Pre-organized structure of viral DNA at the binding-processing site of HIV-1 integrase

The integration of the human immunodeficiency virus type 1 DNA into the host cell genome is catalysed by the viral integrase (IN). The reaction consists of a 3′-processing [dinucleotide released from each 3′ end of the viral long terminal repeat (LTR)] followed by a strand transfer (insertion of the viral genome into the human chromosome). A 17 base pair oligonucleotide d(GGAAAATCTCTAGCAGT), d(ACTGCTAGAGATTTTCC) reproducing the U5-LTR extremity of viral DNA that contains the IN attachment site was analysed by NMR using the classical NOEs and scalar coupling constants in conjunction with a small set of residual dipolar coupling constants (RDCs) measured at the (13)C/(15)N natural abundance. The combination of these two types of parameters in calculations significantly improved the DNA structure determination. The well-known features of A-tracts were clearly identified by RDCs in the first part of the molecule. The binding/cleavage site at the viral DNA end is distinguishable by a loss of regular base stacking and a distorted minor groove that can aid its specific recognition by IN

Structural and dynamic characterization of the upper part of the HIV-1 cTAR DNA hairpin

First strand transfer is essential for HIV-1 reverse transcription. During this step, the TAR RNA hairpin anneals to the cTAR DNA hairpin; this annealing reaction is promoted by the nucleocapsid protein and involves an initial loop–loop interaction between the apical loops of TAR and cTAR. Using NMR and probing methods, we investigated the structural and dynamic properties of the top half of the cTAR DNA (mini-cTAR). We show that the upper stem located between the apical and the internal loops is stable, but that the lower stem of mini-cTAR is unstable. The residues of the internal loop undergo slow motions at the NMR time-scale that are consistent with conformational exchange phenomena. In contrast, residues of the apical loop undergo fast motions. The lower stem is destabilized by the slow interconversion processes in the internal loop, and thus the internal loop is responsible for asymmetric destabilization of mini-cTAR. These findings are consistent with the functions of cTAR in first strand transfer: its apical loop is suitably exposed to interact with the apical loop of TAR RNA and its lower stem is significantly destabilized to facilitate the subsequent action of the nucleocapsid protein which promotes the annealing reaction

An Unusual Helix Turn Helix Motif in the Catalytic Core of HIV-1 Integrase Binds Viral DNA and LEDGF

Background: Integrase (IN) of the type 1 human immunodeficiency virus (HIV-1) catalyzes the integration of viral DNA into host cellular DNA. We identified a bi-helix motif (residues 149–186) in the crystal structure of the catalytic core (CC) of the IN-Phe185Lys variant that consists of the a 4 and a 5 helices connected by a 3 to 5-residue turn. The motif is embedded in a large array of interactions that stabilize the monomer and the dimer. Principal Findings: We describe the conformational and binding properties of the corresponding synthetic peptide. This displays features of the protein motif structure thanks to the mutual intramolecular interactions of the a4 and a5 helices that maintain the fold. The main properties are the binding to: 1- the processing-attachment site at the LTR (long terminal repeat) ends of virus DNA with a Kd (dissociation constant) in the sub-micromolar range; 2- the whole IN enzyme; and 3- the IN binding domain (IBD) but not the IBD-Asp366Asn variant of LEDGF (lens epidermal derived growth factor) lacking the essential Asp366 residue. In our motif, in contrast to the conventional HTH (helix-turn-helix), it is the N terminal helix (a 4) which has the role of DNA recognition helix, while the C terminal helix (a 5) would rather contribute to the motif stabilization by interactions with the a4 helix. Conclusion: The motif, termed HTHi (i, for inverted) emerges as a central piece of the IN structure and function. It coul

Structural insights into the cTAR DNA recognition by the HIV-1 nucleocapsid protein: role of sugar deoxyriboses in the binding polarity of NC

An essential step of the reverse transcription of the HIV-1 genome is the first strand transfer that requires the annealing of the TAR RNA hairpin to the cTAR DNA hairpin. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. Using nuclear magnetic resonance and gel retardation assays, we investigated the interaction between NC and the top half of the cTAR DNA (mini-cTAR). We show that NC(11-55) binds the TGG sequence in the lower stem that is destabilized by the adjacent internal loop. The 5′ thymine interacts with residues of the N-terminal zinc knuckle and the 3′ guanine is inserted in the hydrophobic plateau of the C-terminal zinc knuckle. The TGG sequence is preferred relative to the apical and internal loops containing unpaired guanines. Investigation of the DNA–protein contacts shows the major role of hydrophobic interactions involving nucleobases and deoxyribose sugars. A similar network of hydrophobic contacts is observed in the published NC:DNA complexes, whereas NC contacts ribose differently in NC:RNA complexes. We propose that the binding polarity of NC is related to these contacts that could be responsible for the preferential binding to single-stranded nucleic acids

Specificity of LTR DNA recognition by a peptide mimicking the HIV-1 integrase α4 helix

HIV-1 integrase integrates retroviral DNA through 3′-processing and strand transfer reactions in the presence of a divalent cation (Mg2+ or Mn2+). The α4 helix exposed at the catalytic core surface is essential to the specific recognition of viral DNA. To define group determinants of recognition, we used a model composed of a peptide analogue of the α4 helix, oligonucleotides mimicking processed and unprocessed U5 LTR end and 5 mM Mg2+. Circular dichroism, fluorescence and NMR experiments confirmed the implication of the α4 helix polar/charged face in specific and non-specific bindings to LTR ends. The specific binding requires unprocessed LTR ends—i.e. an unaltered 3′-processing site CA↓GT3′—and is reinforced by Mg2+ (Kd decreases from 2 to 0.8 nM). The latter likely interacts with the ApG and GpT3′ steps of the 3′-processing site. With deletion of GT3′, only persists non-specific binding (Kd of 100 μM). Proton chemical shift deviations showed that specific binding need conserved amino acids in the α4 helix and conserved nucleotide bases and backbone groups at LTR ends. We suggest a conserved recognition mechanism based on both direct and indirect readout and which is subject to evolutionary pressure

Structural determinants of TAR RNA-DNA annealing in the absence and presence of HIV-1 nucleocapsid protein

Annealing of the TAR RNA hairpin to the cTAR DNA hairpin is required for the minus-strand transfer step of HIV-1 reverse transcription. HIV-1 nucleocapsid protein (NC) plays a crucial role by facilitating annealing of the complementary hairpins. To gain insight into the mechanism of NC-mediated TAR RNA–DNA annealing, we used structural probes (nucleases and potassium permanganate), gel retardation assays, fluorescence anisotropy and cTAR mutants under conditions allowing strand transfer. In the absence of NC, cTAR DNA-TAR RNA annealing depends on nucleation through the apical loops. We show that the annealing intermediate of the kissing pathway is a loop–loop kissing complex involving six base-pairs and that the apical stems are not destabilized by this loop–loop interaction. Our data support a dynamic structure of the cTAR hairpin in the absence of NC, involving equilibrium between both the closed conformation and the partially open ‘Y’ conformation. This study is the first to show that the apical and internal loops of cTAR are weak and strong binding sites for NC, respectively. NC slightly destabilizes the lower stem that is adjacent to the internal loop and shifts the equilibrium toward the ‘Y’ conformation exhibiting at least 12 unpaired nucleotides in its lower part

The HIV-1 Integrase α4-Helix Involved in LTR-DNA Recognition Is also a Highly Antigenic Peptide Element

Monoclonal antibodies (MAbas) constitute remarkable tools to analyze the relationship between the structure and the function of a protein. By immunizing a mouse with a 29mer peptide (K159) formed by residues 147 to 175 of the HIV-1 integrase (IN), we obtained a monoclonal antibody (MAba4) recognizing an epitope lying in the N-terminal portion of K159 (residues 147–166 of IN). The boundaries of the epitope were determined in ELISA assays using peptide truncation and amino acid substitutions. The epitope in K159 or as a free peptide (pep-a4) was mostly a random coil in solution, while in the CCD (catalytic core domain) crystal, the homologous segment displayed an amphipathic helix structure (α4-helix) at the protein surface. Despite this conformational difference, a strong antigenic crossreactivity was observed between pep-a4 and the protein segment, as well as K156, a stabilized analogue of pep-a4 constrained into helix by seven helicogenic mutations, most of them involving hydrophobic residues. We concluded that the epitope is freely accessible to the antibody inside the protein and that its recognition by the antibody is not influenced by the conformation of its backbone and the chemistry of amino acids submitted to helicogenic mutations. In contrast, the AA →Glu mutations of the hydrophilic residues Gln148, Lys156 and Lys159, known for their interactions with LTRs (long terminal repeats) and inhibitors (

Mise en evidence de structures locales dans les oligonucleotides derives de la sequence CRE (element receptif a l'AMP cyclique) et reconnaissance ADN-derives cytotoxiques de l'ellipticine

SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : T 78603 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc

Etudes structurales et dynamiques des acides nucléiques impliqués dans le premier transfert de brin lors de la transcription inverse du VIH-1 en présence de la protéine de nucléocapside NCp7

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Etude par RMN et dynamique moléculaire de duplexes d'ADN substrats de la Topoisomérase IIa Humaine

Le mécanisme de Higgs fournit au Modéle Standard une théorie de l'origine de la masse des bosons de jauge et des fermions élémentaires. Le boson de Higgs n'a pas encore été découvert, mais une limite inférieure à sa masse a été posée à 114,4 GeV à 95% de niveau de conance par les collaborations Lep. La recherche du Higgs continue maintenant au Tevatron, un collisionneur proton - anti-proton d'énergie dans le centre de masse de 1,96TeV. À basse masse le canal le plus sensible est la production associée d'un Higgs avec un boson W. Une analyse a été conduite dans le canal de désintégration dans lequel le Higgs donne une paire b b et le W se désintègre en un muon et un neutrino sur 1 fb 1 de données du Run II enregistrées par le détecteur DØ. L'analyse s'appuie sur tous les sous-systèmes du détecteur, tout particulièrement sur le calorimètre qui est un élément essentiel pour la reconstruction du système b b. Une bonne trajectographie et un réseau de neurones d'identication des jets de b fournissent un étiquetage des b performant, ce qui est crucial pour cette analyse. La résolution en énergie des jets étant de première importance lorsqu'on recherche une résonance de deux jets, un travail d'amélioration de la calibration en énergie des jets qui présentent un muon et un neutrino dans leur chaîne de désintégeration a également été conduit. L'analyse WH est eectuée sur des évènements à topologie de type W + 2 jets et W + 3 jets. Des évènements contenant Un muon, de l'énergie transverse manquante et deux ou trois jets sont sélectionnés ; les jets sont ensuite étiquetés selon leur saveur. Les canaux simplement et doublement étiquetés sont analysés séparément an d'obtenir une sensibilité plus importante. L'introduction d'une séparation signal / bruit de fond par réseau de neurones a également permis d'augmenter la sensibilité. Cette recherche d'un boson de Higgs standard a été eectuée pour une masse du Higgs s'étalant entre 100 et 150 GeV. Des limites supérieures sur la section e cace de production multipliée par le rapport d'embranchement sont calculées. Pour une masse du Higgs de 115 GeV, la limite supérieure est de 2,00 pb à 95% de niveau de conance dans le canal WH fleche b b et de 1,37 pb si l'on combine les canaux WH fleche b b et WH fleche eb b, à comparer à la valeur théorique de 0,136 pb. Ces résultats ont été soumis pour publication et sont utilisés dans la combinaison globale des analyses de recherche du Higgs par les collaboration Cdf et DØ.PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF