IDENTIFICATION AND FUNCTIONAL-CHARACTERIZATION OF A HIGH-AFFINITY BEL-1 DNA-BINDING SITE LOCATED IN THE HUMAN FOAMY VIRUS INTERNAL PROMOTER

The transcription of genes carried by primate foamy viruses is depende nt on two distinct promoter elements. These are the long terminal repe at (LTR) promoter, which regulates expression of the viral structural proteins, and a second internal promoter, located towards the 3' end o f the env gene, that directs expression of the viral auxiliary protein s. One of these auxiliary proteins is a potent transcriptional transac tivator, termed Bel-1 in human foamy virus (HFV) and Tas or Taf in the related simian foamy viruses, that is critical for foamy virus replic ation. Previously, it has been demonstrated that the LTR promoter elem ent of HFV contains a DNA binding site for Bel-1 that is critical for transcriptional activation (F. He, W. S. Blair, J. Fukushima, and B. R . Cullen, J. Virol. 70:3902-3908, 1996). Here, we extended this earlie r work by using methylation interference analysis to identify and char acterize the Bel-1 DNA binding sites located in the HFV LTR and intern al promoter elements. Based on these data, we propose a minimal, 25-bp DNA binding site for Bel-1, derived from the HFV internal promoter el ement, and show that this short DNA sequence mediates efficient Bel-1 binding both in vitro and in vivo. We further demonstrate that, as det ermined by both in vitro and in vivo assays, the Bel-1 target site loc ated within the HFV internal promoter binds Bel-1 with a significantly higher affinity than the cap-proximal Bel-1 target site located in th e LTR promoter. This result may provide a mechanistic explanation for the observation that the internal promoter is activated significantly earlier than the LTR promoter during the foamy virus life cycle.