Abstract
Tat activates transcription by interacting with Sp1, NF-kappaB, positive transcription elongation factor b, and trans-activator-responsive element (TAR). Tat and Sp1 play major roles in transcription by protein-protein interactions at human immunodeficiency virus, type 1 (HIV-1) long terminal repeat. Sp1 activates transcription by interacting with cyclin T1 in the absence of Tat. To disrupt the transcription activation by Tat and Sp1, we fused Sp1-inhibiting polypeptides, zinc finger polypeptide, and the TAR-binding mutant Tat (TatdMt) together. A designed or natural zinc finger and Tat mutant fusion was used to target the fusion to the key regulatory sites (GC box and TAR) on the long terminal repeat and nascent short transcripts to disrupt the molecular interaction that normally result in robust transcription. The designed zinc finger and TatdMt fusions were targeted to the TAR, and they potently repressed both transcription and replication of HIV-1. The Sp1-inhibiting POZ domain, TatdMt, and zinc fingers are key functional domains important in repression of transcription and replication. The designed artificial zinc fingers were targeted to the high affinity Sp1-binding site, and by being fused with TatdMt and POZ domain, they strongly block both Sp1-cyclin T1-dependent transcription and Tat-dependent transcription, even in the presence of excess expressed Tat.
Highlights
More than 35 million people worldwide are infected with HIV,1 and most of them will develop AIDS
The molecular mechanism of the regulation of HIV-1 transcription provides a molecular basis for developing novel antiviral agents
TatdMt fusion protein can potently represses transcription of HIV-1—FBI-1 has been shown to bind to the zinc finger DNAbinding domain of Sp1, to prevent Sp1 from recognizing the target GC boxes, and to repress transcription activation by Sp1 [9]
Summary
More than 35 million people worldwide are infected with HIV, and most of them will develop AIDS In the case of HIV-1, the engineered zinc finger proteins were designed to bind a key regulatory DNA sequence within the HIV-1 LTR promoter, including Sp1 sites, the TATA box, and the downstream sequences [2, 7, 8]. They were even more effective when linked with the repression domain of the SID or KRAB transcription repressors [2, 8]. FBI-1 was shown to interact with Tat via its zinc finger and to form an inducer of short transcript-FBI-1-Tat-P-TEFb complex in vivo, which suggested that the FBI-1 protein is in close contact with other regulatory proteins and nucleic acids [10, 11]
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