausibility using Molprobity and QMEAN score servers (table S1). Models were superimposed over the template (1B3T) and RMSD was estimated for each. The qualitative model energy analysis, normalised (QMEANnorm) score of a protein structural model provides a composite scoring function based on several geometrical aspects, both global (for the entire structure) and local (per residue), enabling the discrimination of good and bad models. The human and other primate LCV EBNA1 protein structure models (as indicated) were ...

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“…EBNA1 is required for the maintenance of EBV episomes and the silencing of EBNA1 expression inhibits tumor cell growth. [59][60][61] Thus, the delivery of EBNA1-silencing siRNAs via pEBER2-shRNA vector might prove useful in the development of new anti-EBV therapeutics.…”

Section: Discussionmentioning

confidence: 99%

Utility of Epstein–Barr virus-encoded small RNA promoters for driving the expression of fusion transcripts harboring short hairpin RNAs

et al. 2007

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To induce RNA interference (RNAi), either small interfering RNAs (siRNAs) are directly introduced into the cell or short hairpin RNAs (shRNAs) are expressed from a DNA vector. At present, shRNAs are commonly synthesized by RNA polymerase III (Pol III) promoters of the H1 and U6 RNAs. In this study, we designed and characterized a new set of plasmid vectors driven by promoters of the Epstein-Barr virus (EBV)-encoded small RNAs (EBERs). The EBERs are the most abundant transcript in infected cells and they are transcribed by Pol III. We showed that the EBER promoters were able to drive the expression of shRNA fusion transcripts. siRNAs processed from these fusion transcripts specifically and effectively inhibited the expression of homologous reporter or endogenous genes in various types of cells. The partial EBER sequences in the fusion transcripts did not activate double-stranded RNA-dependent protein kinase or suppress RNAi. In nasopharyngeal carcinoma cells, the EBER2 promoter was stronger than the H1 and U6 promoters in shRNA synthesis, leading to more effective knockdown of the target genes. Taken together, our findings suggest that the EBER promoters fundamentally different from those of H1 and U6 can be used to drive the intracellular expression of shRNAs for effective silencing of target genes in mammalian cells and particularly, in EBV-infected cells.

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Suppression of Epstein-Barr nuclear antigen 1 (EBNA1) by RNA interference inhibits proliferation of EBV-positive Burkitt’s lymphoma cells

Abstract: These findings point to potential therapeutic applications for vector-mediated siRNA delivery to control EBV-associated malignant disorders.

Cited by 59 publications

References 49 publications

Roscovitine Inhibits EBNA1 Serine 393 Phosphorylation, Nuclear Localization, Transcription, and Episome Maintenance

Roscovitine Inhibits EBNA1 Serine 393 Phosphorylation, Nuclear Localization, Transcription, and Episome Maintenance

Modelling the structure of full-length Epstein–Barr virus nuclear antigen 1

Utility of Epstein–Barr virus-encoded small RNA promoters for driving the expression of fusion transcripts harboring short hairpin RNAs

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