Regulation of the HIV promoter/enhancer

Abstract

This unit describes adaptations of two molecular techniques that can be used to study the regulation of HIV expression. The first two protocols describe the chloramphenicol acetyltransferase (CAT) assay, in which the CAT reporter gene is attached to an HIV-1 promoter and CAT activity is measured as an indication of the promoter's activity. The basic protocol is rapid, simple, and suited to analyzing multiple samples. An alternate protocol describes an assay for CAT function that involves separating the reaction products by thin-layer chromatography (TLC). The second basic protocol describes an electrophoretic mobility shift assay for detecting proteins present in cell extracts that can bind to the HIV-1 LTR (long terminal repeat). Such studies are central to current HIV research because it is important to know what agents induce and inhibit (or "down-regulate") HIV transcription.