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Like other complex human retroviruses, Human Foamy Virus (HFV) encodes not only the structural proteins but also several auxiliary proteins, including a potent transcriptional transactivator termed Bel-1, which is critical for viral replication. HFV contains at least two promoter elements that are highly responsive to Bel-1. These are the LTR promoter and the internal promoter, which regulate the expression of the viral structural proteins and the auxiliary proteins, respectively. Although several Bel-1 Response Elements (BREs) have been identified in both promoters by mutational analysis, these show little sequence homology. It has therefore been unclear how Bel-1 is targeted to its response elements, and how Bel-1 participates in the control of the viral early/late transcriptions.
By using methylation interference analysis, we have identified critical residues for Bel-1 binding to the HFV internal promoter, and to a promoter-proximal BRE in the LTR. We subsequently defined a minimal, 25 bp DNA binding site for Bel-1, which mediates efficient Bel-1 binding both in vitro and in vivo. We further demonstrated that the internal promoter Bel-1 target site binds Bel-1 with a significantly higher affinity than the promoter proximal Bel-1 target site located in the LTR. More recently, we have identified three additional, relatively weak Bel-1 binding sites in the LTR. Taken together, these data suggest that activation of the internal promoter may precede activation of the LTR promoter during the HFV life cycle because the internal promoter acts as a more effective Bel-1 binding site when Bel-1 levels are limiting early in infection. After the Bel-1 concentration reaches a threshhold level, the multiple weak Bel-1 binding sites in the LTR may permit a strong transactivation effect through functional synergy, leading to the efficient late transcription of structural proteins. At present, we are using in vivo sequence randomization selection to more precisely define the sequence specificities of Bel-1. Mutational analysis using mammalian reporter gene constructs and live viruses are also underway to test this functional model.
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