Previous studies have shown that the ZII element in the BZLF1 promoter (P1) is responsive to TPA and anti-immunoglobulin induction. In this report, we have studied the DNA/protein complexes formed when ZII is used as a binding site. Twelve distinct DNA/protein complexes were seen in mobility shift experiments using Akata cell nuclear extracts and radiolabeled ZII. Eleven of these complexes were also formed when either BJAB or Raji cell nuclear extracts were used in the binding reaction. Six DNA/protein complexes were affected by mutations in the core TGACATCA motif of ZII which abolish responsiveness to TPA, anti-immunoglobulin treatment, and HHV6 transactivation. The relative sizes of the proteins in the DNA/protein complexes were determined by UV crosslinking. Four distinct specific binding proteins affected by core mutations in ZII were identified as ATFa, ATF1, ATF2, and c-jun. Overexpression of ATF1 in cotransfection experiments caused transactivation of the wild-type P1 promoter but had no effect on a promoter containing a mutant ZII element. An ATF1 mutant with a deleted DNA binding domain failed to transactivate P1. Overexpression of c-jun, ATFa, or ATF2 had no effect on the wild-type or mutant P1 promoter. Our results suggest that ATF1 interacts with the ZII element and may be involved in Epstein-Barr virus reactivation.

Smubp-2 is a novel transcription factor that was first identified through its interaction with the immunoglobulin Smu region (Mizuta et al., 1993) and has been cloned by virtue of its binding to two 12-O-tetradecanoylphorbol-13-acetate-responsive elements in the Epstein-Barr virus immediate-early BZLF1 promoter (Gulley et al., 1997). In this report, we examined the effect of Smubp-2 overexpression on BZLF1 prom oter activity. Overexpression of Smubp-2 in the B lymphocyte cell line BJAB caused repression of the BZLF1 gene promoter. A 14-bp region that partially overlaps with a 12-O-tetradecanoylphorbol-13-acetate-responsive element was required for maximal repression by Smubp-2, but some repression was also seen with a minimal promoter containing only the BZLF1 promoter TATA box and an initiation site. A 30-bp fragment containing the 14-bp region could transfer Smubp-2-mediated repression to heterologous promoters. Smubp-2 was found to associate with the basal transcription factor TATA binding protein (TBP) and to disrupt the formation of a stable TBP-TFIIA-DNA complex on the BZLF1 promoter TATA box and the adenovirus E1B promoter TATA box. Repression of the BZLF1 promoter by overexpressed Smubp-2 was rescued by overexpression of the basal factor TFIIA. These results suggest that complete repression of the BZLF1 promoter by Smubp-2 involves disruption of a functional TBP-TFIIA-TATA box complex and requires the -93 bp-to--79 bp region of the promoter.

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