In vitro and in vivo binding of human immunodeficiency virus type 1 Tat protein and Sp1 transcription factor

Recent genetic experiments have suggested that tat transactivation of the human immunodeficiency virus type 1 (HIV-1) long terminal repeat requires functional upstream enhancer sequences--Sp1 sites, in particular. In these experiments, HeLa cell nuclear extracts were passed over affinity matrices containing chemically synthesized or bacterially expressed HIV-1 Tat. Assay of material that bound to and eluted from the Tat matrices revealed the presence of the Sp1 transcription factor. Other transcription factors (Oct and NF-kappa B) also bound to Tat matrices but with less efficiency--in parallel with the lower capacities of these binding motifs to confer Tat responsiveness on a basal HIV-1 promoter compared with Sp1 sites. Passage of nuclear extracts over matrices containing other neutral proteins, including bovine serum albumin, ovalbumin, and lysozyme, revealed no or reduced binding. Cross-linking experiments indicated that the purified Sp1 and Tat proteins can form multimeric complexes in the absence of other proteins. The region of Tat responsible for Sp1 binding was localized to a region encompassing residues 30 to 62. Immunoprecipitation experiments with HIV-1-infected T lymphocytes indicated coimmunoprecipitation of Tat and Sp1. These experiments extend previous genetic experiments and suggest a direct interaction between Tat and Sp1 during transactivation.

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