The specificity of the CRM1-Rev nuclear export signal interaction is mediated by RanGTP

Nuclear export of intron-containing human immunodeficiency virus type 1 (HI V-1) RNA is mediated by the viral Rev protein that contains both an RNA bin ding domain specific for the viral Rev response element (RRE) and a nuclear export signal (NES). The cellular CRM1 (Exportin1) protein functions as a nuclear export receptor for proteins carrying a Rev-like NES in a process t hat also requires the GTP bound form of the Ran GTPase. Using purified reco mbinant factors, we show by co-precipitation, gel mobility shift and protei n footprinting assays that full-length Rev protein interacts directly with CRM1 in vitro independently of both the integrity of the characteristic leu cine residues of the NES and the presence of the cytotoxin leptomycin B (LM B), Addition of RanGTP induces the formation of an RRE-Rev-CRM1-RanGTP comp lex that is sensitive to LMB, NES mutations, and Ran being charged with GTP . Within this complex, CRM1 is readily cross-linked to Cys(89) near the NES of Rev. By protein footprinting, we demonstrate that the NES of Rev and tw o regions in CRM1 become inaccessible to endoproteinases upon binding sugge sting that these regions are involved in protein-protein interactions. Our data are consistent with a model in which CRM1 is the nuclear export recept or for the Rev-RRE ribonucleoprotein complex and that RanGTP binds to a pre formed Rev-CRM1 complex and specifies a functional interaction with the NES .