The luciferase value (RLU) obtained with empty pSG5 and the reporter construct was set to 100%. mimics an epitope shared with cellular proteins that is used for conversation with target structures. With monoclonal antibodies against the altered RG-repeat, we indeed identified cellular homologues that apparently have the same surface structure as methylated EBNA2. With the SDMA-specific antibodies, we precipitated the Sm protein D3 (SmD3) which, like EBNA2, binds via its SDMA-modified RG-repeat to SMN. With the ADMA-specific antibodies, we precipitated the heterogeneous ribonucleoprotein K (hnRNP K). Specific binding of the ADMA- antibody to hnRNP K was exhibited using expressed/ADMA-methylated hnRNP K. In addition, we show that EBNA2 and hnRNP K form a complex in EBV- infected B-cells. Finally, hnRNP K, when co-expressed with EBNA2, strongly enhances viral latent membrane protein 2A (LMP2A) expression by an unknown mechanism as we did not detect a direct association of hnRNP K with DNA-bound EBNA2 in gel shift experiments. Our data support the notion that this methylated surface of EBNA2 mimics the surface structure of cellular proteins to interfere with or co-opt their functional properties. Introduction The Epstein-Barr computer virus (EBV) is usually associated with various human malignancies [1] and growth-transforms primary human B-lymphocytes which are the correlate of EBV-associated post-transplant lymphoproliferative disease (PTLD) (for review, see [2]). In EBV-transformed lymphocytes, 11 so-called latent genes are expressed. Of these, only the nuclear antigens EBNA1, -2, -3a, -3c and the latent LDN-214117 membrane LDN-214117 protein LMP1 are necessary for transformation (reviewed in [3]). EBNA2 is usually a multifunctional transcriptional activator that does not bind directly to DNA but is usually tethered to promoter elements by interacting with DNA-bound transcription factors. For example, it associates through a Trp-Trp-Pro (WWP325) motif at position 323C325 (see Figure 1) with the DNA-bound repressor RBPj [4], [5], [6]. EBNA2 is the viral functional homologue to the cellular transmembrane receptor Notch which also activates gene expression via RBPj (reviewed in [7]). Binding of EBNA2 or Notch converts the repressor RBPj to the transcriptionally active form. Figure 1 shows a schematic representation of EBNA2. A computer virus encoding an EBNA2 protein with a mutation in the WWP-motif is unable to immortalise B-lymphocytes and does not activate the viral oncogene LMP1 [8]. In addition to RBPj, EBNA2 binds to a variety of basal transcription factors [2] and forms complexes with proteins involved in RNA metabolism like the DEAD-box protein DDX20 (DP103/Gemin3) [9] or the survival of motor neurons (SMN) protein [10], [11]. The binding of EBNA2 to a variety of other host proteins is usually reflected by its presence in high molecular weight complexes of different composition [12], [13], [14]. Adjacent to the WWP-motif, EBNA2 contains an Arginine-Glycine (RG-) repeat element at aa 339C354 with methylated arginine residues [10], [15]. The deletion of the RG-repeat results in a five-fold higher ability of EBNA2 to stimulate LMP1 expression, but a recombinant computer virus featuring this deletion in EBNA2 has a reduced transforming activity and needs an extended time span to induce outgrowth of transformed cell clones [16]. The EBNA2A protein from type A isolates was originally shown to confer a higher transforming capacity than EBNA2B derived from type B isolates of Mst1 EBV [17]. Recently, it was exhibited that this RG- repeat, among other C-terminal sequences, is usually important to confer the higher transforming activity of EBNA2A vs. EBNA2B [18]. Open in a separate window Physique 1 Schematic representation of the Epstein-Barr computer virus nuclear antigen 2 (EBNA2).EBNA2 of the standard B95.8 strain (accession number: AJ507799) of EBV consists of 487 amino acids (aa) present in an A-type virus. The N-terminal dimerization domain name (Dim) is located next to a poly-Proline stretch (Pro). The variable region (variable) differs between the LDN-214117 A-type viruses and B-type viruses. B-type viruses have a reduced transformation potential. The binding site for RBPj (WWP) is located around a Trp-Trp-Pro motif at aa 323C325. The adjacent Arginine-Glycine repeat (RG) between aa 339C354 confers binding to the survival of motor neurons (SMN) protein and represents the second nuclear localization signal (NLS) in addition to the canonical NLS found at the extreme C-terminus between aa 468C487. The C-terminal acidic transactivation domain name (TAD) between aa 424C468 interacts with various basal transcription factors. Methylation is usually a post-translational modification that affects protein-protein interactions [19]. Methylation at arginine residues [20] may lead to three known forms in higher eukaryotes: -NG MonoMethyl-arginine (MMA),.