Institut für Mikrobiologie und Hygiene, Abteilung Virologie, Haus 47, Universitätskliniken, 66421 Homburg/Saar, Germany1
Author for correspondence: Friedrich Grässer. Fax +49 6841 162 3980. e-mail graesser{at}uniklinik-saarland.de
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Abstract |
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Within 487 residues, EBNA2A contains six lysine (K) residues (positions 335, 357, 359, 363, 366 and 480), of which residues 366 and 480 are conserved within EBNA2A, EBNA2B and the HVP homologue, while K359 is conserved between EBNA2A and EBNA2B (Ling et al., 1993 ). EBNA2A has a calculated molecular mass of about 52 kDa but migrates with an apparent molecular mass of 85 kDa in SDSPAGE. This discordant behaviour cannot be attributed to phosphorylation (Grässer et al., 1992
) but could be due to the presence of about 30% proline residues. Another possibility might be a covalent modification via lysine residues of EBNA2A with molecules like ubiquitin (Bradbury, 1992
), SUMO (Mahajan et al., 1997
; Saitoh & Hinchey, 2000
) or a SUMO-related protein(s) (Hochstrasser, 2000
). In particular, modification of certain proteins by SUMO-1 is necessary for subcellular localization (Mahajan et al., 1997
); for instance, modification by a SUMO-related protein might be used to direct EBNA2 to certain target proteins or even to the nucleus. To test for SUMO-1 modification, EBNA2 was immunoprecipitated from EBV-positive Raji cells using EBV-negative BJAB cells as a control (Kremmer et al., 1995
); the resulting blot, however, only yielded a signal with EBNA2-specific monoclonal antibodies (mAbs) but not with a commercially available SUMO-1 specific antiserum (Santa Cruz). Likewise, a SUMO-1-specific goat serum (a kind gift of Frauke Melchior, Max-Planck Institute, Munich) did not detect a signal of SUMO-modified EBNA2 when EBNA2 immunopurified from Raji cell extracts was tested (data not shown). Since at least three additional SUMO-1-related proteins (Melchior, 2000
) that might also modify EBNA2 exist, we chose to mutate the lysine residues of EBNA2 to study this question.
To demonstrate that the mutation of lysine residues does not change the apparent molecular mass of EBNA2A, the lysine residues of EBNA2 were mutated to arginine, either individually or in combination, as shown in Fig. 1(a), and tested for expression in 293gp cells and in EBV-negative BJAB and BL41 cells. In addition, a serine to alanine exchange within the C-terminal NLS (S479,485
A) was generated. Mutation was achieved by site-directed mutagenesis, which introduced a new restriction site in addition to the desired mutation, using Pfu Turbo DNA Polymerase (Stratagene). Mutations were verified by DNA sequencing. EBNA2 was expressed from vectors pSG5 (Stratagene) or pEGFP-C1 (Clontech), either untagged or with an enhanced green fluorescent protein (EGFP) tag, respectively. Briefly, 106 293gp cells were transfected with 5 µg of expression vector by the calcium phosphate method, 107 B lymphocytes were electroporated with 10 µg EBNA2 plasmid, 4 µg reporter plasmid and 1 µg pEGFP vector (when appropriate) at 950 µF and 250 V with a Bio-Rad Gene Pulser, exactly as described previously (Voss et al., 2001
). Western blot analysis of transfected 293gp cells using a set of non-fusion EBNA2 constructs (Fig. 1b
) showed that all constructs expressed EBNA2 to comparable levels, while the EBNA2EGFP fusion proteins varied somewhat in their expression level. As shown in Fig. 1(c)
, none of these constructs, including the EBNA2 K335480R construct (all lysine residues mutated to arginine), exhibited a change in mobility following SDSPAGE, indicating that secondary modifications, when present, were not conferred to EBNA2 through the lysine residues, i.e. linking SUMO-like peptides or ubiquitin through isopeptide bonds. In the EGFPEBNA2 K335480R construct, all lysine residues were mutated and the N-terminal NH2 group of EBNA2 was also masked by fusion to EGFP, thus excluding a theoretical modification through the N-terminal NH2 group.
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EBNA2 transactivation was tested either with the EBNA2-responsive, viral LMP1 promoter from position -634 to +40, pgLRS(-634)CAT (Sjøblom et al., 1998 ), the LMP1 promoter LLO from position -327 to +40 (Laux et al., 1994b
) or a multimerized RBP-J
-binding site in front of a minimal promoter driving the firefly luciferase gene pGA981-16 (Strobl et al., 1997
) in conjunction with the appropriate control reporter plasmids. We assayed both the non-fusion EBNA2 mutants as well as the EGFPEBNA2 mutants for transactivation in BJAB cells; in the former case, a plasmid expressing EGFP was co-transfected to normalize FACS analysis for transfection efficiency. The amount of EBNA2 used was chosen to be below the saturation level, as a titration curve had shown that maximal activity of EBNA2 was reached at 1012 µg DNA/107 cells (data not shown). Thus, 107 BJAB cells were transfected by electroporation with 2 µg promoter plasmid, 4 µg EGFPEBNA2 vector or 4 µg EBNA2 construct in conjunction with 1 µg EGFP expression plasmid. After 48 h, 106 cells were harvested and luciferase or CAT activity was determined. With the remaining cells, FACS or Western blot analysis was performed to estimate transfection efficiency. Both kinds of constructs yielded comparable results. Because normalization can be carried out without co-transfection of the EGFP vector, a larger series of transfections employing the EBNA2EGFP constructs was performed to allow for better statistical analysis.
Activation of the LMP1 promoter by EBNA2 is mediated through both RBP-J and Spi-1/Spi-B. We chose to test the influence of the lysine residues first using a promoter construct that features 12 RBP-J
-binding sites in front of a minimal promoter together with the luciferase gene (Strobl et al., 1997
). The activation by wild-type EBNA2 was set to a relative level of 100% and compared to the activity obtained with the mutants as shown in Fig. 3(a)
. Exchange of only the conserved lysine residues, K480 or K366, slightly decreased activation. Since these two mutants were expressed at a lower level than wild-type EBNA2, this reduction of specific transactivation potential was relatively small, in line with previous results that deletion of the C-terminal NLS had been shown to increase the expression of the LMP1 protein (Cohen et al., 1991
). In contrast, all combinations that included the K335R mutation stimulated the expression of the LMP1 promoter as compared to wild-type EBNA2. When all but the C-terminal lysine residue were mutated, we observed a 1·5-fold stimulation in the expression of the LMP1 promoter and the mutation with all lysine residues (EBNA2K335480R) resulted in an approximately 2·5-fold stimulation.
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In summary, only the lysine to arginine mutation at position 480 and the serine to arginine mutations at positions 479 and 485 resulted in a discernible effect on subcellular localization. Of the other conserved lysine residues at positions 359 and 366, mutation of either residue had no effect on the functions of the protein, while mutation at position 335 increased activity when we used a promoter containing 12 RBP-J-sites; this might indicate that this lysine residue (position 335) somehow affects binding to RBP-J
.
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Acknowledgments |
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References |
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Received 3 October 2001;
accepted 18 January 2002.
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