EBNA3C coactivation with EBNA2 requires a SUMO homology domain.
Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is critical for EBV immortalization of infected B lymphocytes and can coactivate the EBV LMP1 promoter with EBNA2. EBNA3C amino acids 365 to 545 are necessary and sufficient for coactivation and are required for SUMO-1 and SUMO-3 interaction. We found that EBNA3C but not EBNA3CDelta343-545 colocalized with SUMO-1 in nuclear bodies and was modified by SUMO-2, SUMO-3, and SUMO-1. EBNA3C amino acids 545 to 628 and amino acids 30 to 365 were also required for EBNA3C sumolation and nuclear body localization but were dispensable for coactivation, indicating that EBNA3C sumolation is not required for coactivation. Furthermore, EBNA3C amino acids 476 to 992 potently coactivated with EBNA2 but EBNA3C amino acids 516 to 922 lacked activity, indicating that amino acids 476 to 515 are critical for coactivation. EBNA3C amino acids 476 to 515 include DDDVIEV(507-513), which are similar to SUMO-1 EEDVIEV(84-90). EBNA3C m1 and m2 point mutations, DDD(507-509) mutated to AAA and DVIEVID(509-513) mutated to AVIAVIA, respectively, diminished SUMO-1 and SUMO-3 interaction in directed yeast two-hybrid and glutathione S-transferase pulldown assays. Furthermore, EBNA3C m1 and m2 did not coactivate the LMP1 promoter with EBNA2. Overexpression of wild-type SUMO-1, SUMO-3, and the SUMO-conjugating enzyme UBC9 coactivated the LMP1 promoter with EBNA2. Since EBNA2 activation is dependent on p300/CBP, the possible effect of EBNA3C on p300-mediated transcription was assayed. EBNA3C potentiated transcription of p300 fused to a heterologous DNA binding domain, whereas EBNA3C m1 and m2 did not. All of these data are consistent with a model in which EBNA3C upregulates EBNA2-mediated gene activation by binding to a sumolated repressor and inhibiting repressive effects on p300/CBP and other transcription factor(s) at EBNA2-regulated promoters.
Rosendorff
Adam
A
Virology Program and Department of Medicine, Channing Laboratory, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
Illanes
Diego
D
David
Gregory
G
Lin
Jeffrey
J
Kieff
Elliott
E
Johannsen
Eric
E
D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D019309:Epstein-Barr Virus Nuclear Antigens / Q000378:metabolism*; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D025842:SUMO-1 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D020798:Two-Hybrid System Techniques; D014763:Viral Matrix Proteins / Q000378:metabolism; D014764:Viral Proteins
D000956:Antigens, Viral; C402641:EBNA-2 protein, Human herpesvirus 4; C498774:EBNA-3C, epstein-barr virus; C037662:EBV-associated membrane antigen, Epstein-Barr virus; D019309:Epstein-Barr Virus Nuclear Antigens; D025842:SUMO-1 Protein; D025841:Small Ubiquitin-Related Modifier Proteins; D014763:Viral Matrix Proteins; D014764:Viral Proteins