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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.

SUBMITTER: Mundigala H

PROVIDER: S-EPMC4066764 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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

Mundigala Hansini H Michaux Jonathan B JB Feig Andrew L AL Ennifar Eric E Rueda David D

Nucleic acids research 20140509 11

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 ...[more]

PMID: 24813449

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Project description:INTRODUCTION: Riboswitches are cis-acting regulatory RNA elements prevalently located in the leader sequences of bacterial mRNA. An adenine sensing riboswitch cis-regulates adeninosine deaminase gene (add) in Vibrio vulnificus. The structural mechanism regulating its conformational changes upon ligand binding mostly remains to be elucidated. In this open framework it has been suggested that the ligand stabilizes the interaction of the distal "kissing loop" complex. Using accurate full-atom molecular dynamics with explicit solvent in combination with enhanced sampling techniques and advanced analysis methods it could be possible to provide a more detailed perspective on the formation of these tertiary contacts. METHODS: In this work, we used umbrella sampling simulations to study the thermodynamics of the kissing loop complex in the presence and in the absence of the cognate ligand. We enforced the breaking/formation of the loop-loop interaction restraining the distance between the two loops. We also assessed the convergence of the results by using two alternative initialization protocols. A structural analysis was performed using a novel approach to analyze base contacts. RESULTS: Contacts between the two loops were progressively lost when larger inter-loop distances were enforced. Inter-loop Watson-Crick contacts survived at larger separation when compared with non-canonical pairing and stacking interactions. Intra-loop stacking contacts remained formed upon loop undocking. Our simulations qualitatively indicated that the ligand could stabilize the kissing loop complex. We also compared with previously published simulation studies. DISCUSSION AND CONCLUSIONS: Kissing complex stabilization given by the ligand was compatible with available experimental data. However, the dependence of its value on the initialization protocol of the umbrella sampling simulations posed some questions on the quantitative interpretation of the results and called for better converged enhanced sampling simulations.

Dataset Information

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

Publications

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

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