UNIT 12.7 Regulation of the HIV Promoter/Enhancer Most techniques used in molecular biology can be applied to the study of human immunodeficiency virus (HIV). This unit describes adaptations of such molecular tech- niques that can be used to study the regulation of HIV expression. The first two protocols (see Basic Protocol 1 and Alternate Protocol 1) describe the chloramphenicol acetyltrans- ferase (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. Basic Protocol 1 is rapid, simple, and suited to analyzing multiple samples. Alternate Protocol 1 describes an assay for CAT function that involves separating the reaction products by thin-layer chromatography (TLC). In addition, a luciferase-based reporter assay that allows a rapid quantitative analysis of long terminal repeat (LTR)–driven transcription is described (see Altenate Protocol 2). The second basic protocol describes an electrophoretic mobility shift assay (EMSA) for detecting proteins present in cell extracts that can bind to the HIV-1 LTR (see Basic Protocol 2). Moreover, this unit describes a simple assay based on DNA affinity chromatography to identify protein binding to LTR cis sequences (see Basic Protocol 3). Such studies are central to current HIV research, because it is important to know what agents induce and inhibit (or “down-regulate”) HIV transcription. The reader is encouraged to consult Chapter 10 in this manual or Current Protocols in Molecular Biology (Ausubel et al., 1993) for a description of other common molecular biological techniques that may be applied to HIV research (in particular, see Chapters 9 and 12 for descriptions of CAT assays and mobility shift assays, respectively). CAUTION: In performing molecular studies of HIV, unless all experimental manipula- tions are done in a biosafety level 3 (BL-3) facility, great care must be taken that the virus is totally inactivated (UNIT 12.1). Even after inactivation, HIV-derived materials should be handled in a BL-1 or BL-2 facility. NOTE: All incubations of cells are performed in a humidified 37°C, 5% CO 2 incubator. NOTE: All phosphate-buffered saline used in this unit is prepared without calcium and magnesium. BASIC PROTOCOL 1 ANALYSIS OF PROMOTER ACTIVITY BY THE CHLORAMPHENICOL ACETYLTRANSFERASE (CAT) ASSAY The chloramphenicol acetyltransferase (CAT) assay provides a sensitive method to study the regulation of the HIV-1 LTR, including effects of stimulatory or inhibitory agents, utilizing a plasmid construct that contains the CAT gene driven by the HIV LTR. Eukaryotic cells do not contain a CAT gene; therefore, introduction of this gene into eukaryotic cells by transfection provides a reporter gene to detect activation of the HIV LTR by cellular factors. Generally, the construct is introduced into T cells or mono- cyte/macrophage cells—usually cell lines, because they are transfected more efficiently than primary cells. This system may then be used to test the stimulatory or inhibitory effects of cytokines or pharmacologic agents. CAT enzyme is very stable and can be quantitated simply using a sensitive enzymatic assay. This basic protocol provides a rapid, quantitative, one-step method for measuring CAT activity and requires less manipulation than other methods. Supplement 54 Contributed by Camillo Palmieri, Francesca Trimboli, Giuseppe Scala, Ileana Quinto, and Peter B. Bressler Current Protocols in Immunology (2003) 12.7.1-12.7.18 Copyright © 2003 by John Wiley & Sons, Inc. 12.7.1 Detection and Analysis of HIV