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. 1998 Jul;72(7):6034-9.
doi: 10.1128/JVI.72.7.6034-6039.1998.

Functional replacement of the intracellular region of the Notch1 receptor by Epstein-Barr virus nuclear antigen 2

T Sakai  1 , Y Taniguchi, K Tamura, S Minoguchi, T Fukuhara, L J Strobl, U Zimber-Strobl, G W Bornkamm, T Honjo
Affiliations

Functional replacement of the intracellular region of the Notch1 receptor by Epstein-Barr virus nuclear antigen 2

T Sakai et al. J Virol. 1998 Jul.

Abstract

The intracellular region (RAMIC) of the mouse Notch1 receptor interacts with RBP-J/CBF-1, which binds to the DNA sequence CGTGGGAA and suppresses differentiation by transcriptional activation of genes regulated by RBP-J. Epstein-Barr virus nuclear antigen 2 (EBNA2) is essential for immortalization of human B cells by the virus. EBNA2 is a pleiotropic activator of viral and cellular genes and is targeted to DNA at least in part by interacting with RBP-J. We found that EBNA2 and the Notch1 RAMIC compete for binding to RBP-J, indicating that their interaction sites on RBP-J overlap at least partially. EBNA2 and Notch1 RAMIC transactivated the same set of viral and host promoters, i.e., the EBNA2 response element of the Epstein-Barr virus TP1 and the HES-1 promoter. Furthermore, EBNA2 functionally replaced the Notch1 RAMIC by suppressing differentiation of C2C12 myoblast progenitor cells.

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Figures

FIG. 1
FIG. 1
Interaction regions of RBP-J with EBNA2 and RAM23. (A) Positions of the point and insertional mutations in RBP-J (3) are shown by ellipses. N (residues 212 to 227) and C (residues 275 to 323) regions shown by bars are DNA binding regions and are slightly enlarged compared with those from a previous study (3). Residue numbers are shown at the bottom. The top line shows DNA binding activity relative to that of the wild type. The second and third lines indicate the relative interacting abilities of the mutants with RAM23 and EBNA2, respectively. Both yeast two-hybrid assays and coprecipitation experiments using GST-fusion proteins were carried out to measure interaction activities. Closed ellipse, less than 5% of that of the wild type; shaded ellipse, less than 15% of that of the wild type; open ellipse, more than 50% of that of the wild type. (B) In vitro interactions of 35S-labeled products of RBP-J wild type (lane 1), luciferase (lane 2, negative control), or RBP-J mutants (lanes 3 to 6) with GST-RAM23, GST-EBNA2, or GST (pGEX) vector. The same amounts of the wild type and mutant forms of RBP-J were used.
FIG. 2
FIG. 2
Competition of RAM23 and EBNA2 for their interaction with RBP-J. (A) GST-RAM23 coprecipitates 35S-labeled RBP-J but not 35S-labeled EBNA2. Added samples in each lane are shown above. 35S-labeled in vitro-translated RBP-J (lane 1) or EBNA2 (lane 2) was clearly distinguished by size. Samples in lanes 3 to 8 were coprecipitated as described previously (22). (B) GST-EBNA2 coprecipitates 35S-RBP-J but not 35S-RAM23. Added samples in each lane are shown above. Samples in lanes 3 to 6 were coprecipitated. (C) EMSA for ternary complex formation among EBNA2, RAM23, and RBP-J. EMSA was carried out as previously described (7) with 2 ng of 32P-labeled Epstein-Barr virus Cp promoter probe (16). Added samples other than DNA probe are listed above. Arrowheads indicate the sample added at the end. Sequential addition of both GST-EBNA2 and GST-RAM23 did not change the mobility of the supershifted band with either GST-RAM23 or GST-EBNA2 alone (lanes 7 and 8). Neither GST-RAM23 nor GST-EBNA2 formed a complex with the probe (data not shown). Note that the complex of RBP-J and GST-EBNA2 migrates slightly faster than that of RBP-J and GST-RAM23. S, supershifted band.
FIG. 2
FIG. 2
Competition of RAM23 and EBNA2 for their interaction with RBP-J. (A) GST-RAM23 coprecipitates 35S-labeled RBP-J but not 35S-labeled EBNA2. Added samples in each lane are shown above. 35S-labeled in vitro-translated RBP-J (lane 1) or EBNA2 (lane 2) was clearly distinguished by size. Samples in lanes 3 to 8 were coprecipitated as described previously (22). (B) GST-EBNA2 coprecipitates 35S-RBP-J but not 35S-RAM23. Added samples in each lane are shown above. Samples in lanes 3 to 6 were coprecipitated. (C) EMSA for ternary complex formation among EBNA2, RAM23, and RBP-J. EMSA was carried out as previously described (7) with 2 ng of 32P-labeled Epstein-Barr virus Cp promoter probe (16). Added samples other than DNA probe are listed above. Arrowheads indicate the sample added at the end. Sequential addition of both GST-EBNA2 and GST-RAM23 did not change the mobility of the supershifted band with either GST-RAM23 or GST-EBNA2 alone (lanes 7 and 8). Neither GST-RAM23 nor GST-EBNA2 formed a complex with the probe (data not shown). Note that the complex of RBP-J and GST-EBNA2 migrates slightly faster than that of RBP-J and GST-RAM23. S, supershifted band.
FIG. 3
FIG. 3
Transactivation of the viral and host genes by EBNA2 or RAMIC. COS7 cells were transfected with a luciferase reporter plasmid alone (-) or together with various expression vectors [pEF-BOSneo-RAM23, pEF-BOSneoRBP(R218H), pSG5-EBNA2, pEF-BOSneo-RAMIC] as indicated. A total of 1.0 μg of pSG5-EBNA2 (A and C) or pEF-BOSneo-RAMIC (B and D) per 3.5-cm-diameter dish was introduced into COS7 cells together with a reporter plasmid ERE-TP1-luc (pGa981-6) (A and B) or HES1-luc (C and D) and increasing amounts (1 or 2 μg/dish) of pEF-BOSneo-RAM23 or pEF-BOSneoRBP(R218H) DNA as indicated by bottoms of arrowheads. The luciferase reporter plasmid/pSG5-EBNA2/pEF-BOSneo-RAMIC ratio was 1/2/2 for all cotransfection experiments. Each transfection assay was carried out in comparison with a negative control reporter, ptk-luc177 (28) against pGa981-6 or pGV-B vector (TOYO-INKI Inc.) against HES1-luc. Error bars indicate standard deviations.
FIG. 4
FIG. 4
Suppression of myogenic differentiation of C2C12 cells by RAMIC and EBNA2 mutants. RAMIC (A), EBNA2 (B), and EBNA2 mutant proteins (C and D) are stained green and differentiated cells, judged by myoglobin expression, are stained red. Note that nuclei of transfected cells that underwent differentiation look yellow because of overlap with the red staining of myoglobin. Green nuclei indicate expression of RAMIC or EBNA2 or EBNA 2 mutants without differentiation into myotube. Differentiation of C2C12 cells is suppressed strongly with RAMIC (A) and EBNA2 (B). No or weak, if any, differentiation suppression was observed for EBNA2 WW323SS (C) and EBNA2 ΔHincII-StuI (D).
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