Epstein–Barr virus nuclear antigen 1 is a DNA- binding protein with strong RNA-binding activity Chih-Chung Lu, 1 Chia-Wei Wu, 1 Shin C. Chang, 1 Tzu-Yi Chen, 1 Chwan-Ren Hu, 1 3 Ming-Yi Yeh, 1 Jen-Yang Chen 1,2 and Mei-Ru Chen 1 Correspondence Mei-Ru Chen mrc@ha.mc.ntu.edu.tw 1 Graduate Institute of Microbiology, College of Medicine, No. 1, Jen-Ai Road, 1st Section, National Taiwan University, Taipei, Taiwan 2 National Health Research Institutes, Taipei, Taiwan Received 25 April 2004 Accepted 14 June 2004 Epstein–Barr virus (EBV) nuclear antigen 1 (EBNA-1) plays key roles in both the regulation of gene expression and the replication of the EBV genome in latently infected cells. To characterize the RNA-binding activity of EBNA-1, it was demonstrated that EBNA-1 binds efficiently to RNA homopolymers that are composed of poly(G) and weakly to those composed of poly(U). All three RGG boxes of EBNA-1 contributed additively to poly(G)-binding activity and could mediate RNA binding when attached to a heterologous protein in an RNA gel mobility-shift assay. In vitro-transcribed EBV and non-EBV RNA probes revealed that EBNA-1 bound to most RNAs examined and the affinity increased as the content of G and U increased, as demonstrated in competition assays. Among these probes, the 59 non-coding region (NCR) (nt 131–278) of hepatitis C virus RNA appeared to be the strongest competitor for EBNA-1 binding to the EBV-encoded small nuclear RNA 1 (EBER1) probe, whereas a mutant 59 NCR RNA with partially disrupted secondary structure was a weak competitor. Furthermore, the interaction of endogenous EBNA-1 and EBER1 in EBV-infected cells was demonstrated by a ribonucleoprotein immunoprecipitation assay. These results revealed that EBNA-1 is a DNA-binding protein with strong binding activity to a relatively broad spectrum of RNA and suggested an additional biological impact of EBNA-1 through its ability to bind to RNA. INTRODUCTION Epstein–Barr virus (EBV), a ubiquitous human gamma- herpesvirus, infects most of the world’s population. EBV infections usually result in latency; Epstein–Barr virus nuclear antigen 1 (EBNA-1) is the only viral protein that is detectable in all EBV-associated malignancies (Kieff, 1996). EBNA-1 initiates DNA replication at the latent replication origin, oriP, and maintains EBV DNA molecules in cells by binding to a specific DNA sequence in oriP (Middleton & Sugden, 1992). In addition to modulation of EBV latent promoters, transient expression of EBNA-1 is sufficient to induce the recombinase-activating genes RAG1, RAG2 (Srinivas & Sixbey, 1995) and CD25 (interleukin 2 receptor a-chain; Kube et al., 1999) in cell lines. However, the mechanism whereby EBNA-1 regulates the expression of these cellular genes remains unknown. EBNA-1 is a 641 aa DNA-binding protein (Fig. 1) that binds to multiple sites within the family of repeat (FR) and dyad symmetry (DS) elements that comprise the 1?8 kbp boundary of oriP (Rawlins et al., 1985). The DNA-binding and dimerization domains of EBNA-1 have been mapped to the C-terminal 459–607 aa (Chen et al., 1993) and the crystal structure of EBNA-1 reveals a glycine-rich region that is responsible for direct DNA recognition (Bochkarev et al., 1996). The N-terminal 50 aa region of EBNA-1 shares 50 % sequence similarity with the N-terminus of the human ribosomal protein S2, but the functional significance remains obscure (Yates & Camiolo, 1988). EBNA-1 also contains three RGG-like motifs, repeated at aa 33–56, 330–350 and 354–377, that have been hypothesized to be responsible for RNA binding (Snudden et al., 1994). Investigations seeking proteins that interact with EBNA-1 have identified cellular proteins involved in RNA pro- cessing, including p32 (Wang et al., 1997; Chen et al., 1998) and EBNA-1-binding protein 2 (EBP2; Shire et al., 1999). The cellular protein p32, which is also known as SF2- associated protein or TAP (HIV Tat-associated protein), was first isolated from HeLa cells as a protein that was associated tightly with the essential splicing factor ASF/ SF2 (Krainer et al., 1990). ASF/SF2 is a member of the SR family of splicing factors and can stimulate constitutive splicing and regulate alternative RNA splicing factors positively or negatively, depending on where they bind to the pre-mRNA (Fu, 1995; Manley & Tacke, 1996). p32 was shown to inhibit the phosphorylation of ASF/SF2 and thereby prevent a stable interaction between ASF/SF2 and 3Deceased. 0008-0239 G 2004 SGM Printed in Great Britain 2755 Journal of General Virology (2004), 85, 2755–2765 DOI 10.1099/vir.0.80239-0