Regulation of the EBNA1 Epstein-Barr Virus Protein by Serine Phosphorylation and Arginine Methylation

Abstract

The Epstein-Barr virus (EBV) EBNA1 protein is important for the replication and mitotic segregation of EBV genomes in latently infected cells and also activates the transcription of some of the viral latency genes. A Gly-Arg-rich region between amino acids 325 and 376 is required for both the segregation and transcriptional activation functions of EBNA1. Here we show that this region is modified by both arginine methylation and serine phosphorylation. Mutagenesis of the four potentially phosphorylated serines in this region indicated that phosphorylation of multiple serines contributes to the efficient segregation of EBV-based plasmids by EBNA1, at least in part by increasing EBNA1 binding to hEBP2. EBNA1 was also found to bind the arginine methyltransferases PRMT1 and PRMT5. Multiple arginines in the 325-376 region were methylated in vitro by PRMT1 and PRMT5, as was an N-terminal Gly-Arg-rich region between amino acids 41 and 50. EBNA1 was also shown to be methylated in vivo, predominantly in the 325-376 region. Treatment of cells with a methylation inhibitor or down-regulation of PRMT1 altered EBNA1 localization, resulting in the formation of EBNA1 rings around the nucleoli. The results indicate that EBNA1 function is influenced by both serine phosphorylation and arginine methylation.The EBNA1 protein of Epstein-Barr virus (EBV) enables the persistence of the episomal viral genome in latently infected, cycling B lymphocytes, which can lead to malignant transformation under some circumstances. EBNA1 makes several contributions to EBV latent infection. First, EBNA1 is required for the initiation of DNA replication from the EBV latent origin, oriP (69). This involves the binding of EBNA1 to the dyad symmetry (DS) element of oriP and recruitment of cellular replication initiation proteins (9, 13, 55). Second, EBNA1 is required for the stable segregation of the viral genomes during cell division. Segregation requires EBNA1 binding to the family of repeats (FR) element of oriP and attachment of EBNA1 to the host mitotic chromosomes through an interaction with the cellular hEBP2 protein present on the mitotic chromosomes (25,26,28,66). Third, through binding to the FR element, EBNA1 can activate the transcription of other EBV latency genes, although the mechanism of this activation is not known (19,52). Finally, EBNA1 can counteract the stabilization of p53 by USP7 that occurs in response to DNA damage, thereby decreasing apoptosis and increasing cell survival (30, 54).Several functionally important regions of EBNA1 have been defined. The replication, segregation, and transcriptional activation functions of EBNA1 all require the DNA binding and dimerization domain, located near the C terminus between amino acids 459 and 607 (6, 50, 68), in order to bind an 18-bp recognition site present in multiple copies in the oriP DS and FR elements (51) (Fig. 1A). However, this domain is not sufficient for any EBNA1 function (8,39,67). The replication function also requires sequences in the N-terminal half of EBNA1, which likely ...

Viral DNA tethering domains complement replication-defective mutations in the p12 protein of MuLV Gag

“…Factors that could alter the transport of the PIC to the nucleus did not overcome the p12-PM14 defect. Insertion of nuclear localization sequences (NLS), including the classical SV40 T antigen (TAg) NLS and the nonclassical KSHV LANA NLS (LANA [24][25][26][27][28][29][30][31][32] ), did not rescue MuLV p12-PM14, and neither showed tethering to the mitotic chromatin (Fig. S2).…”

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

“…Such regions could provide molecular flexibility, allowing for rapid association/dissociation, promiscuity in partner interactions and increased availability to modification (66)(67)(68). Proteins with signalling or transcriptional regulatory functions appear to be enriched with intrinsically disordered segments, probably because this permits a greater repertoire of specific interactions (69).…”

Mitotic chromosome interactions of Epstein-Barr nuclear antigen 1 (EBNA1) and human EBNA1-binding protein 2 (EBP2)

“…In addition, segregation of FR-containing plasmids in yeast was also achieved by expressing a single protein in which the EBP2 chromosomebinding domain was fused to the EBNA1 DNA-binding domain (Kapoor and Frappier, 2003). The importance of EBP2 for EBNA1-mediated segregation in human cells was suggested by the close correlation of effects of point mutations in residues 325-376 on plasmid-maintenance activity and EBP2 binding (Shire et al, 2006), and further confirmed by the finding that silencing of EBP2 in four different human cells lines (including EBV-positive cells) led to a substantial decrease in the association of EBNA1 with metaphase chromosomes (Kapoor et al, 2005). As expected, this loss of EBNA1 from the mitotic chromosomes was accompanied by a similar loss of oriP plasmids from the chromosomes (Kapoor et al, 2005).…”

“…We have previously shown that this interaction can be detected by yeast two-hybrid assay and that residues 325-376 are important for this interaction (Shire et al, 1999;Shire et al, 2006). We have now used a more sensitive version of this assay based on LexA fusion proteins, to evaluate whether EBNA1 N-terminal sequences also contribute to EBP2 interactions (Fig.…”

“…Two-hybrid assays were performed as described (Shire et al, 2006). Briefly, S. cerevisiae strain L40a (MATa trp1 leu2 his3 LYS2::lexA-HIS3 URA3::lexA-lacZ) was transformed with one of the pLexA.EBNA1 constructs and with pACTII expressing hEBP2 (fused to the GAL4 activation domain) or with empty pACTII (negative control).…”