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 r equires functional upstream enhancer sequences-Spl sites, in particula r. In these experiments, HeLa cell nuclear extracts were passed over a ffinity matrices containing chemically synthesized or bacterially expr essed HIV-1 Tat. Assay of material that bound to and eluted from the T at matrices revealed the presence of the Sp1 transcription factor. Oth er transcription factors (Oct and NF-kappaB) also bound to Tat matrice s but with less efficiency-in parallel with the lower capacities of th ese binding motifs to confer Tat responsiveness on a basal HIV-1 promo ter compared with Sp1 sites. Passage of nuclear extracts over matrices containing other neutral proteins, including bovine serum albumin, ov albumin, and lysozyme, revealed no or reduced binding. Cross-linking e xperiments indicated that the purified Spl and Tat proteins can form m ultimeric complexes in the absence of other proteins. The region of Ta t responsible for Sp1 binding was localized to a region encompassing r esidues 30 to 62. Immunoprecipitation experiments with HIV-1-infected T lymphocytes indicated coimmunoprecipitation of Tat and Sp1. These ex periments extend previous genetic experiments and suggest a direct int eraction between Tat and Sp1 during transactivation.