Poster abstracts

Poster number 96 submitted by Hansini Mundigala

The HIV-1 DIS RNA Dimerizes Through a Bent Kissing Complex Intermediate

Hansini R Mundigala (Department of Chemistry, Wayne State University, Detroit, MI 48202), Jonathan Michaux (Department of Chemistry, Wayne State University, Detroit, MI 48202), A.L.Feig (Department of Chemistry, Wayne State University, Detroit, MI 48202), E.Ennifar (Architecture et Ractivitde lARN, Universit de Strasbourg, Institut de Biologie Molculaire et Cellulaire du CNRS, F-67084 Strasbourg, France), Karin Musier-Forsyth (Department of Chemistry and Biochemistry, Center for RNA Biology, and Center for Retrovirus Research,The Ohio State University, Columbus, 43210), David Rueda (Department of Chemistry, Wayne State University, Detroit, MI 48202 and Department of Medicine, Section of Virology, Imperial College, London W12 0NN, UK)

Abstract:
The HIV-1 Dimerization Initiation Sequence (DIS) is a conserved hairpin motif in the 5’-UTR of its RNA genome. DIS plays an important role in genome dimerization by formation of a “kissing complex” (KC) between two homologous hairpins. Dimerization of HIV-1 genomic RNA is facilitated by Nucleocapsid protein, a small, basic protein with two zinc-finger domains Under-standing the kinetics of this interaction is key to exploiting DIS as a possible HIV drug target. Here, we present a single-molecule Förster Resonance Energy Transfer (smFRET) study of dimerization kinetics. Our data show the real-time formation and dissociation dynamics of individual kissing complexes, as well as the rearrangement of the kissing complexes into extended duplexes. Interestingly, the single-molecule trajectories reveal the presence of a previously unobserved bent intermediate required for extended duplex formation. Mutation of universally con-served residue A272 leads to dramatically altered behavior, showing that this adenosine is essential for the formation of this intermediate,. The intermediate state is stabilized by Mg²⁺, but not by K⁺ cations. We propose a 3D model of a possible bent intermediate and a minimal dimerization pathway consisting of three steps including two obligatory intermediates (kissing complex and bent intermediate) and driven by Mg²⁺ ions. We have also shown that certain aminoglycosides with high ionic charge (Neomycin, Paromomycin and Lividomycin) mimic the function of Mg²⁺. This displacement results in a stabilized KC and stalling the dimerization pathway at KC stage. HIV-1 nucleocapsid protein (NCp7) facilitates formation of the extended RNA duplex by shifting the KC and monomer RNA equilibrium toward the KC. Furthermore, experiments with NCp7 support the conclusion that the proposed newly observed bent dimer conformation is an on-path obligatory intermediate.

Keywords: smFRET, Aminoglycosides, Nucleocapsid Protein