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Phosphorylation sites of Epstein-Barr virus EBNA1 regulate its function.

ABSTRACT: Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and a risk factor for developing a variety of lymphomas and carcinomas. EBV nuclear antigen 1 (EBNA1) is the only viral protein found in all EBV-related malignancies. It plays a key role in establishing and maintaining the altered state of cells transformed with EBV. EBNA1 is required for a variety of functions, including gene regulation, replication and maintenance of the viral genome, but the regulation of EBNA1's functions is poorly understood. We demonstrate that phosphorylation affects the functions of EBNA1. By using electron-transfer dissociation tandem mass spectrometry, ten specific phosphorylated EBNA1 residues were identified. A mutant derivative preventing the phosphorylation of all ten phosphosites retained the unusually long half-life and the ability to translocate into the nucleus of wild-type EBNA1. This phosphorylation-deficient mutant, however, had a significantly reduced ability to activate transcription and to maintain EBV's plasmids in cells.

SUBMITTER: Duellman SJ

PROVIDER: S-EPMC2786306 | biostudies-literature | 2009 Sep

REPOSITORIES: biostudies-literature

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Phosphorylation sites of Epstein-Barr virus EBNA1 regulate its function.

Duellman Sarah J SJ Thompson Katie L KL Coon Joshua J JJ Burgess Richard R RR

The Journal of general virology 20090513 Pt 9

Epstein-Barr virus (EBV) is the causative agent of infectious mononucleosis and a risk factor for developing a variety of lymphomas and carcinomas. EBV nuclear antigen 1 (EBNA1) is the only viral protein found in all EBV-related malignancies. It plays a key role in establishing and maintaining the altered state of cells transformed with EBV. EBNA1 is required for a variety of functions, including gene regulation, replication and maintenance of the viral genome, but the regulation of EBNA1's func ...[more]

PMID: 19439552

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Project description:The Epstein-Barr Virus (EBV) Nuclear Antigen 1 (EBNA1) protein is required for the establishment of EBV latent infection in proliferating B-lymphocytes. EBNA1 is a multifunctional DNA-binding protein that stimulates DNA replication at the viral origin of plasmid replication (OriP), regulates transcription of viral and cellular genes, and tethers the viral episome to the cellular chromosome. EBNA1 also provides a survival function to B-lymphocytes, potentially through its ability to alter cellular gene expression. To better understand these various functions of EBNA1, we performed a genome-wide analysis of the viral and cellular DNA sites associated with EBNA1 protein in a latently infected Burkitt lymphoma B-cell line. Chromatin-immunoprecipitation (ChIP) combined with massively parallel deep-sequencing (ChIP-Seq) was used to identify cellular sites bound by EBNA1. Sites identified by ChIP-Seq were validated by conventional real-time PCR, and ChIP-Seq provided quantitative, high-resolution detection of the known EBNA1 binding sites on the EBV genome at OriP and Qp. We identified at least one cluster of unusually high-affinity EBNA1 binding sites on chromosome 11, between the divergent FAM55 D and FAM55B genes. A consensus for all cellular EBNA1 binding sites is distinct from those derived from the known viral binding sites, suggesting that some of these sites are indirectly bound by EBNA1. EBNA1 also bound close to the transcriptional start sites of a large number of cellular genes, including HDAC3, CDC7, and MAP3K1, which we show are positively regulated by EBNA1. EBNA1 binding sites were enriched in some repetitive elements, especially LINE 1 retrotransposons, and had weak correlations with histone modifications and ORC binding. We conclude that EBNA1 can interact with a large number of cellular genes and chromosomal loci in latently infected cells, but that these sites are likely to represent a complex ensemble of direct and indirect EBNA1 binding sites.

Project description:BackgroundThe Epstein-Barr virus (EBV)-encoded EBNA1 protein is expressed in all EBV-associated tumours, including undifferentiated nasopharyngeal carcinoma (NPC), where it is indispensable for viral replication, genome maintenance and viral gene expression. EBNA1's transcription factor-like functions also extend to influencing the expression of cellular genes involved in pathways commonly dysregulated during oncogenesis, including elevation of AP-1 activity in NPC cell lines resulting in enhancement of angiogenesis in vitro. In this study we sought to extend these observations by examining the role of EBNA1 upon another pathway commonly deregulated during carcinogenesis; namely NF-kappaB.ResultsIn this report we demonstrate that EBNA1 inhibits the canonical NF-kappaB pathway in carcinoma lines by inhibiting the phosphorylation of IKKalpha/beta. In agreement with this observation we find a reduction in the phosphorylation of IkappaBalpha and reduced phosphorylation and nuclear translocation of p65, resulting in a reduction in the amount of p65 in nuclear NF-kappaB complexes. Similar effects were also found in carcinoma lines infected with recombinant EBV and in the EBV-positive NPC-derived cell line C666-1. Inhibition of NF-kappaB was dependent upon regions of EBNA1 essential for gene transactivation whilst the interaction with the deubiquitinating enzyme, USP7, was entirely dispensable. Furthermore, in agreement with EBNA1 inhibiting p65 NF-kappaB we demonstrate that p65 was exclusively cytoplasmic in 11 out of 11 NPC tumours studied.ConclusionsInhibition of p65 NF-kappaB in murine and human epidermis results in tissue hyperplasia and the development of squamous cell carcinoma. In line with this, p65 knockout fibroblasts have a transformed phenotype. Inhibition of p65 NF-kappaB by EBNA1 may therefore contribute to the development of NPC by inducing tissue hyperplasia. Furthermore, inhibition of NF-kappaB is employed by viruses as an immune evasion strategy which is also closely linked to oncogenesis during persistent viral infection. Our findings therefore further implicate EBNA1 in playing an important role in the pathogenesis of NPC.

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Phosphorylation sites of Epstein-Barr virus EBNA1 regulate its function.

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Phosphorylation sites of Epstein-Barr virus EBNA1 regulate its function.

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