STRUCTURAL DYNAMICS OF HIV-1 REV AND ITS COMPLEXES WITH RRE AND 5S RNA

The Rev protein of the human immunodeficiency virus type 1 (HIV-1) has been studied by time-resolved fluorescence spectroscopy, The single t ryptophan residue of Rev, Trp45, located within the arginine-rich RNA- binding domain of the protein, was utilized as an intrinsic spectrosco pic probe, In addition, five peptides spanning different lengths of th e arginine-rich domain, each containing the tryptophan residue, and tw o C-terminal deletion mutants of Rev, Rev M9 Delta 14 and Rev M11 Delt a 14, were examined. Rev M9 Delta 14 lacks residues 68-112 whereas Rev M11 Delta 14 is missing residues 92-112 of the C-terminus of Rev. The fluorescence decay of Trp45 in wild-type Rev was resolved into four d iscrete Lifetime components, and decay-associated spectra (DAS) were o btained for each component. The fluorescence decays of all five peptid es and Rev M9 Delta 14 were resolved into three lifetime components, T he fluorescence decay of Rev M11 Delta 14 was resolved into four compo nents similar to those found for wild-type Rev, These results indicate that the activation domain (residues 78-93), present in wild-type Rev and Rev M11 Delta 14, induced a unique tryptophan environment, charac terized by a short-lived, blue-shifted emission, attributed to higher order assembly of Rev, In addition, fluorescence anisotropy decay data obtained for wild-type Rev and the two C-terminal deletion mutants al so indicate that the activation domain mediates self-association of Re v, Based an the anisotropy decay results for wild-type Rev, the distri bution of oligomers is independent of stilt concentration, The average fluorescence lifetime of Trp45 was reduced upon complexation of Rev w ith a 40-mer fragment of the Rev response element containing the minim al element for Rev binding (F8-RRE), and the emission was blue-shifted In addition, the local rotation of the tryptophan side chain was bloc ked in the prtotein-RRE complex. These results indicate that Trp45 dir ectly interacts with the RRE. Rev is also shown to bind to 5S RNA, res ulting in very similar changes in the time-resolved tryptophan fluores cence to those observed upon complexation of Rev with F8-RRE.