Biomedical subjects

H De Rocquigny

Publications and source records attributed to H De Rocquigny.

14 recordsLinked to original sources

PubMed2000

Generation of monoclonal antibodies specifically directed against the proximal zinc finger of HIV type 1 NCp7.

The HIV-1 NCp7 contains two spatially close zinc fingers, required for the production of infectious particles. To investigate in more detail the function of the zinc finger domain, monoclonal antibodies were generated with a cyclic analog of the NCp7 proximal zinc finger. This analog was shown to bind zinc ions and to preserve the highly folded structure of the native peptide (Dong C-Z et al.: J Am Chem Soc 1995;117:2726-2731). We report here two monoclonal antibodies (2B10 and 4D3), which are the first monoclonal antibodies directed against CCHC NCp7 zinc fingers. Dot-blot experiments revealed that a few nanograms of synthetic NCp7 can be detected on a nitrocellulose membrane. Whereas 2B10 appears specific for an epitope located in sequence 19-27 of NCp7, 4D3 appears to be structurally specific. Immunocomplex affinities were evaluated, using BIAcore technology, to be up to 1 and 10 nM, respectively, for 2B10 and 4D3 in 100 mM NaCl. These antibodies were able to recognize NCp7 in the Gag polyprotein precursor and were shown to immunoprecipitate NCp7 from a cell supernatant. Moreover, NCp7-Vpr interaction mediated by the zinc fingers is inhibited by 2B10, emphasizing the role of these domains in the protein-protein complex. These results indicate that 2B10 and 4D3 behave as useful tools for studying both NC protein functions during the course of virion morphogenesis and the role played by its zinc finger domain at various steps in the retroviral life cycle.

Amino Acid Sequence↗

PubMed1999

Cleavage of L-leucine-containing dipeptides by Clostridium butyricum.

The ability of Clostridium butyricum cultures to hydrolyze three L-leucine-containing dipeptides (Leu-Leu, Leu-Gly and Gly-Leu) in a synthetic minimal medium is demonstrated by using gas chromatography coupled with mass spectrometry. The 13C nuclear magnetic resonance and a labeled dipeptide L-[1-13C]Leu-Gly were used to confirm this activity.

Carbon Isotopes↗

PubMed1997

Direct activation of protein phosphatase-2A0 by HIV-1 encoded protein complex NCp7:vpr.

The effects of HIV-1 encoded proteins NCp7, vpr and NCp7:vpr complex on the activity of protein phosphatase-2A0 have been tested. We report that NCp7 is an activator of protein phosphatase-2A0 and that vpr activated protein phosphatase-2A0 only slightly. We also report that NCp7 and vpr form a tight complex which becomes a more potent activator of protein phosphatase-2A0 than NCp7 alone. The ability of NCp7 to activate protein phosphatase-2A0 is regulated by vpr. The C-terminal portion of vpr prevents NCp7 from activating protein phosphatase-2A0 while the N-terminal portion of vpr potentiates the effect of NCp7 on the activity of protein phosphatase-2A0. Our findings indicate that vpr may be acting as a targeting subunit which directs NCp7 to activate protein phosphatase-2A0. In view of the fact that protein phosphatase-2A functions as an inhibitor of G0 to M transition of the cell cycle and is involved in other key cellular processes such as the control of RNA transcription, the results presented in this report may explain how HIV-1 causes cell cycle arrest which may lead to CD4+ T cell depletion and also how it disturbs normal cellular processes of its host cell.

Animals↗

PubMed1997

Ordered aggregation of ribonucleic acids by the human immunodeficiency virus type 1 nucleocapsid protein.

The nucleocapsid protein NCp7, which is the major genomic RNA binding protein of human immunodeficiency virus type 1, plays an important role in several key steps of the viral life cycle. Many of the NCp7 activities, notably the nucleic acid annealing and the genomic RNA wrapping ones, are thought to be linked to a nonspecific binding of NCp7 to its nucleic acid targets. The mechanism of these activities is still debated but several clues are in favor of an intermediate aggregation of nucleic acids by NCp7. To check and characterize the nucleic acid aggregating properties of NCp7, we investigated the interaction of NCp7 with the model RNA homopolymer, polyA, by quasielastic light scattering and optical density measurements. The ordered growth of monodisperse large particles independently of the nucleic acid size and the almost complete covering of polyA by NCp7 strongly suggested an ordered aggregation mechanism. The aggregate kinetics of growth in the optimum protein concentration range (> or = 2 microM) were governed by a so-called Ostwald ripening mechanism limited by transfer of NCp7-covered polyA complexes from small to large aggregates. The aggregation process was strongly dependent on both Na+ and Mg2+ concentrations, the optimum concentrations being in the physiological range. Similar conclusions held true when polyA was replaced by 16S + 23S ribosomal RNA, suggesting that the NCp7 aggregating properties were only poorly dependent on the nucleic acid sequence and structure. Finally, as in the NCp7 annealing activities, the basic regions of NCp7, but not the zinc fingers, were found critical in nucleic acid aggregation. Taken together, our data indicate that NCp7 is a highly efficient nucleic acid aggregating agent and strengthen the hypothesis that aggregation may constitute a transient step in various NCp7 functions.

Amino Acid Sequence↗

PubMed1997

Synthesis and biological activities of fluorescent acridine-containing HIV-1 nucleocapsid proteins for investigation of nucleic acid-NCp7 interactions.

Specific interactions between the 72-amino acid nucleocapsid protein NCp7 of the human immunodeficiency virus, type 1 and the genomic RNA are essential for virus replication. Studies on the mechanism of action of NCp7 require a direct visualization of its complexes with nucleic acids and the determination of binding affinities. To facilitate these investigations, fluorescent NCp7 derivatives were developed by introduction in the NCp7 sequence of a non-natural amino acid, (S)-beta-(9-acridinyl)alanine (Aca) obtained by a chiral synthetic method. Three fluorescent NCp7 derivatives were obtained by introducing this amino acid at different positions. As shown by NMR, the three-dimensional structure of NCp7 is not altered by introduction of Aca. The fluorescent peptides were found to be as potent as their precursors in interacting with nucleic acids and in promoting HIV-1 genomic RNA dimerization. Moreover, because of their fluorescent properties, these NCp7s can be used at submicromolar concentrations to directly visualize and quantify protein-nucleic acid interactions in solution or after gel electrophoresis. This could facilitate the development of new antiviral agents aimed at inhibiting the functions of NCp7 and studies on the intracellular traffic of NCp7 within the preintegration complex.

Acridines↗

PubMed1996

NCp7 activates HIV-1Lai RNA dimerization by converting a transient loop-loop complex into a stable dimer.

Nucleocapsid protein 7 (NCp7), the human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein, was shown to strongly potentiate the dimerization of the retroviral genomic RNA. This process involves the interaction of two retroviral RNA monomer subunits near their 5'-ends. A region located upstream from the splice donor site was recently identified as being responsible for the formation of dimeric HIV-1 RNA. This region appeared to be confined within a stem-loop structure, with an autocomplementary sequence in the loop. In an in vitro study of spontaneous dimer formation, we reported that the 77-402 RNA transcript forms two distinct dimers differing in their thermostability: D37 and D55. We identified D37 as a "kissing" complex structure, formed via a loop-loop interaction between the two monomers, and D55 as a double stranded structure involving all nucleotides of the stem-loop via canonical base pairing. In this report, we have characterized the role of NCp7 in the HIV-1Lai RNA dimerization process by using in vitro dimerization assays with RNA transcripts of different lengths and dimer thermal dissociation. Our results show that the nucleocapsid protein NCp7 activates RNA dimerization very likely through interaction with the kissing complex and converts it into a stable dimer. Furthermore, this NCp7-promoted conversion only occurs if the 240-280 stem-loop structure is present in HIV-1Lai RNA molecules and contains the autocomplementary G257CGCGC262 sequence. This study suggests that, under physiological conditions, an NCp7-mediated RNA conformational change is involved in the maturation of the HIV-1 RNA dimer.

Base Sequence↗

PubMed1996

Zinc binding to the HIV-1 nucleocapsid protein: a thermodynamic investigation by fluorescence spectroscopy.

The HIV-1 nucleocapsid protein, NCp7, is characterized by two CCHC zinc finger motifs which have been shown to stoichiometrically bind zinc in mature virions. Moreover, this binding of zinc proves to be critical in various NCp7 functions, especially in the encapsidation process. To further understand the central role of zinc binding to NCp7, we closely investigated the zinc binding properties of NCp7 and various deleted or substituted derivatives. To this end, the fluorescence of wither the naturally occurring Trp37 or the conservatively substituted Trp16 was used to monitor the binding of zinc to the N- and C-terminal finger motifs, respectively. At pH 7.5, the NCp7 proximal motif was found to bind zinc strongly with 2.8 x 10(14) M-1 binding constant about five times higher than the NCp7 distal motif. Moreover, the binding of zinc to one finger motif decreased the affinity of the second one, and this negative cooperativity was shown to be related to the spatial proximity of the zinc-saturated finger motifs. The binding seemed to be almost equally driven by entropy and enthalpy, and the binding information was essentially encoded by the finger motifs themselves whereas the other parts of the protein only played a marginal stabilization role. As expected, the Cys and His residues of the CCHC motifs were critical and competition between protons and zinc ions to these residues induced a steep pH-dependence of the zinc binding constants to both sites. Taken together, our data provide further evidence for the nonequivalence of the two NCp7 finger motifs.

Amino Acid Sequence↗

PubMed1994

Spatial proximity of the HIV-1 nucleocapsid protein zinc fingers investigated by time-resolved fluorescence and fluorescence resonance energy transfer.

The three-dimensional structure of peptides encompassing the two zinc-saturated finger motifs of the nucleocapsid protein NCp7 of HIV-1 has been reported by several groups. Whereas the folded structures of the finger motifs were in good agreement, discrepancies existed concerning their spatial relationship since the fingers were found either close to each other [Morellet, N., Jullian, N., De Rocquigny, H., Maigret, B., Darlix, J. L., & Roques, B. P. (1992) Embo J. 11, 3059-3065] or independently folded [Omichinski, J. G., Clore, G. M., Sakaguchi, K., Appella, E., & Gronenborn, A. M. (1991) FEBS Lett. 292, 25-30, Summers, M. F., Henderson, L. E., Chance, M. R., Bess, J. W., Jr., South, T. L., Blake, P. R., Sagi, I., Perez-Alvarado, G., Sowder, R.C., III, Hare, D.R., & Arthur, L. O. (1992) Protein Sci. 1, 563-574]. As in the interacting finger model, Phe16 in the NH2-terminal finger and Trp37 in the COOH-terminal finger were found to be spatially close, the fluorescence properties of the aromatic residues at positions 16 and 37 in the wild-type and two conservatively substituted (12-53) NCp7 peptides were investigated and compared with those of three negative control derivatives where the finger motifs were not in close contact. Direct distance measurements by Tyr-Trp fluorescence resonance energy transfer of the former derivatives yielded a 7-12 A interchromophore distance range which is clearly inconsistent with the 12.5-18 A range measured for the negative controls and thus a random orientation of the zinc finger motifs.(ABSTRACT TRUNCATED AT 250 WORDS)

Algorithms↗

PubMed1994

Monoclonal antibody-mediated inhibition of RNA binding and annealing activities of HIV type 1 nucleocapsid protein.

Retroviral nucleocapsid (NC) proteins are highly basic, with one or two zinc fingers, and are required for virion formation, genomic RNA dimerization and packaging, and replication primer tRNA annealing to the viral RNA. We report here the first characterization of monoclonal antibodies directed against a retroviral nucleocapsid protein and their use to study the structure-function relationships of the nucleocapsid protein NCp7 of HIV-1. Four anti-NCp7 monoclonal antibodies (MAbs) have been generated by using NCp7 of HIV-1. The epitope targets of these MAbs were mapped using ELISA and BIAcore techniques. Whereas three of them are specific for epitopes located in the N and C termini of NCp7, the fourth one appears to be conformation specific. Interestingly, only two of these MAbs, the conformation-specific one and the MAb recognizing an N-terminal epitope are able to inhibit the RNA-binding and annealing activities of NCp7 as well as strong-stop DNA synthesis in vitro. The binding of the two other MAbs onto NCp7 either has no effect or enhances the NCp7-RNA interactions. These MAbs also display a differential recognition of the Gag polyprotein precursor, which makes them useful tools for studying NC protein maturation in the course of virion morphogenesis.

Amino Acid Sequence↗

PubMed1993

Two short basic sequences surrounding the zinc finger of nucleocapsid protein NCp10 of Moloney murine leukemia virus are critical for RNA annealing activity.

The 56 amino acid nucleocapsid protein (NCp10) of Moloney Murine Leukemia Virus, contains a CysX2CysX4HisX4Cys zinc finger flanked by basic residues. In vitro NCp10 promotes genomic RNA dimerization, a process most probably linked to genomic RNA packaging, and replication primer tRNA(Pro) annealing to the initiation site of reverse transcription. To characterize the amino-acid sequences involved in the various functions of NCp10, we have synthesized by solid phase method the native protein and a series of derived peptides shortened at the N- or C-terminus with or without the zinc finger domain. In the latter case, the two parts of the protein were linked by a Glycine - Glycine spacer. The in vitro studies of these peptides show that nucleic acid annealing activities of NCp10 do not require a zinc finger but are critically dependent on the presence of specific sequences located on each side of the CCHC domain and containing proline and basic residues. Thus, deletion of 11R or 49PRPQT, of the fully active 29 residue peptide 11RQGGERRRSQLDRDGGKKPRGPRGPRPQT53 leads to a complete loss of NCp10 activity. Therefore it is proposed that in NCp10, the zinc finger directs the spatial recognition of the target RNAs by the basic domains surrounding the zinc finger.

Amino Acid Sequence↗

PubMed1993

Interactions between HIV-1 nucleocapsid protein and viral DNA may have important functions in the viral life cycle.

In the virion core of retroviruses, the genomic RNA is tightly associated with nucleocapsid (NC) protein molecules, forming the nucleocapsid structure. NC protein, a highly basic protein with two zinc fingers, is indispensable for RNA dimerization, encapsidation and the initiation of reverse transcription in avian, murine and human retroviruses. Here we show that NC protein of HIV-1 (NCp7) and NCp7 mutants bind to DNA fragments representing proviral DNA sequences, forming stable complexes. NCp7 and NCp7 mutants form high molecular weight complexes with large DNA fragments and show cooperativity in binding to the DNAs. It appears that the conserved basic residues, and not the zinc fingers, are important for complex formation. In addition, NCp7 and several NCp7 mutants protect DNAs from nuclease digestion while the DNA ends appear to be poorly protected. NCp7 has been found to bind to strong stop cDNA, the initial product of reverse transcription, and to promote the annealing of this cDNA to HIV-1 RNA corresponding to the 3' end of the genome. In addition, NCp7 slightly stimulates an in vitro IN cleavage assay. These results indicate that the interactions between NCp7 and proviral DNA may be important during provirus synthesis and/or prior to integration.

Amino Acid Sequence↗

PubMed1993

Basic amino acids flanking the zinc finger of Moloney murine leukemia virus nucleocapsid protein NCp10 are critical for virus infectivity.

Nucleocapsid (NC) protein NCp10 of Moloney murine leukemia virus is encoded by the 3' domain of gag and contains a zinc finger surrounded by basic amino acids. During virion assembly, NC protein is necessary for core formation and the NC zinc finger is required for the packaging of the genomic RNA dimer. In vitro NCp10 has RNA-binding and -annealing activities critical for virus infectivity, since NCp10 promotes dimerization of viral RNA containing the Psi packaging element and annealing of replication primer tRNA(Pro) to the initiation site of reverse transcription (primer-binding site). To investigate the role of the basic amino acids flanking the NCp10 zinc finger, neutral residues were substituted for the basic amino acids and the effects of these mutations in vivo on virus assembly and infectivity and in vitro on the RNA-annealing activity of NCp10 were analyzed. Here we report that the substitution of 1 or 2 neutral amino acids for the basic residues did not impair the production of mature virions but that infectivity was either moderately or strongly attenuated. When more than 2 basic residues were replaced by neutral amino acids, viruses were poorly infectious because of a severe defect in genomic RNA dimer packaging and initiation of reverse transcription. In vitro NCp10-derived peptides with similar mutations were chemically synthesized and were found to be either fully or partially active or completely inactive. These data indicate that the basic residues flanking the zinc finger of NCp10 are required for the production of infectious Moloney murine leukemia virus virions.

3T3 Cells↗

PubMed1992

Viral RNA annealing activities of human immunodeficiency virus type 1 nucleocapsid protein require only peptide domains outside the zinc fingers.

The nucleocapsid (NC) of human immunodeficiency virus type 1 consists of a large number of NC protein molecules, probably wrapping the dimeric RNA genome within the virion inner core. NC protein is a gag-encoded product that contains two zinc fingers flanked by basic residues. In human immunodeficiency virus type 1 virions, NCp15 is ultimately processed into NCp7 and p6 proteins. During virion assembly the retroviral NC protein is necessary for core formation and genomic RNA encapsidation, which are essential for virus infectivity. In vitro NCp15 activates viral RNA dimerization, a process most probably linked in vivo to genomic RNA packaging, and replication primer tRNA(Lys,3) annealing to the initiation site of reverse transcription. To characterize the domains of human immunodeficiency virus type 1 NC protein necessary for its various functions, the 72-amino acid NCp7 and several derived peptides were synthesized in a pure form. We show here that synthetic NCp7 with or without the two zinc fingers has the RNA annealing activities of NCp15. Further deletions of the N-terminal 12 and C-terminal 8 amino acids, leading to a 27-residue peptide lacking the finger domains, have little or no effect on NC protein activity in vitro. However deletion of short sequences containing basic residues flanking the first finger leads to a complete loss of NC protein activity. It is proposed that the basic residues and the zinc fingers cooperate to select and package the genomic RNA in vivo. Inhibition of the viral RNA binding and annealing activities associated with the basic residues flanking the first zinc finger of NC protein could therefore be used as a model for the design of antiviral agents.

Amino Acid Sequence↗

PubMed1992

Determination of the structure of the nucleocapsid protein NCp7 from the human immunodeficiency virus type 1 by 1H NMR.

The retroviral gag nucleocapsid protein NCp7 (72 amino acids) of HIV-1 (LAV strain), which contains two successive zinc fingers of the Cys-X2-Cys-X4-His-X4-Cys form linked by a stretch of basic residues, promotes viral RNA dimerization and encapsidation and activates annealing of the primer tRNA to the initiation site of reverse transcription. The structure of NCp7 and other shorter fragments was studied by 600 MHz 1H nuclear magnetic resonance (NMR) in aqueous solution to account for its various biological properties. Complete sequence specific 1H NMR assignments of the 13-51 residues of NCp7 encompassing the two zinc fingers was achieved by two-dimensional NMR experiments and the three-dimensional structure of (13-51)NCp7 was deduced from DIANA calculations, using nuclear Overhauser effects as constraints. The structure of the zinc complexed form of NCp7 is characterized by a kink at the Pro31 level in the basic Arg29-Ala-Pro-Arg-Lys-Lys-Gly35 RNA binding linker leading to a proximity of the Lys14-Cys18 to the Gly35-Cys39 sequences, which belong to the folded proximal and distal zinc fingers, respectively. Accordingly, the aromatic residues Phe16 and Trp37 were found to be spatially close. The Lys33 and Lys34 side-chains involved in viral RNA dimerization were solvent exposed. The N- and C-terminal sequences of NCp7 behave as flexible independent domains. The proposed structure of NCp7 might be used to rationally design new anti-viral agents aimed at inhibiting its functions.

Amino Acid Sequence↗