title: Thermodynamics of Rev-RNA interactions in HIV-1 Rev-RRE assembly.
abstract: The HIV-1 protein Rev facilitates the nuclear export of intron-containing viral mRNAs by recognizing a structured RNA site, the Rev-response-element (RRE), contained in an intron. Rev assembles as a homo-oligomer on the RRE using its α-helical arginine-rich-motif (ARM) for RNA recognition. One unique feature of this assembly is the repeated use of the ARM from individual Rev subunits to contact distinct parts of the RRE in different binding modes. How the individual interactions differ and how they contribute toward forming a functional complex is poorly understood. Here we examine the thermodynamics of Rev-ARM peptide binding to two sites, RRE stem IIB, the high-affinity site that nucleates Rev assembly, and stem IA, a potential intermediate site during assembly, using NMR spectroscopy and isothermal titration calorimetry (ITC). NMR data indicate that the Rev-IIB complex forms a stable interface, whereas the Rev-IA interface is highly dynamic. ITC studies show that both interactions are enthalpy-driven, with binding to IIB being 20-30 fold tighter than to IA. Salt-dependent decreases in affinity were similar at both sites and predominantly enthalpic in nature, reflecting the roles of electrostatic interactions with arginines. However, the two interactions display strikingly different partitioning between enthalpy and entropy components, correlating well with the NMR observations. Our results illustrate how the variation in binding modes to different RRE target sites may influence the stability or order of Rev-RRE assembly and disassembly, and consequently its function.
authors.affiliation: Department of Biochemistry and Biophysics and ‡Department of Pharmaceutical Chemistry, University of California , San Francisco, United States.
authors.forename: Bhargavi
authors.initials: B
authors.lastname: Jayaraman
authors.affiliation: Department of Biochemistry and Biophysics and ‡Department of Pharmaceutical Chemistry, University of California , San Francisco, United States.
authors.forename: David
authors.initials: D
authors.lastname: Mavor
authors.affiliation: Department of Biochemistry and Biophysics and ‡Department of Pharmaceutical Chemistry, University of California , San Francisco, United States.
authors.forename: John D
authors.initials: JD
authors.lastname: Gross
authors.affiliation: Department of Biochemistry and Biophysics and ‡Department of Pharmaceutical Chemistry, University of California , San Francisco, United States.
authors.forename: Alan D
authors.initials: AD
authors.lastname: Frankel
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002151:Calorimetry; D015752:Genes, env / Q000235:genetics; D015497:HIV-1 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D054730:Protein Interaction Domains and Motifs; D012367:RNA, Viral / Q000737:chemistry* / Q000235:genetics; D013816:Thermodynamics; D019065:Virus Assembly / Q000235:genetics / Q000502:physiology; D054321:rev Gene Products, Human Immunodeficiency Virus / Q000737:chemistry* / Q000235:genetics
chemical_list: D012367:RNA, Viral; D054321:rev Gene Products, Human Immunodeficiency Virus; C514860:rev protein, Human Immunodeficiency Virus-1