EBNA3C coactivation with EBNA2 requires a SUMO homology domain.

On infection of human B lymphocytes, Epstein-Barr virus (EBV) expresses six nuclear (EBNAs) and two integral membrane (LMPs) proteins, resulting in lymphoproliferation (for review, see reference 23). EBNA2 upregulates transcription of cell and viral promoters through association with a cellular DNA-specific transcription factor, RBP-Jκ (CBF-1) (15, 23). EBNA2's effects are upregulated by EBNALP (17, 23). EBNA3A, EBNA3B, and EBNA3C are encoded by three related genes that are in tandem in the EBV genome; their transcription is regulated by EBNA2 and EBNALP. Like EBNA2, EBNA3A, EBNA3B, and EBNA3C associate at a high level with RBP-Jκ, limiting transcription from some promoters, such as the EBNA Cp promoter, and coactivating transcription of other promoters, such as the LMP1 promoter (24, 26, 36-38, 52, 57-59). EBNA3C is a potent coactivator of the LMP1 promoter with EBNA2 in transient assays and can be critically important in maintaining LMP1 levels, as is evident from the low LMP1 levels in growth-arrested Raji cells (1-3, 24, 59). In Raji cells, a Burkitt's lymphoma cell line infected with an EBV episome lacking the EBNA3C open reading frame, expression of EBNA3C restores LMP1 levels. EBNA3C domains can also affect transcription alone and when fused to the GAL4 DNA binding domain (5, 24, 26, 35, 58, 59). EBNA3C amino acids 365 to 545 are necessary and sufficient for coactivation of the LMP1 promoter with EBNA2 (24), whereas EBNA3C amino acids 280 to 525 repress promoter activity when fused to the GAL4 DNA binding domain (5). The common roles of EBNA3A, EBNA3B, and EBNA3C in transcriptional regulation do not explain the need for three distinct genes and the unique requirement for EBNA3C and EBNA3A for lymphoblastoid cell (LCL) outgrowth (50, 51). Some clues to EBNA3C function have come from the identification of interacting cell proteins implicated in transcriptional regulation and cell survival. These include DP103 (gemin3), the metastatic tumor suppressor Nm-23H1, CtBP, Rint-1, and prothymosin α (7, 16, 24, 42, 43, 51). Other clues come from differential effects of EBNA3C versus EBNA3A and EBNA3B in EBNA2 coactivation (24). SUMO-1 and SUMO-3 interact with EBNA3C in yeast two-hybrid screens. EBNA3C amino acids 365 to 545, which are necessary and sufficient for coactivation of the LMP1 promoter with EBNA2, are also required for binding SUMO-1 and SUMO-3 (24). SUMO-1 is a 101-amino-acid ubiquitinlike protein which is conjugated to target proteins in a series of enzymatic steps requiring a heterodimeric E1 activation complex (SAE1/SAE2) and an E2-like protein (UBC9). E3 ligases for SUMO include ring finger proteins that differ in target protein specificity (10, 13, 14, 34, 41, 47, 56). Unlike polyubiquitination, which generally targets proteins for degradation, sumolation can affect intracellular localization, protein stability, and transcriptional effects (reviewed in references 56 and 30). SUMO-1 modification of the promyelocytic leukemia protein PML is required for targeting to nuclear bodies (also known as ND10 bodies) and for recruitment of other NB proteins such as Sp100 and Daxx. SUMO-1 modification of IκBα increases its half-life by preventing ubiquitination and inhibits NF-κB signaling. SUMO-1 modification of HDAC-1 is required for repression, while sumolation of the transcription factor Sp3 represses activation (8, 9, 20, 39, 60). SUMO-1 has 48% identity with SUMO-2 and 46% identity with SUMO-3, whereas SUMO-2 and SUMO-3 are 97% identical to each other. In contrast to SUMO-1, which lacks an internal sumolation consensus motif, SUMO-2 and SUMO-3 can form polymeric chains. Unique functions for SUMO-2 and SUMO-3 have not been clearly delineated (40, 48). Since EBNA3C transcriptional activation is mediated by amino acids 365 to 545, which are required for binding to SUMO, EBNA3C interaction with SUMO and modification by SUMO could be important for EBNA3C transcriptional effects. The experiments reported here investigated this hypothesis.