Development of a nuclear export signal trapping method for isolating genes with HIV rev activity

Abstract

We have developed a method for nuclear export signal trapping (NEST) to isolate functional Rev clones from various types of libraries such as libraries of Rev mutants. The expression libraries are cotransfected into COS cells together with a novel Rev-dependent immunoselectable CD28 expression plasmid, pCMV128-CD28. CD28-positive cells are recovered by FACS or by immune precipitation with magnetic beads, and the low-molecular-weight extra chromosomal DNA is recovered, amplified for Rev-containing DNA by PCR and recloned into expression plasmids. The resulting clones are enriched for functional Rev clones. These can be recovered efficiently after several repetitive NEST cycles. This technique 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 export capabilities.