Technical Reference #274

274.

A synthetic HIV-1 Rev Inhibitor Interfereing with the CRM1-mediated Nuclear Export Dirk Daelemans; Elena Afonina; Jakob Nilsson; Gudrun Werner; Jørgen Kjems§; Erik De Clercq; George N. Pavlakis; and Anne-Mieke Vandamme, Katholieke Universiteit Leuven, PNAS, 99(274), (2002)
Link To Paper

Abstract:
The HIV-1 Rev protein is an essential regulator of the HIV-1 mRNAexpression that promotes the export of unspliced and partiallyspliced mRNA. The export receptor for the leucine-rich nuclearexport signal (NES) of Rev has recently been recognized as C

Materials & Methods:
Plasmids and Cells. pBrev-GFP pBrev14-GFP and pBrev38-GFP plasmids produce fusion proteins of Rev Rev14 and Rev38 respectively fused to the enhanced version of the GFP emitting green light (GFPsg25; ref. 22). In the Rev14 mutant amino acids 14–16 (RTV) are mutated to EED (23). To obtain the mutant pBrev38-GFP the amino acids 38–44 were deleted. pBrev-blue fluorescent protein (BFP) expresses the Rev protein fused to the enhanced BFP emitting blue light (22). hCRM1-GFP was a kind gift of Barbara Felber (National Cancer Institute Frederick MD). pTat-GFP-NES expresses a Tat-GFP hybrid protein containing the Rev NES (24). pCMV-Luc contains the firefly luciferase reporter gene under control of the CMV immediate early promoter. pEF-Rev expresses the HIV-1 Rev protein from the EF1- promoter. pCMVgagLucRRE contains the firefly luciferase reporter gene fused to the p17 gag sequences and the RRE flanked by the HIV-1 major splice sites and driven by the CMV promoter.

Microscopic Technique
Confocal Microscopy, Laser Scanning

Cell Type(s)
pRev14-GFP-transfected