title: HIV-1 DIS stem loop forms an obligatory bent kissing intermediate in the dimerization pathway.
abstract: The HIV-1 dimerization initiation sequence (DIS) is a conserved palindrome in the apical loop of a conserved hairpin motif in the 5'-untranslated region of its RNA genome. DIS hairpin plays an important role in genome dimerization by forming a 'kissing complex' between two complementary hairpins. Understanding the kinetics of this interaction is key to exploiting DIS as a possible human immunodeficiency virus (HIV) drug target. Here, we present a single-molecule Förster resonance energy transfer (smFRET) study of the dimerization reaction kinetics. Our data show the real-time formation and dissociation dynamics of individual kissing complexes, as well as the formation of the mature extended duplex complex that is ultimately required for virion packaging. Interestingly, the single-molecule trajectories reveal the presence of a previously unobserved bent intermediate required for extended duplex formation. The universally conserved A272 is essential for the formation of this intermediate, which is stabilized by Mg(2+), but not by K(+) cations. We propose a 3D model of a possible bent intermediate and a minimal dimerization pathway consisting of three steps with two obligatory intermediates (kissing complex and bent intermediate) and driven by Mg(2+) ions.
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, MI 48236, USA.
authors.forename: Hansini
authors.initials: H
authors.lastname: Mundigala
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, MI 48236, USA.
authors.forename: Jonathan B
authors.initials: JB
authors.lastname: Michaux
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, MI 48236, USA david.rueda@imperial.ac.uk.
authors.forename: Andrew L
authors.initials: AL
authors.lastname: Feig
authors.affiliation: Architecture et Réactivité de l'ARN, Université de Strasbourg, Institut de Biologie Moléculaire et Cellulaire du CNRS, F-67084 Strasbourg, France david.rueda@imperial.ac.uk.
authors.forename: Eric
authors.initials: E
authors.lastname: Ennifar
authors.affiliation: Department of Chemistry, Wayne State University, Detroit, MI 48236, USA Department of Medicine, Section of Virology, Imperial College, London W12 0NN, UK Single Molecule Imaging Group, MRC Clinical Sciences Center, Imperial College, London W12 0NN, UK david.rueda@imperial.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Rueda
mesh_terms: D019281:Dimerization; D031541:Fluorescence Resonance Energy Transfer; D015497:HIV-1 / Q000235:genetics*; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D012367:RNA, Viral / Q000737:chemistry*
chemical_list: D012367:RNA, Viral; D008274:Magnesium