Structural dynamics of HIV-1 Rev and its complexes with RRE and 5S RNA

W.-C. Lam, D.P. Millar, J.M. Seifert, F. Amberger, C. Graf, M. Auer

Research output: Contribution to journalArticlepeer-review


The Rev protein of the human immunodeficiency virus type 1 (HIV-1) has been studied by time-resolved fluorescence spectroscopy. The single tryptophan residue of Rev, Trp45, located within the arginine-rich RNA- binding domain of the protein, was utilized as an intrinsic spectroscopic probe. In addition, five peptides spanning different lengths of the arginine- rich domain, each containing the tryptophan residue, and two C-terminal deletion mutants of Rev, Rev M9Δ14 and Rev M11Δ14, were examined. Rev M9Δ14 lacks residues 68-112 whereas Rev M11Δ14 is missing residues 92-112 of the C-terminus of Rev. The fluorescence decay of Trp45 in wild-type Rev was resolved into four discrete lifetime components, and decay-associated spectra (DAS) were obtained for each component. The fluorescence decays of all five peptides and Rev M9Δ14 were resolved into three lifetime components. The fluorescence decay of Rev M11Δ14 was resolved into four components 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 M11Δ14, induced a unique tryptophan environment, characterized 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 also indicate that the activation domain mediates self-association of Rev. Based on the anisotropy decay results for wild-type Rev, the distribution of oligomers is independent of salt concentration. The average fluorescence lifetime of Trp45 was reduced upon complexation of Rev with a 40-mer fragment of the Rev response element containing the minimal element for Rev binding (F8-RRE), and the emission was blue-shifted. In addition, the local rotation of the tryptophan side chain was blocked in the protein-RRE complex. These results indicate that Trp45 directly interacts with the RRE. Rev is also shown to bind to 5S RNA, resulting in very similar changes in the time-resolved tryptophan fluorescence to those observed upon complexation of Rev with F8-RRE.
Original languageEnglish
Pages (from-to)1800-1809
Number of pages10
Issue number7
Publication statusPublished - 17 Feb 1998


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