Poster abstracts

Poster number 59 submitted by Hansini Mundigala

Single-Molecule Studies of HIV-1 Dimerization Initiation Sequence Kissing Interaction and its Resolution to a Stable Extended Duplex

Hansini Mundigala (Department of Chemistry,Wayne State University,Detroit. MI 48202), Jonathan Michaux (Department of Chemistry,Wayne State University,Detroit. MI 48202), Andrew Feig (Department of Chemistry,Wayne State University,Detroit. MI 48202), David Rueda (Department of Chemistry,Wayne State University,Detroit. MI 48202)

Abstract:
The Dimerization Initiation Sequence (DIS) is a conserved hairpin motif on the 5’ UTR of the HIV genome[1]. This hairpin structure plays an important role in genome dimerization by formation of a “kissing complex” between two homologous DIS sequences [2]. Understanding the kinetics of this interaction is key to exploiting DIS as a possible drug target against HIV[3]. We have developed a novel single-molecule Fluorescence Resonance Energy Transfer (smFRET) assay to study the formation kinetics of the DIS kissing complex in solution. Our data show with unprecedented clarity, the formation and dissociation dynamics of single kissing complexes as well as formation of the extended duplex conformation. We will present a complete kinetic analysis including the effect of Mg²⁺ and K⁺ ions. Our data suggests the presence of an alternative pathway for the extended duplex formation through an intermediate dimer. The observed alternative pathway and the direct pathway have similar probabilities under physiological monovalent and divalent ionic conditions. It is observed that high Mg²⁺ favors the newly observed alternative pathway. The formation of this intermediate dimer may be the result the H⁺ and Mg²⁺ dependent conformational dynamics of purines flanking the complementary nucleotides in DIS sequence [4,5]. The mechanistic insights gained from these experiments would represent significant progress in understanding the HIV-1 dimerization mechanism.

References:
1.Goto, T., M. Nakai, and K. Ikuta, The life-cycle of human immunodeficiency virus type 1. Micron, 1998. 29(2-3): p. 123-38.
2.Skripkin, E., et al., Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro. Proc Natl Acad Sci U S A, 1994. 91(11): p. 4945-9.
3.Berkhout, B. and J.L. van Wamel, Role of the DIS hairpin in replication of human immunodeficiency virus type 1. J Virol, 1996. 70(10): p. 6723-32.
4.Mihailescu, M.R. and J.P. Marino, A proton-coupled dynamic conformational switch in the HIV-1 dimerization initiation site kissing complex. Proc Natl Acad Sci U S A, 2004. 101(5): p. 1189-94.
5.Paillart, J.C., et al., Non-canonical interactions in a kissing loop complex: the dimerization initiation site of HIV-1 genomic RNA. J Mol Biol, 1997. 270(1): p. 36-49.

Keywords: HIV-1 DIS, smFRET, Dimerization