We have developed a novel eukaryotic expression library screening method (NEST: Nuclear Export Signal Trap) for isolating cellular factors involved in the REV mechanism of HIV autoregulation. Our initial use of this system focused on isolating REV clones with random mutations in the Rev effector domain which interacts with critical cellular factors to mediate mucleo-cytoplasmic export of HIV RNAs. The advantage of our system is that it allows the selection of large numbers of functional clones, permitting an assessment of the """"""""space"""""""" of permissable amino acid substitutions. We found that the nuclear export signal tolerates a much wider range of amino-acid substitutions than previously expected, forcing a re-evaluation of nuclear export consensus signals. In other studies we have investigated the regulation of host cell genes by HIV infection of primary human macrophages. Using commercial gene grids we have screened in parallel primary human macrophages either infectred with HIV or uninfected. From these we have identified hundreds of genes which appear to be regulated by HIV infection. We are now confirming these by """"""""TAQ-Man"""""""" PCR analysis, focusing first on genes which are involved in apoptosis. Currently we are doing similar studies on macrophages treated with HIV tat protein.
