Hugues de Rocquigny

University of Strasbourg, Strasburg, Alsace, France

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Publications (66)284.61 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: The Gag precursor of HIV-1, formed of the four proteic regions matrix (MA), capsid (CA), nucleocapsid (NC) and p6, orchestrates virus morphogenesis. This complex process relies on three major interactions, NC-RNA acting as a scaffold, CA-CA and MA-membrane that targets assembly to the plasma membrane (PM). The characterization of the molecular mechanism of retroviral assembly has extensively benefited from biochemical studies and more recently an important step forward was achieved with the use of fluorescence-based techniques and fluorescently-labelled viral proteins. In this review, we summarize the findings obtained with such techniques, notably quantitative-based approaches, which highlight the role of the NC region in Gag assembly.
    Virus research. 07/2014;
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    ABSTRACT: This review aims at briefly presenting a retrospect on the retroviral nucleocapsid protein (NC), from an unspecific nucleic acid binding protein (NABP) to an all-in-one viral protein with multiple key functions in the early and late phases of the retrovirus replication cycle, notably reverse transcription of the genomic RNA and viral DNA integration into the host genome, and selection of the genomic RNA together with the initial steps of virus morphogenesis, respectively. In this context we will discuss the notion that NC protein has a flexible conformation and is thus a member of the growing family of intrinsically disordered proteins (IDP) where disorder may account, at least in part, for its functions as a nucleic acid (NA) chaperone and possibly as a protein chaperone vis à vis the viral DNA polymerase during reverse transcription. Lastly, we will briefly review the development of new anti-retroviral/AIDS compounds targeting HIV-1 NC because it represents an ideal target due to its multiple roles in the early and late phases of virus replication and its high degree of conservation.
    Virus research. 06/2014;
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    ABSTRACT: The HIV-1 transactivator of transcription (Tat) protein is thought to stimulate reverse transcription (RTion). The Tat protein and, more specifically, its (44-61) domain were recently shown to promote the annealing of complementary DNA sequences representing the HIV-1 transactivation response element TAR, named dTAR and cTAR, that plays a key role in RTion. Moreover, the kinetic mechanism of the basic Tat(44-61) peptide in this annealing further revealed that this peptide constitutes a representative nucleic acid annealer. To further understand the structure-activity relationships of this highly conserved domain, we investigated by electrophoresis and fluorescence approaches the binding and annealing properties of various Tat(44-61) mutants. Our data showed that the Tyr47 and basic residues of the Tat(44-61) domain were instrumental for binding to cTAR through stacking and electrostatic interactions, respectively, and promoting its annealing with dTAR. Furthermore, the annealing efficiency of the mutants clearly correlates with their ability to rapidly associate and dissociate the complementary oligonucleotides and to promote RTion. Thus, transient and dynamic nucleic acid interactions likely constitute a key mechanistic component of annealers and the role of Tat in the late steps of RTion. Finally, our data suggest that Lys50 and Lys51 acetylation regulates Tat activity in RTion.
    Nucleic Acids Research 10/2013; · 8.81 Impact Factor
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    ABSTRACT: The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in non-permissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (A3G) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55Gag. Recently, we showed that oligomerization of Vif into high-molecular mass complexes induces Vif folding and influences its binding to high affinity RNA binding sites present in the HIV genomic RNA. To get further insight into the role of Vif multimerization in viral assembly and A3G repression, we used fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays (FRET/FLIM) to investigate Vif-Vif interactions in living cells. By using two N-terminal tagged Vif proteins, we show that Vif-Vif interactions occur in living cells. This oligomerization is strongly reduced when the putative Vif multimerization domain (161PPLP164) is mutated, indicating that this domain is crucial, but that regions outside this motif also participate to Vif oligomerization. When co-expressed together with Pr55Gag, Vif is largely relocated to the cell membrane, where Vif oligomerization also occurs. Interestingly, wild-type A3G strongly interferes with Vif multimerization, contrary to an A3G mutant that does not bind to Vif. These findings confirm that Vif oligomerization occurs in living cells partly through its C-terminal motif and suggest that A3G may target and perturb the Vif oligomerization state to limit its functions in the cell.
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    ABSTRACT: The HIV-1 viral infectivity factor (Vif) is a small basic protein essential for viral fitness and pathogenicity. Vif allows productive infection in non-permissive cells, including most natural HIV-1 target cells, by counteracting the cellular cytosine deaminases APOBEC3G (A3G) and A3F. Vif is also associated with the viral assembly complex and packaged into viral particles through interactions with the viral genomic RNA and the nucleocapsid domain of Pr55(Gag). Recently, we showed that oligomerization of Vif into high-molecular mass complexes induces Vif folding and influences its binding to high affinity RNA binding sites present in the HIV genomic RNA. To get further insight into the role of Vif multimerization in viral assembly and A3G repression, we used fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays (FRET/FLIM) to investigate Vif-Vif interactions in living cells. By using two N-terminal tagged Vif proteins, we show that Vif-Vif interactions occur in living cells. This oligomerization is strongly reduced when the putative Vif multimerization domain ((161)PPLP(164)) is mutated, indicating that this domain is crucial, but that regions outside this motif also participate to Vif oligomerization. When co-expressed together with Pr55(Gag), Vif is largely relocated to the cell membrane, where Vif oligomerization also occurs. Interestingly, wild-type A3G strongly interferes with Vif multimerization, contrary to an A3G mutant that does not bind to Vif. These findings confirm that Vif oligomerization occurs in living cells partly through its C-terminal motif and suggest that A3G may target and perturb the Vif oligomerization state to limit its functions in the cell.
    Journal of Virology 04/2013; · 5.08 Impact Factor
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    ABSTRACT: During reverse transcription, the HIV-1 RNA is converted by the reverse transcriptase (RT) into proviral DNA. RT is assisted by the HIV-1 nucleocapsid (NCp7) protein that notably increases the ability of RT to synthesize DNA through pause sites. Using single molecule FRET, we monitored the NCp7 effect on the binding of RT to nucleic acid sequences corresponding to two different pause sites. NCp7 was found to modify the distribution of RT orientations on the oligonucleotides and decrease the residence time of RT on one of the pause sites. These results give direct insight into the NCp7 molecular mechanism in reverse transcription.
    Single Molecule Spectroscopy and Superresolution Imaging VI, edited by Jörg Enderlein, Ingo Gregor, Zygmunt Karol Gryczynski, Rainer Erdmann, Felix Koberling, Proc. of SPIE, USA; 02/2013
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    ABSTRACT: HIV-1 nucleocapsid protein (NC) is involved in the rearrangement of nucleic acids occurring in key steps of reverse transcription. The protein, through its two zinc fingers, interacts preferentially with unpaired guanines in single-stranded sequences. In mini-cTAR stem-loop, which corresponds to the top half of the cDNA copy of the transactivation response element of the HIV-1 genome, NC was found to exhibit a clear preference for the TGG sequence at the bottom of mini-cTAR stem. To further understand how this site was selected among several potential binding sites containing unpaired guanines, we probed the intrinsic dynamics of mini-cTAR using (13)C relaxation measurements. Results of spin relaxation time measurements have been analyzed using the model-free formalism and completed by dispersion relaxation measurements. Our data indicate that the preferentially recognized guanine in the lower part of the stem is exempt of conformational exchange and highly mobile. In contrast, the unrecognized unpaired guanines of mini-cTAR are involved in conformational exchange, probably related to transient base-pairs. These findings support the notion that NC preferentially recognizes unpaired guanines exhibiting a high degree of mobility. The ability of NC to discriminate between close sequences through their dynamic properties contributes to understanding how NC recognizes specific sites within the HIV genome.
    PLoS ONE 01/2012; 7(6):e38905. · 3.53 Impact Factor
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    ABSTRACT: The core protein of hepatitis c virus (HCV) is a structural protein with potent RNA chaperoning activities mediated by its hydrophilic N-terminal domain D1, which is thought to play a key role in HCV replication. To further characterize the core chaperoning properties, we studied the interactions between core D1 and the conserved HCV 3'X genomic region required for genome replication. To this end, we monitored the real-time annealing kinetics of native and mutated fluorescently labelled 16-nt palindromic sequence (DLS) and 27-nt Stem Loop II (SL2) from X with their respective complementary sequences. Core D1 and peptides consisting of the core basic domains were found to promote both annealing reactions and partly switch the loop-loop interaction pathway, which predominates in the absence of peptide, towards a pathway involving the stem termini. The chaperone properties of the core D1 peptides were found to be mediated through interaction of their basic clusters with the oligonucleotide phosphate groups, in line with the absence of high affinity site for core on HCV genomic RNA. The core ability to facilitate the interconversion between different RNA structures may explain how this protein regulates RNA structural transitions during HCV replication.
    Nucleic Acids Research 11/2011; 40(6):2540-53. · 8.81 Impact Factor
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    ABSTRACT: Addition of Vpr C-terminus to various cell types provokes cell apoptosis. This property was recently shown useful to develop inhibitors of cell proliferation. In that context, we investigated the cellular uptake of rhodamine- and fluorescein-labeled Vpr(52-96) peptides to understand the mechanism of Vpr C-terminus entry into cells. Dynamic light scattering data indicated that this peptide spontaneously formed polydispersed aggregates in cell culture medium. The fluorescently labeled Vpr(52-96) peptide was efficiently internalized, appearing either as large fluorescent patches in the cytoplasm or in a more diffuse form throughout the cell. Using isothermal titration calorimetry, we demonstrated that Vpr(52-96) can tightly associate with heparin, a glycosaminoglycan analog of heparan sulphate, suggesting a central role of the ubiquitous cell surface-associated heparan sulphate proteoglycans for the internalization of Vpr C-terminus. Fluorescently-labeled transferrin and methyl-β-cyclodextrin showed that the Vpr C-terminus was mediated through clathrin- and caveolae/raft-dependent endocytosis. We found that Vpr C-terminus uptake was partly blocked at 4°C suggesting the importance of membrane fluidity for Vpr C-terminus entry. In fact, atomic force microscopy and liposome leakage further indicated that the Vpr peptide can destabilize and disrupt model membrane bilayers, suggesting that this mechanism may contribute to the passive entry of the peptide. Finally, using fluorescence lifetime imaging, we found that the Vpr(52-96) peptide was stable in cells for at least 48h, probably as a consequence of the poor accessibility of the peptide to proteolytic enzymes in aggregates.
    Biochimie 06/2011; 93(10):1647-58. · 3.14 Impact Factor
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    ABSTRACT: Synthesis of the HIV-1 viral DNA by reverse transcriptase involves two obligatory strand transfer reactions. The second strand transfer corresponds to the annealing of the (-) and (+) DNA copies of the primer binding site (PBS) sequence which is chaperoned by the nucleocapsid protein (NCp7). NCp7 modifies the (+)/(-)PBS annealing mechanism by activating a loop-loop kissing pathway that is negligible without NCp7. To characterize in depth the dynamics of the loop in the NCp7/PBS nucleoprotein complexes, we investigated the time-resolved fluorescence parameters of a (-)PBS derivative containing the fluorescent nucleoside analogue 2-aminopurine at positions 6, 8 or 10. The NCp7-directed switch of (+)/(-)PBS annealing towards the loop pathway was associated to a drastic restriction of the local DNA dynamics, indicating that NCp7 can 'freeze' PBS conformations competent for annealing via the loops. Moreover, the modifications of the PBS loop structure and dynamics that govern the annealing reaction were found strictly dependent on the integrity of the zinc finger hydrophobic platform. Our data suggest that the two NCp7 zinc fingers are required to ensure the specificity and fidelity of the second strand transfer, further underlining the pivotal role played by NCp7 to control the faithful synthesis of viral HIV-1 DNA.
    Nucleic Acids Research 05/2011; 39(15):6633-45. · 8.81 Impact Factor
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    ABSTRACT: During formation of HIV particles, the Gag polyproteins are thought to interact with Vpr proteins to promote their encapsidation in the nascent particles. To directly visualize and monitor the formation of the Gag-Vpr complexes and correlate their formation with Vpr oligomerization, we used two photon lifetime imaging microscopy (FLIM) and time laps microscopy on HeLa cells expressing eGFP-labelled Vpr and tetracystein-tagged Gag proteins detected by the biarsenical ReAsH labelling reagent. Using these fluorescent microscopy approaches, we found that Gag proteins interact directly with Vpr proteins, which results in their transfer from the nuclear to the plasma membrane. Vpr oligomerization was found critical for both its interaction with Gag proteins and its transfer to the plasma membrane. Moreover, mutations in the C-terminal domain of Gag abolished the interaction with Vpr and its redistribution to the plasma membrane, indicating that this domain was critical for interaction with Vpr. Taken together, these data show that the Gag polyproteins interact through their C-terminal domain with Vpr oligomers, promoting the redistribution of the latter at the plasma membrane and probably their incorporation into nascent viral particles.
    Proc SPIE 02/2011;
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    ABSTRACT: The multifunctional HCV core protein consists of a hydrophilic RNA interacting D1 domain and a hydrophobic D2 domain interacting with membranes and lipid droplets. The core D1 domain was found to possess nucleic acid annealing and strand transfer properties. To further understand these chaperone properties, we investigated how the D1 domain and two peptides encompassing the D1 basic clusters chaperoned the annealing of complementary canonical nucleic acids that correspond to the DNA sequences of the HIV-1 transactivation response element TAR and its complementary cTAR. The core peptides were found to augment cTAR-dTAR annealing kinetics by at least three orders of magnitude. The annealing rate was not affected by modifications of the dTAR loop but was strongly reduced by stabilization of the cTAR stem ends, suggesting that the core-directed annealing reaction is initiated through the terminal bases of cTAR and dTAR. Two kinetic pathways were identified with a fast pre-equilibrium intermediate that then slowly converts into the final extended duplex. The fast and slow pathways differed by the number of base pairs, which should be melted to nucleate the intermediates. The three peptides operate similarly, confirming that the core chaperone properties are mostly supported by its basic clusters.
    Nucleic Acids Research 02/2010; 38(11):3632-42. · 8.81 Impact Factor
  • Hiv Therapy. 01/2010; 4(2):179-198.
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    ABSTRACT: During HIV-1 assembly, the viral protein R (Vpr) is incorporated into newly made viral particles via an interaction with the C-terminal domain of the Gag polyprotein precursor Pr55(Gag). Vpr has been implicated in the nuclear import of newly made viral DNA and subsequently in its transcription. In addition, Vpr can affect the cell physiology by causing G(2)/M cell cycle arrest and apoptosis. Vpr can form oligomers, but their roles have not yet been investigated. We have developed fluorescence lifetime imaging microscopy-fluorescence resonance energy transfer-based assays to monitor the interaction between Pr55(Gag) and Vpr in HeLa cells. To that end, we used enhanced green fluorescent protein-Vpr that can be incorporated into the virus and tetracysteine (TC)-tagged Pr55(Gag)-TC. This TC motif is tethered to the C terminus of Pr55(Gag) and does not interfere with Pr55(Gag) trafficking and the assembly of virus-like particles (VLPs). Results show that the Pr55(Gag)-Vpr complexes accumulated mainly at the plasma membrane. In addition, results with Pr55(Gag)-TC mutants confirm that the (41)LXXLF domain of Gag-p6 is essential for Pr55(Gag)-Vpr interaction. We also report that Vpr oligomerization is crucial for Pr55(Gag) recognition and its accumulation at the plasma membrane. On the other hand, Pr55(Gag)-Vpr complexes are still formed when Pr55(Gag) carries mutations impairing its multimerization. These findings suggest that Pr55(Gag)-Vpr recognition and complex formation occur early during Pr55(Gag) assembly.
    Journal of Virology 11/2009; 84(3):1585-96. · 5.08 Impact Factor
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    ABSTRACT: Due to its highly conserved zinc fingers and its nucleic acid chaperone properties which are critical for HIV-1 replication, the nucleocapsid protein (NC) constitutes a major target in AIDS therapy. Different families of molecules targeting NC zinc fingers and/or inhibiting the binding of NC with its target nucleic acids have been developed. However, their limited specificity and their cellular toxicity prompted us to develop a screening assay to target molecules able to inhibit NC chaperone properties, and more specifically the initial NC-promoted destabilization of the nucleic acid secondary structure. Since this destabilization is critically dependent on the properly folded fingers, the developed assay is thought to be highly specific. The assay was based on the use of cTAR DNA, a stem–loop sequence complementary to the transactivation response element, doubly labelled at its 5′ and 3′ ends by a rhodamine 6G fluorophore and a fluorescence quencher, respectively. Addition of NC(12-55), a peptide corresponding to the zinc finger domain of NC, to this doubly-labelled cTAR, led to a partial melting of the cTAR stem, which increases the distance between the two labels and thus, restores the rhodamine 6G fluorescence. Thus, positive hits were detected through the decrease of rhodamine 6G fluorescence. An “in-house” chemical library of 4800 molecules was screened and five compounds with IC50 values in the micromolar range have been selected. The hits were shown by mass spectrometry and fluorescence anisotropy titration to prevent binding of NC(12-55) to cTAR through direct interaction with the NC folded fingers, but without promoting zinc ejection. These non-zinc ejecting NC binders are a new series of anti-NC molecules that could be used to rationally design molecules with potential anti-viral activities.
    Biochimie 05/2009; · 3.14 Impact Factor
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    ABSTRACT: We present a new methodology for site-specific sensing of peptide-oligonucleotide (ODN) interactions using a solvatochromic fluorescent label based on 3-hydroxychromone (3HC). This label was covalently attached to the N-terminus of a peptide corresponding to the zinc finger domain of the HIV-1 nucleocapsid protein (NC). On interaction with target ODNs, the labeled peptide shows strong changes in the ratio of its two emission bands, indicating an enhanced screening of the 3HC fluorophore from the bulk water by the ODN bases. Remarkably, this two-color response depends on the ODN sequence and correlates with the 3D structure of the corresponding complexes, suggesting that the 3HC label monitors the peptide-ODN interactions site-specifically. By measuring the two-color ratio, we were also able to determine the peptide-ODN-binding parameters and distinguish multiple binding sites in ODNs, which is rather difficult using other fluorescence methods. Moreover, this method was found to be more sensitive than the commonly used steady-state fluorescence anisotropy, especially in the case of small ODNs. The described methodology could become a new universal tool for investigating peptide-ODN interactions.
    Nucleic Acids Research 02/2009; 37(3):e25. · 8.81 Impact Factor
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    ABSTRACT: The human immunodeficiency virus type 1 (HIV-1) encodes several regulatory proteins, notably Vpr which influences the survival of the infected cells by causing a G2/M arrest and apoptosis. Such an important role of Vpr in HIV-1 disease progression has fuelled a large number of studies, from its 3D structure to the characterization of specific cellular partners. However, no direct imaging and quantification of Vpr-Vpr interaction in living cells has yet been reported. To address this issue, eGFP- and mCherry proteins were tagged by Vpr, expressed in HeLa cells and their interaction was studied by two photon fluorescence lifetime imaging microscopy and fluorescence correlation spectroscopy. Results show that Vpr forms homo-oligomers at or close to the nuclear envelope. Moreover, Vpr dimers and trimers were found in the cytoplasm and in the nucleus. Point mutations in the three alpha helices of Vpr drastically impaired Vpr oligomerization and localization at the nuclear envelope while point mutations outside the helical regions had no effect. Theoretical structures of Vpr mutants reveal that mutations within the alpha-helices could perturb the leucine zipper like motifs. The DeltaQ44 mutation has the most drastic effect since it likely disrupts the second helix. Finally, all Vpr point mutants caused cell apoptosis suggesting that Vpr-mediated apoptosis functions independently from Vpr oligomerization. We report that Vpr oligomerization in HeLa cells relies on the hydrophobic core formed by the three alpha helices. This oligomerization is required for Vpr localization at the nuclear envelope but not for Vpr-mediated apoptosis.
    Retrovirology 10/2008; 5:87. · 5.66 Impact Factor
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    ABSTRACT: The nucleocapsid protein (NC) plays an important role in HIV-1, mainly through interactions with the genomic RNA and its DNA copies. Though the structures of several complexes of NC with oligonucleotides (ODNs) are known, detailed information on the ODN dynamics in the complexes is missing. To address this, we investigated the steady state and time-resolved fluorescence properties of 2-aminopurine (2Ap), a fluorescent adenine analog introduced at positions 2 and 5 of AACGCC and AATGCC sequences. In the absence of NC, 2Ap fluorescence was strongly quenched in the flexible ODNs, mainly through picosecond to nanosecond dynamic quenching by its neighboring bases. NC strongly restricted the ODN flexibility and 2Ap local mobility, impeding the collisions of 2Ap with its neighbors and thus, reducing its dynamic quenching. Phe(16)-->Ala and Trp(37)-->Leu mutations largely decreased the ability of NC to affect the local dynamics of 2Ap at positions 2 and 5, respectively, while a fingerless NC was totally ineffective. The restriction of 2Ap local mobility was thus associated with the NC hydrophobic platform at the top of the folded fingers. Since this platform supports the NC chaperone properties, the restriction of the local mobility of the bases is likely a mechanistic component of these properties.
    Nucleic Acids Research 02/2008; 36(3):885-96. · 8.81 Impact Factor
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    ABSTRACT: The nucleocapsid protein (NC) plays seminal roles in HIV replication, thus representing a major drug target. NC functions rely on its two zinc-fingers and flanking basic residues. Zinc ejectors inhibit NC functions, but with limited specificity. New classes of molecules competing with NC or its viral nucleic acid and enzyme partners are reviewed here.
    Mini Reviews in Medicinal Chemistry 02/2008; 8(1):24-35. · 2.87 Impact Factor
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    ABSTRACT: The retroviral nucleocapsid proteins (NCs) are small proteins with either one or two conserved zinc fingers flanked by basic domains. NCs play key roles during reverse transcription by chaperoning the obligatory strand transfers. In HIV-1, the first DNA strand transfer relies on the NCp7-promoted destabilization and subsequent annealing of the transactivation response element, TAR with its complementary cTAR sequence. NCp7 chaperone activity relies mainly on its two folded fingers. Since NCs with a unique zinc finger are encoded by gammaretroviruses such as the canonical Moloney murine leukemia virus (MoMuLV), our objective was to characterize, by fluorescence techniques, the binding and chaperone activities of the NCp10 protein of MoMuLV to the TAR sequences of HIV-1. The unique finger and the flanking 12-25 and 40-48 domains of NCp10 were found to bind and destabilize cTAR stem-loop almost as efficiently as the homologous NCp7 protein. The flanking domains were essential for properly positioning the finger and, notably, the Trp35 residue onto cTAR. Thus, the binding and destabilization determinants scattered on the two NCp7 fingers are encoded by the unique finger of NCp10 and its flanking domains. NCp10 also activates the cTAR/TAR annealing reaction, but less efficiently than NCp7, suggesting that the two NCp7 fingers promote in concert the rate-limiting nucleation of the duplex. Due to its ability to mimic NCp7, the simple structure of NCp10 might be useful to design peptidomimetics aimed at inhibiting HIV replication.
    Biochemistry 01/2008; 46(50):14650-62. · 3.38 Impact Factor

Publication Stats

2k Citations
284.61 Total Impact Points

Institutions

  • 2009–2013
    • University of Strasbourg
      • Laboratoire de Biophotonique et Pharmacologie
      Strasburg, Alsace, France
  • 1992–2011
    • French National Centre for Scientific Research
      • Institute for Molecular and Cellular Biology (IBMC)
      Lutetia Parisorum, Île-de-France, France
  • 2007
    • Unité Inserm U1077
      Caen, Lower Normandy, France
  • 2000
    • Jewish General Hospital
      Montréal, Quebec, Canada
  • 1998
    • Observatoire de Paris
      Lutetia Parisorum, Île-de-France, France
  • 1996–1998
    • McGill University Health Centre
      Montréal, Quebec, Canada
  • 1993–1996
    • French Institute of Health and Medical Research
      Lutetia Parisorum, Île-de-France, France
  • 1993–1995
    • Ecole normale supérieure de Lyon
      Lyons, Rhône-Alpes, France
  • 1992–1993
    • Université René Descartes - Paris 5
      Lutetia Parisorum, Île-de-France, France