Rev Redistribution Assay

• Designed to assay compounds for their ability to inhibit nuclear export of Rev
• Coupled to EGFP for easy monitoring of the cellular translocation event
• Robust cell-based assay for use in high content analysis and fluorescence microscope applications

• Biologically relevant data: Compounds tested in a cellular environment
• Validated: Functionally tested cells provided with an optimized assay protocol
• Easy to use: Just plate cells, add compounds, and image

The human immunodeficiency virus type 1 (HIV-1) regulatory protein, Rev, is an RNA-binding protein essential for the expression of viral structural proteins and productive infection. Rev contains a nuclear export signal (NES) in its C-terminal domain and a nuclear localization signal (NLS) in its N-terminal domain. The NES and NLS are necessary for shuttling between nucleus and cytoplasm and are therefore crucial for the transport of unspliced and singly spliced viral transcripts. Nuclear export of Rev protein is dependent on the classical NES/Crm1 pathway that regulates the continuous shuttling of many proteins between the nucleus and the cytoplasm.

In the Rev Redistribution assay the Rev sequence is fused to the NES of the human protein kinase inhibitor alpha in order to induce a clear cytoplasmic localization of Rev in untreated cells. Nuclear accumulation of Rev can be obtained by specific inhibitors of Crm1-mediated nuclear export such as Ratjadone A.

Figure 2. Concentration response curves in the Rev assay. Concentration response was measured in 9 point half log dilution series (n=8). The EC₅₀ of Leptomycin B is ~0.3 nM, and the EC50 of Ratjadone A is ~0.6 nM Cells were incubated with compound for 1 hr. Cells were then fixed and membrane translocation was measured using the Cellomics ArrayScan V^TI Reader and the Redistribution V3 BioApplication. % activity was calculated relative to the positive (30 nM Ratjadone A) and negative control (0.25% DMSO).