Optimal Transactivation by Epstein-Barr Nuclear Antigen 1 Requires the UR1 and ATH1 Domains

Abstract

Epstein-Barr nuclear antigen 1 (EBNA1) is essential for Epstein-Barr virus to immortalize naïve B cells. EBNA1 transactivates viral promoters for genes that are necessary for immortalization when it is bound to a cluster of 20 cognate binding sites, termed the family of repeats. A region of EBNA1 from amino acids (aa) 40 to 89, termed linking region 1 (LR1), has been identified previously as being sufficient for transactivation. LR1 contains two domains that are conserved in the EBNA1 orthologs of other gamma herpesviruses. The first of these, termed unique region 1 (UR1), corresponds to aa 65 to 89 of EBNA1. UR1 is necessary for transactivation and contains a conserved recognition site for cyclic AMP-dependent protein kinase (PKA), corresponding to serine 78 of EBNA1. We have pharmacologically modulated PKA activity to determine if PKA controls EBNA1's ability to transactivate. Our results indicate that PKA activators and inhibitors do not affect transactivation by EBNA1. In addition, site-directed mutagenesis demonstrates that transactivation is not influenced by the phosphorylation status of serine 78 in the UR1 domain. The second conserved domain within LR1 is a glycine-arginine repeat, corresponding to aa 40 to 54 of EBNA1. This domain, termed ATH1, functions as an AT-hook, a DNA-binding motif found in architectural transcription factors such as HMGA1a. We demonstrate that deletion of the ATH1 domain decreases EBNA1 transactivation ability, which is consistent with a transcriptional role for ATH1. Furthermore, transactivation is restored when ATH1 is replaced by equivalent AT-hook motifs from HMGA1a. Our data strongly indicate a role for AT-hooks in EBNA1's ability to transactivate, a function necessary for EBV to immortalize naïve B-cells. Latent infection by Epstein-Barr virus (EBV) is associatedwith several diseases and malignancies including infectious mononucleosis, Burkitt's lymphoma, nasopharyngeal carcinoma, Hodgkin's disease, and lymphoproliferative diseases in immunocompromised hosts (36). Infection of naïve human B cells by EBV results in their immortalization. A subset of EBV genes is required to immortalize B cells, including the nuclear proteins EBNA1, EBNA2, EBNA3A, EBNA3C, and the membrane protein LMP1 (36). Upon binding to a set of 20 cognate binding sites, termed the family of repeats (FR), EBNA1 exerts two functions that are necessary for EBV to immortalize naïve human B cells. First, it facilitates stable replication and partitioning of EBV genomes in proliferating, latently infected cells, and, second, it activates viral promoters used to express itself and the other genes required to immortalize naïve B cells (3).Analyses conducted using derivatives of EBNA1 have revealed a region of EBNA1 from amino acid (aa) 40 to 89, termed linking region 1 (LR1), that is sufficient for transactivation when fused to the DNA-binding domain (DBD) of EBNA1 (23). Consistent with this observation, a derivative of EBNA1 with two copies of LR1 (2ϫLR1) fused to the DBD, activates transcription to levels hig...

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

“…Introduction of zinc ions into the dimer model predicts stable binding to Cys79 and Cys82 (conserved in all homologues), which could facilitate self-association of an array of EBNA1 dimers ( figure 8E). This is consistent with the observation that these residues are required for cooperative transactivation requiring zinc (24) and that deletion of LR1 from hu-EBNA1 significantly impairs the transactivation function (23).…”

“…The N-terminal LR1 (residues 33-89) can be subdivided into two regions: a Gly and Arg repeating unit (GR1: residues 33 to 53), which allows the protein to associate with ATrich DNA ("AT hook") and a unique region with multiple Gly then Arg residues, containing potential Ser phosphorylation sites and two highly conserved Cys residues. Both sub-regions are required for transactivation by the protein (18,23,24). LR2 or GR2 (residues 327-377) is also a Gly and Arg repeating unit.…”

Contributions of the EBNA1 Protein of Epstein-Barr Virus Toward B-Cell Immortalization and Lymphomagenesis

“…In addition to DBD, two other EBNA1 domains are necessary to activate FR-dependent transcription: 1) The AT-hooks (ATH1/ATH2); and 2) UR1. Deletion of either ATH1 or ATH2 reduces transactivation by about 50% Singh, Aras et al 2009); deletion of both ATH1 and ATH2 reduced transactivation to 5% of wild-type EBNA1 (ibid).…”

“…For this reason, the GR repeats of EBNA1 are referred to as AT-hook 1 and 2 (ATH1, ATH2) in this review. Deletion of ATH1 or ATH2 reduces the capacity of EBNA1 to transactivate EBV promoters and to support genome replication/segregation (Sears, Ujihara et al 2004;Singh, Aras et al 2009). Deletion of both ATH1 and ATH2 eliminates both functions (Sears, Kolman et al 2003).…”

“…Therefore, an association between EBNA1's AT-hooks and specific nucleic acids, such as AT-rich DNA or G-quadruplex RNA, is necessary for EBNA1's functions. While initially considered to be a single positively-charged domain, it is now appreciated that LR1 contains two distinct domains: 1) ATH1; and 2) A short unique region (UR1) that lies between ATH1 and GAr ( Figure 2) (Kennedy and Sugden 2003;Singh, Aras et al 2009). Deletion mutagenesis has revealed that UR1 is essential for EBNA1 to transactivate, but is not required for EBNA1 to support genome replication/segregation (Kennedy and Sugden 2003).…”