HIV-1 DIS stem loop forms an obligatory bent kissing intermediate in the dimerization pathway

Nucleic Acids Res. 2014 Jun;42(11):7281-9. doi: 10.1093/nar/gku332. Epub 2014 May 9.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Dimerization
  • Fluorescence Resonance Energy Transfer
  • HIV-1 / genetics*
  • Magnesium / chemistry
  • Models, Molecular
  • Mutation
  • Nucleic Acid Conformation
  • RNA, Viral / chemistry*

Substances

  • RNA, Viral
  • Magnesium