We have developed a novel eukaryotic expression library screening method for isolating cellular factors involved in the Rev mechanism of HIV autoregulation, one of two key viral autoregulatory pathways required for replication and transmission. We have determined the best mode of transfection (electroporation) and levels of transfecting DNA to achieve the single hit targeting required by the method and have evaluated several methods for recovering positive clones (magnetic beads and FACS). Although two initial library screens were unsuccessful, we have subsequently developed the technology to the point where we can detect one clone in >107 independent clones. We have created a vastly improved, normalized library (C0t fractionated to enrich for rare clones) consisting of shorter fragments and are currently screening this library. During the last year we have extended our analysis of the RNA target sequence for the Rev protein, the RRE. Using site directed mutagenesis and in vivo functional assays we determined that the RRE can have two alternative structures and that both structures are likely to be represented in various strains of HIV in vivo. We have resumed an earlier study of host genes differentially regulated by HIV infection of macrophages. We have determined that one clone is upregulated by 10 to 100 fold by HIV infection of primary human macrophages. We are currently characterizing this response.
