Determination of the Promoter Activity of HIV-1 Using the Chloramphenicol Acetyltransferase Reporter Gene Assay

RNA transcripts are produced from the 5' long terminal repeat (LTR) of human immunodeficiency virus type-1 (HIV-1) under the complex control of viral regulatory sequences (1-7). The LTR from a prototypical, laboratory-adapted HIV-1 (HXB2) can be divided into modulatory, core promoter, and transactivating regions (Fig. 1). The modulatory regions include elements with limited homology to AP-1 enhancer sequences at positions -347 to -329 relative to the cap site (8), two NF-AT sites at positions -292 to -255 (9), a USF site at positions -159 to -173 (10-12), a TCF-lα site at positions -139 to -124 (13), and two NF-kB enhancer elements at positions -104 to -81 (14). The core positive strand promoter is composed of three Spl binding sites located at positions -78 through -47 and a TATA box at positions -28 to -24 (15,16). A 59-base pair region which confers transcriptional transactivating potential to the core promoter elements by the viral Tat protein is located within the R region of the LTR at positions +1 to +59 (17,18). This transactivating region (TAR) folds into alternate RNA stem-loop structures that are found in the 5' termini of all viral transcripts. The TAR element mediates a substantial increase in transcriptional initiation and elongation through interactions with the viral Tat protein and other cellular factors (3,19-22). Fig. 1. LTR/gag leader region of HIV-1. A schematic drawing of the LTR/gag leader region of HIV-1 is shown. The positions of cis-acting regulatory sequences are given on the heavy line and genomic positions relative to the initiation of positive strand transcription (shown by the arrow) are given on the thinner line below. The functional and structural regions of the HIV-1 LTR are given at the bottom of the figure. The schematic is not drawn to scale. PBS, primer binding site; SD 1, splice donor 1 (major 5' splice donor); y, packaging signal.