DEVELOPMENT OF A NUCLEAR EXPORT SIGNAL TRAPPING METHOD FOR ISOLATING GENES WITH HIV REV ACTIVITY

We have developed a method for nuclear export signal trapping (NEST) t o isolate functional Rev clones from various types of libraries such a s libraries of Rev mutants. The expression libraries are cotransfected into COS cells together with a novel Rev-dependent immunoselectable C D28 expression plasmid, pCMV 128-CD28. CD28-positive cells are recover ed by FACS or by immune precipitation with magnetic beads, and the low -molecular-weight extra chromosomal DNA is recovered, amplified for Re v-containing DNA by PCR and recloned into expression plasmids. The res ulting clones are enriched for functional Rev clones. These can be rec overed efficiently after several repetitive NEST cycles. This techniqu e may be usefully applied to study various regions of Rev, such as the RNA binding domain and the nuclear export signal, or effector domain and potentially to the isolation of cellular factors with nuclear expo rt capabilities.