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

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.