Rev Nuclear Export Redistribution Assay (BHK-21)

• Designed to assay compounds for their ability to modulate nuclear export
• 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 a 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.

Nuclear accumulation of Rev can be obtained by specific inhibitors of Crm1-mediated nuclear export such as Ratjadone A. The activity of Ratjadone A in the assay is approximately equivalent to the activity obtained with the natural export inhibitor Leptomycin B. Ratjadone A is used as reference compound in the assay and compounds are tested for their ability to induce nuclear accumulation of Rev. The assay can also be used as a selectivity assay for nuclear translocation assays such as FKHR or MK2 to deselect compounds that are general export inhibitors.

Figure 1. Nuclear translocation of Rev-EGFP. Cells expressing Rev-EGFP (DMSO control, left panel) were treated with 50 nM Ratjadone A for 1 hr (right panel). Arrows indicate nuclear accumulation of Rev-EGFP detected by the image analysis algorithm.

Figure 2. Concentration response curves in the Rev_BHK assay. Concentration response was measured in 9 point half log dilution series (n=2). The EC₅₀ of both Leptomycin B and Ratjadone A is ~3 nM Cells were incubated with compound for 1 hr. Cells were then fixed and nuclear translocation was measured using the IN Cell Analyzer 3000 (GE Healthcare). % activity was calculated relative to the positive (50 nM Ratjadone A) and negative control (0.25% DMSO).