The Rev axis of HIV is required for active viral replication and emergence from microbiological latency. We study it on two levels. Firstly we study the viral components, the Rev protein and its RNA target sequence, the RRE. Using quantitative band shift analysis and computer-generated quantitative equilibrium models we have recently determined that sequence-specific information in the RNA contributes not just to the initial Rev/RRE interaction but also to subsequent steps in the multimerization of Rev protein along the RRE. This work is in press. Secondly we study the cellular factors that the Rev axis uses to achieve the nuclear-cytoplasmic transport of unspliced viral RNAs. We are pursuing two approaches, one biochemical and one genetic. The biochemical approach has detected several cellular proteins that specifically bind the effector domain of Rev while it is multimerized along the RRE. The purification of these proteins for microsequencing awaits only the arrival of appropriate chromatographic equipment. Most recently we have focused primarily on the genetic approach. We have developed a Rev/RRE-dependent selectable marker and are now making cDNA libraries in which cellular genes are fused to RNA-binding-positive, effector-domain-negative Rev mutants. Cellular genes which can transport other molecules out of the nucleus will be specifically targeted to the selectable marker RNAs and will thus be selected by our system. We have successfully developed an appropriate eukaryotic expression/screening system. Using model libraries we have demonstrated the ability of this system to isolate one positive clone in 106. We are now preparing to screen these libraries and characterize the resulting clones. We will also use this system to select randomized mutants of the Rev effector domain.
