Nucleophosmin Contributes to the Transcriptional Activation Function of the Epstein-Barr Virus EBNA1 Protein

Abstract

The Epstein-Barr virus (EBV) EBNA1 protein plays important roles in latent infection, including transcriptional activation of EBV latency genes by binding to the family-of-repeats (FR) element. Through a proteomic approach, we previously identified an interaction between EBNA1 and the histone chaperone nucleophosmin. Here we show that the EBNA1-nucleophosmin interaction is direct and requires the Gly-Arg-rich sequences that contribute to transactivation. Additionally, nucleophosmin is recruited by EBNA1 to the FR element and is required for EBNA1-mediated transcriptional activation. E pstein-Barr virus (EBV) is a gammaherpesvirus that infects greater than 95% the of world's adult population. The EBV infectious cycle is composed of lytic and latent phases, each characterized by the expression of specific viral genes. Upon primary infection, EBV establishes latency and the viral genome is circularized and maintained as nuclear episomes that are assembled into nucleosomes similar to cellular chromatin (1, 2). The EBV EBNA1 protein plays important roles in replicating and maintaining the EBV episomes, as well as in transactivating the expression of itself and other EBV latency genes important for cell immortalization (reviewed in reference 3). The transcriptional activation function of EBNA1 involves its binding to a cluster of 20 recognition sites in the family-of-repeats (FR) element of the latent origin of replication, oriP. The FR element also enhances reporter gene expression in an EBNA1-dependent manner. Two EBNA1 regions have been shown to be critical for transcriptional activation; an N-terminal sequence mapping to amino acids 61 to 83 and a central Gly-Arg-rich region (amino acids 325 to 376) (4-6). The amino acid 61-to-83 region mediates an interaction with Brd4, an acetylated-histone-binding protein known to contribute to the transcriptional activation of both cellular and viral genes (7-10). The central Gly-Arg region contributes to interactions with the histone chaperones NAP1 and TAF-I (11, 12).Recently, we conducted a series of EBNA1 proteomic experiments in the context of EBV-infected cells and found that, in addition to NAP1 and TAF-I, EBNA1 also associates with the histone chaperone nucleophosmin (NPM1; also called B23) (13). The EBNA1-NPM1 interaction was confirmed by coimmunoprecipitation and was unaffected by RNase treatment, showing that is not RNA mediated (13). NPM1 has many cellular functions, including, ribosome biogenesis, histone assembly, regulation of DNA integrity, cell proliferation, and regulation of the tumor suppressors p53 and ARF (14-18). In addition, NPM1 has been shown to contribute to the transcription of both cellular and viral genes and to functionally interact with several viral proteins, including EBV EBNA2, HIV-1 Rev and Tat, and the hepatitis virus ␦ antigen (19)(20)(21)(22). The NPM1-EBNA2 interaction was shown to be important for EBNA2-dependent transcription and transformation in lymphoblastoid cells (22).To investigate the significance of the EBNA1-NPM1 interaction, ...

Ebna1

“…The related nucleosome assembly proteins, NAP1, TAF-Iβ (also called SET), and nucleophosmin, also interact with the EBNA1 325-376 sequence and are known to affect transcription in multiple ways (Holowaty et al 2003c;Wang and Frappier 2009;Park and Luger 2006;Frappier 2012, 2013). A role for NAP1, TAF-Iβ, and nucleophosmin in EBNA1-mediated transcriptional activation is supported by the finding that each protein is recruited to the FR element by EBNA1 and that EBNA1 transactivation activity is decreased upon depleting any of these proteins Malik-Soni and Frappier 2013). Depletion of nucleophosmin had the biggest effect on EBNA1-mediated transcriptional activation, suggesting, either that the EBNA1-nucleophosmin interaction is the most important for transcription function, or that nucleophosmin is more limiting in the cell than NAP1 or TAF-Iβ (Malik-Soni and Frappier 2013).…”

Epstein-Barr Virus EBNA1 Protein Regulates Viral Latency through Effects on let-7 MicroRNA and Dicer

“…To verify the contribution of EBNA1 to let-7a regulation, we tested whether silencing of EBNA1 in EBV-positive NPC cells would lower let-7a levels. To this end, C666-1 and HONE1-Akata cells were transfected with either AllStar negative-control siRNA or siRNA that we have previously shown specifically targets EBNA1 (15,58), and downregulation of EBNA1 was confirmed by Western blotting (Fig. 2B).…”

“…The transactivation activity of EBNA1 has been mapped to two EBNA1 regions: amino acids 61 to 83 in the N terminus and an internal Gly-Arg-rich sequence (amino acids 325 to 376) which is also essential for segregation function (11-13). The transcriptional activation activity of the region from amino acids 61 to 83 appears to involve an interaction with the acetylated histone reader protein Brd4 (14), while transactivation by the Gly-Arg sequence involves interactions with the related nucleosome assembly proteins, NAP1, TAFI-␤, and nucleophosmin (15)(16)(17).…”

Ribosome Protein L4 is essential for Epstein–Barr Virus Nuclear Antigen 1 function

“…Despite a 2.4-Å resolution crystal structure of the EBNA1 DNA binding domain bound to its cognate DNA element (4), mechanistic insights into EBNA1 and oriP-mediated episome maintenance mainly come from genetic studies using EBV recombinants and biochemical studies of EBNA1's association with cell proteins, including CTCF, Bromodomain Protein 4 (BRD4), Nucleosome Assembly Protein 1 (NAP1), the cell Origin Recognition Complex, and the Mini Chromosome Maintenance complex (5-8). Recent studies indicate that EBNA1 may use complex strategies for episome maintenance (9-16).EBNA1-associated ribosome biogenesis factors Nucleophosmin (NPM1) and Nucleolin (NCL) have been implicated in EBNA1 and oriP-dependent functions (17,18). Other viruses also use ribosomal proteins (RPs), such as RPL4, RPS19, and RPL40, to enhance virus protein translation (19-21).…”

“…EBNA1-associated ribosome biogenesis factors Nucleophosmin (NPM1) and Nucleolin (NCL) have been implicated in EBNA1 and oriP-dependent functions (17,18). Other viruses also use ribosomal proteins (RPs), such as RPL4, RPS19, and RPL40, to enhance virus protein translation (19-21).…”