Two nonconsensus sites in the Epstein-Barr virus oncoprotein EBNA3A cooperate to bind the co-repressor carboxyl-terminal-binding protein (CtBP).
CtBP (carboxyl-terminal binding protein) has been shown to be a highly conserved co-repressor of transcription that is important in development, cell cycle regulation, and transformation. Viral proteins E1A and EBNA3C and all the various Drosophila and vertebrate transcription factors to which CtBP has been reported to bind contain a conserved "PXDLS" CtBP-interaction domain. Here we show that EBNA3A binds CtBP both in vitro and in vivo but that this interaction does not require a near consensus (98)PLDLR(102) motif in the NH(2) terminus of EBNA3A. However, further deletion and mutation analysis revealed that CtBP interacts with this viral protein through a cryptic, bipartite motif located in the COOH terminus of EBNA3A. The two components of this binding domain are similar to the canonical PXDLS motif but do not include the highly conserved, and normally critical, first proline residue. These nonconsensus sites, (857)ALDLS(861) and (886)VLDLS(890), synergize to produce very efficient binding to CtBP. Interaction with CtBP was shown to be important in the repression of transcription by EBNA3A and in the ability of EBNA3A to cooperate with activated Ras to immortalize and transform primary rat embryo fibroblasts. Similar bipartite sequences can be found in other viral and cellular proteins that can interact with CtBP, including the retinoblastoma-interacting protein-methyltransferase RIZ, the oncoprotein EVI1, and Marek's disease virus transforming protein Meq.
Hickabottom
Mark
M
Department of Virology and Ludwig Institute for Cancer Research, Imperial College of Science Technology and Medicine, Faculty of Medicine, Wright-Fleming Institute, Norfolk Place, London W2 1PG, United Kingdom.
Parker
Gillian A
GA
Freemont
Paul
P
Crook
Tim
T
Allday
Martin J
MJ
D000429:Alcohol Oxidoreductases; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D015153:Blotting, Western; D002461:Cell Line, Transformed; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D015536:Down-Regulation; D019309:Epstein-Barr Virus Nuclear Antigens / Q000737:chemistry*; D005347:Fibroblasts / Q000378:metabolism; D017353:Gene Deletion; D017930:Genes, Reporter; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry* / Q000378:metabolism; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D011392:Proline / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D001616:beta-Galactosidase / Q000378:metabolism
D000956:Antigens, Viral; D004268:DNA-Binding Proteins; C100369:Eco-Q protein, Gallid herpesvirus 2; D019309:Epstein-Barr Virus Nuclear Antigens; D009856:Oncogene Proteins, Viral; D010750:Phosphoproteins; C411723:lymphocryptovirus nuclear antigen 3A; D011392:Proline; D000429:Alcohol Oxidoreductases; C096794:C-terminal binding protein; D005982:Glutathione Transferase; D001616:beta-Galactosidase