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J Biol Chem, Vol. 273, Issue 43, 28219-28228, October 23, 1998

Identification of Limiting Steps for Efficient Trans-activation of HIV-1 Promoter by Tat in Saccharomyces cerevisiae

Laurent Daviet, Florence Bois, Pier-Luigi Battisti^¶, and Anne Gatignol^¶

From  Unité 332 and ^¶ CJF 97-03, INSERM, Institut Cochin de Génétique Moléculaire, 22, rue Méchain, 75014 Paris, France

Cellular context is an important determinant for the activity of Tat, the trans-activator of human immunodeficiency virus (HIV). We have investigated HIV-1 promoter expression and trans-activation in Saccharomyces cerevisiae to provide clues about the limiting steps for Tat activity in this organism. A minimal 43-nucleotide HIV promoter (HIV43) has the activity of a weak yeast promoter in the presence or absence of various enhancer binding sites (bs), whereas the entire long terminal repeat is not expressed. None of these constructs could be trans-activated by Tat. Fusion proteins Gal4 binding domain (BD)-Tat48 and Gal4BD-Tat72 are active with different efficiencies on various yeast promoters that have Gal4 bs. They have 70 and 50% of Gal4 wild type activity on hybrid HIV promoters fused to Gal4 bs only in the presence of AP1 bs. This study shows that trans-activation of the HIV-1 promoter by Tat occurs in yeast when Tat is targeted to the promoter and a functional enhancer activity is present. Sp1 function and Tat transfer from the RNA to the promoter are two major elements for in vivo trans-activation of HIV-1 that are defective in S. cerevisiae but can be replaced by functional equivalents.

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