Characterization of cellular proteins recognizing the HIV enhancer using a microscale DNA-affinity precipitation assay

Abstract

Numerous nucleic acid sequence motifs have been identified as transcriptional regulatory elements, and proteins involved in the control of gene replication and transcription have been successfully purified using nucleic acid affinity procedures. We have developed an assay that allows direct characterization of cellular proteins binding to the enhancer element of the human immunodeficiency virus (HIV), which is analogous to immunoprecipitation techniques. In extracts of H9 cells, a human CD4+ lymphoblast line clonally selected for its ability to support HIV replication, we find a class of proteins that interact specifically with the HIV enhancer. Reproducible high-resolution, two-dimensional gel electrophoresis has allowed us to resolve these proteins into two major sets. One set is common to H9 cells, two human B-lymphoblast lines, and a phytohaemagglutinin (PHA)-stimulated human CD4+ lymphoblast line (Jurkat). A second set of proteins is found only in H9 cells. Thus, with this assay we have identified HIV enhancer-binding proteins that are constitutive or inducible and that are cell-type specific.