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1.
Sci Rep ; 10(1): 4040, 2020 03 04.
Article de Anglais | MEDLINE | ID: mdl-32132633

RÉSUMÉ

Flaviviridae infections represent a major global health burden. By deciphering mechanistic aspects of hepatitis C virus (HCV)-host interactions, one could discover common strategy for inhibiting the replication of related flaviviruses. By elucidating the HCV interactome, we identified the 17-beta-hydroxysteroid dehydrogenase type 12 (HSD17B12) as a human hub of the very-long-chain fatty acid (VLCFA) synthesis pathway and core interactor. Here we show that HSD17B12 knockdown (KD) impairs HCV replication and reduces virion production. Mechanistically, depletion of HSD17B12 induces alterations in VLCFA-containing lipid species and a drastic reduction of lipid droplets (LDs) that play a critical role in virus assembly. Oleic acid supplementation rescues viral RNA replication and production of infectious particles in HSD17B12 depleted cells, supporting a specific role of VLCFA in HCV life cycle. Furthermore, the small-molecule HSD17B12 inhibitor, INH-12, significantly reduces replication and infectious particle production of HCV as well as dengue virus and Zika virus revealing a conserved requirement across Flaviviridae virus family. Overall, the data provide a strong rationale for the advanced evaluation of HSD17B12 inhibition as a promising broad-spectrum antiviral strategy for the treatment of Flaviviridae infections.


Sujet(s)
17-Hydroxysteroid dehydrogenases/métabolisme , Hepacivirus/physiologie , Hépatite C/enzymologie , Acide oléique/pharmacologie , Réplication virale/effets des médicaments et des substances chimiques , 17-Hydroxysteroid dehydrogenases/génétique , Animaux , Chlorocebus aethiops , Cellules HeLa , Cellules HepG2 , Hépatite C/génétique , Humains , Cellules Vero , Réplication virale/génétique
2.
Traffic ; 18(6): 362-377, 2017 06.
Article de Anglais | MEDLINE | ID: mdl-28295920

RÉSUMÉ

In this study, newly identified host interactors of hepatitis C virus (HCV) proteins were assessed for a role in modulating the innate immune response. The analysis revealed enrichment for components of the nuclear transport machinery and the crucial interaction with NS3/4A protein in suppression of interferon-ß (IFNB1) induction. Using a comprehensive microscopy-based high-content screening approach combined to the gene silencing of nuclear transport factors, we showed that NS3/4A-interacting proteins control the nucleocytoplasmic trafficking of IFN regulatory factor 3 (IRF3) and NF-κB p65 upon Sendai virus (SeV) infection. Notably, importin ß1 (IMPß1) knockdown-a hub protein highly targeted by several viruses-decreases the nuclear translocation of both transcription factors and prevents IFNB1 and IFIT1 induction, correlating with a rapid increased of viral proteins and virus-mediated apoptosis. Here we show that NS3/4A triggers the cleavage of IMPß1 and inhibits nuclear transport to disrupt IFNB1 production. Importantly, mutated IMPß1 resistant to cleavage completely restores signaling, similar to the treatment with BILN 2061 protease inhibitor, correlating with the disappearance of cleavage products. Overall, the data indicate that HCV NS3/4A targeting of IMPß1 and related modulators of IRF3 and NF-κB nuclear transport constitute an important innate immune subversion strategy and inspire new avenues for broad-spectrum antiviral therapies.


Sujet(s)
Protéines de transport/métabolisme , Facteur-3 de régulation d'interféron/métabolisme , Facteur de transcription NF-kappa B/métabolisme , Transduction du signal , Protéines virales non structurales/métabolisme , Caryophérines bêta/métabolisme , Protéines adaptatrices de la transduction du signal/immunologie , Antiviraux/pharmacologie , Hepacivirus , Humains , Protéines et peptides de signalisation intracellulaire , Caryophérines/métabolisme , Inhibiteurs de protéases/pharmacologie
3.
PLoS Pathog ; 13(1): e1006174, 2017 Jan.
Article de Anglais | MEDLINE | ID: mdl-28118396

RÉSUMÉ

[This corrects the article DOI: 10.1371/journal.ppat.1005772.].

4.
Cell Rep ; 17(2): 425-435, 2016 10 04.
Article de Anglais | MEDLINE | ID: mdl-27705791

RÉSUMÉ

Functional genomic analysis of gene expression in mice allowed us to identify a quantitative trait locus (QTL) linked in trans to the expression of 190 gene transcripts and in cis to the expression of only two genes, one of which was Ypel5. Most of the trans-expression QTL genes were interferon-stimulated genes (ISGs), and their expression in mouse macrophage cell lines was stimulated in an IFNB1-dependent manner by Ypel5 silencing. In human HEK293T cells, YPEL5 silencing enhanced the induction of IFNB1 by pattern recognition receptors and phosphorylation of TBK1/IKBKE kinases, whereas co-immunoprecipitation experiments revealed that YPEL5 interacted physically with IKBKE. We thus found that the Ypel5 gene (contained in a locus linked to a network of ISGs in mice) is a negative regulator of IFNB1 production and innate immune responses that interacts functionally and physically with TBK1/IKBKE kinases.


Sujet(s)
Protéines du cycle cellulaire/génétique , I-kappa B Kinase/génétique , Interféron bêta-1a/génétique , Interférons/génétique , Protein-Serine-Threonine Kinases/génétique , Animaux , Cellules HEK293 , Humains , Macrophages/métabolisme , Souris , Liaison aux protéines , Locus de caractère quantitatif/génétique , Éléments de régulation transcriptionnelle/génétique
5.
PLoS Pathog ; 12(7): e1005772, 2016 07.
Article de Anglais | MEDLINE | ID: mdl-27454487

RÉSUMÉ

Spliceosomal SNRNP200 is a Ski2-like RNA helicase that is associated with retinitis pigmentosa 33 (RP33). Here we found that SNRNP200 promotes viral RNA sensing and IRF3 activation through the ability of its amino-terminal Sec63 domain (Sec63-1) to bind RNA and to interact with TBK1. We show that SNRNP200 relocalizes into TBK1-containing cytoplasmic structures upon infection, in contrast to the RP33-associated S1087L mutant, which is also unable to rescue antiviral response of SNRNP200 knockdown cells. This functional rescue correlates with the Sec63-1-mediated binding of viral RNA. The hindered IFN-ß production of knockdown cells was further confirmed in peripheral blood cells of RP33 patients bearing missense mutation in SNRNP200 upon infection with Sendai virus (SeV). This work identifies a novel immunoregulatory role of the spliceosomal SNRNP200 helicase as an RNA sensor and TBK1 adaptor for the activation of IRF3-mediated antiviral innate response.


Sujet(s)
Immunité innée/immunologie , Facteur-3 de régulation d'interféron/immunologie , ARN viral/immunologie , Petites ribonucléoprotéines nucléaires/immunologie , Maladies virales/immunologie , Technique de Western , Test ELISA , Technique d'immunofluorescence , Techniques de knock-down de gènes , Humains , Immunoprécipitation , Séquençage par oligonucléotides en batterie , Réaction de polymérisation en chaîne , Protein-Serine-Threonine Kinases/immunologie , Transduction du signal/immunologie , Splicéosomes/immunologie
6.
Mol Cell Proteomics ; 13(1): 184-203, 2014 Jan.
Article de Anglais | MEDLINE | ID: mdl-24169621

RÉSUMÉ

More than 170 million people worldwide are infected with the hepatitis C virus (HCV), for which future therapies are expected to rely upon a combination of oral antivirals. For a rapidly evolving virus like HCV, host-targeting antivirals are an attractive option. To decipher the role of novel HCV-host interactions, we used a proteomics approach combining immunoprecipitation of viral-host protein complexes coupled to mass spectrometry identification and functional genomics RNA interference screening of HCV partners. Here, we report the proteomics analyses of protein complexes associated with Core, NS2, NS3/4A, NS4B, NS5A, and NS5B proteins. We identified a stringent set of 98 human proteins interacting specifically with one of the viral proteins. The overlap with previous virus-host interaction studies demonstrates 24.5% shared HCV interactors overall (24/98), illustrating the reliability of the approach. The identified human proteins show enriched Gene Ontology terms associated with the endoplasmic reticulum, transport proteins with a major contribution of NS3/4A interactors, and transmembrane proteins for Core interactors. The interaction network emphasizes a high degree distribution, a high betweenness distribution, and high interconnectivity of targeted human proteins, in agreement with previous virus-host interactome studies. The set of HCV interactors also shows extensive enrichment for known targets of other viruses. The combined proteomic and gene silencing study revealed strong enrichment in modulators of HCV RNA replication, with the identification of 11 novel cofactors among our set of specific HCV partners. Finally, we report a novel immune evasion mechanism of NS3/4A protein based on its ability to affect nucleocytoplasmic transport of type I interferon-mediated signal transducer and activator of transcription 1 nuclear translocation. The study revealed highly stringent association between HCV interactors and their functional contribution to the viral replication cycle and pathogenesis.


Sujet(s)
Hepacivirus/génétique , Interactions hôte-pathogène/génétique , Protéomique , Protéines virales/biosynthèse , Génomique , Humains , Spectrométrie de masse , Protéines membranaires/biosynthèse , Protéines membranaires/métabolisme , Interférence par ARN
7.
J Virol ; 87(21): 11704-20, 2013 Nov.
Article de Anglais | MEDLINE | ID: mdl-23986595

RÉSUMÉ

Hepatitis C virus (HCV) orchestrates the different stages of its life cycle in time and space through the sequential participation of HCV proteins and cellular machineries; hence, these represent tractable molecular host targets for HCV elimination by combination therapies. We recently identified multifunctional Y-box-binding protein 1 (YB-1 or YBX1) as an interacting partner of NS3/4A protein and HCV genomic RNA that negatively regulates the equilibrium between viral translation/replication and particle production. To identify novel host factors that regulate the production of infectious particles, we elucidated the YB-1 interactome in human hepatoma cells by a quantitative mass spectrometry approach. We identified 71 YB-1-associated proteins that included previously reported HCV regulators DDX3, heterogeneous nuclear RNP A1, and ILF2. Of the potential YB-1 interactors, 26 proteins significantly modulated HCV replication in a gene-silencing screening. Following extensive interaction and functional validation, we identified three YB-1 partners, C1QBP, LARP-1, and IGF2BP2, that redistribute to the surface of core-containing lipid droplets in HCV JFH-1-expressing cells, similarly to YB-1 and DDX6. Importantly, knockdown of these proteins stimulated the release and/or egress of HCV particles without affecting virus assembly, suggesting a functional YB-1 protein complex that negatively regulates virus production. Furthermore, a JFH-1 strain with the NS3 Q221L mutation, which promotes virus production, was less sensitive to this negative regulation, suggesting that this HCV-specific YB-1 protein complex modulates an NS3-dependent step in virus production. Overall, our data support a model in which HCV hijacks host cell machinery containing numerous RNA-binding proteins to control the equilibrium between viral RNA replication and NS3-dependent late steps in particle production.


Sujet(s)
Hepacivirus/physiologie , Interactions hôte-pathogène , ARN viral/métabolisme , Protéines virales non structurales/métabolisme , Réplication virale , Protéine-1 de liaison à la boîte Y/métabolisme , Lignée cellulaire , Extinction de l'expression des gènes , Hépatocytes/composition chimique , Humains , Structures macromoléculaires , Spectrométrie de masse , Protéines mutantes/génétique , Protéines mutantes/métabolisme , Mutation faux-sens , Liaison aux protéines , Multimérisation de protéines , Protéines virales non structurales/génétique
8.
PLoS Pathog ; 9(6): e1003416, 2013.
Article de Anglais | MEDLINE | ID: mdl-23785285

RÉSUMÉ

To identify new regulators of antiviral innate immunity, we completed the first genome-wide gene silencing screen assessing the transcriptional response at the interferon-ß (IFNB1) promoter following Sendai virus (SeV) infection. We now report a novel link between WNT signaling pathway and the modulation of retinoic acid-inducible gene I (RIG-I)-like receptor (RLR)-dependent innate immune responses. Here we show that secretion of WNT2B and WNT9B and stabilization of ß-catenin (CTNNB1) upon virus infection negatively regulate expression of representative inducible genes IFNB1, IFIT1 and TNF in a CTNNB1-dependent effector mechanism. The antiviral response is drastically reduced by glycogen synthase kinase 3 (GSK3) inhibitors but restored in CTNNB1 knockdown cells. The findings confirm a novel regulation of antiviral innate immunity by a canonical-like WNT/CTNNB1 signaling pathway. The study identifies novel avenues for broad-spectrum antiviral targets and preventing immune-mediated diseases upon viral infection.


Sujet(s)
Glycoprotéines/immunologie , Immunité innée , Protéines et peptides de signalisation intracellulaire/immunologie , Infections à respirovirus/immunologie , Virus Sendai/immunologie , Protéines de type Wingless/immunologie , Voie de signalisation Wnt/immunologie , Protéines adaptatrices de la transduction du signal , Protéines de transport/immunologie , Protéines de transport/métabolisme , Lignée cellulaire , Protéine-58 à domaine DEAD , DEAD-box RNA helicases/immunologie , DEAD-box RNA helicases/métabolisme , Femelle , Régulation de l'expression des gènes/génétique , Régulation de l'expression des gènes/immunologie , Étude d'association pangénomique , Glycoprotéines/métabolisme , Humains , Interféron bêta/immunologie , Interféron bêta/métabolisme , Protéines et peptides de signalisation intracellulaire/métabolisme , Mâle , Interférence par ARN , Protéines de liaison à l'ARN , Récepteurs immunologiques , Infections à respirovirus/métabolisme , Infections à respirovirus/anatomopathologie , Virus Sendai/métabolisme , Protéines de type Wingless/métabolisme
9.
Results Immunol ; 3: 17-25, 2013.
Article de Anglais | MEDLINE | ID: mdl-24600555

RÉSUMÉ

In addition to their classical antigen presenting functions, MHC class II molecules potentiate the TLR-triggered production of pro-inflammatory cytokines. Here, we have addressed the effect of Tollip and MARCH1 on the regulation of MHC II trafficking and TLR signaling. Our results show that MARCH1-deficient mice splenocytes are impaired in their capacity to produce pro-inflammatory cytokines in response to poly(I:C) and that TLR3 and MHC II molecules interact in the endocytic pathway. Knocking down Tollip expression in human CIITA(+) HeLa cells increased expression of HLA-DR but reduced the proportion of MHC II molecules associated with the CLIP peptide. Truncation of the HLA-DR cytoplasmic tails abrogated the effect of Tollip on MHC class II expression. While overexpression of Tollip did not affect HLA-DR levels, it antagonized the function of co-transfected MARCH1. We found that Tollip strongly reduced MARCH1 protein levels and that the two molecules appear to compete for binding to MHC II molecules. Altogether, our results demonstrate that Tollip regulates MHC class II trafficking and that MARCH1 may represent a new Tollip target.

10.
Curr Opin Virol ; 2(5): 622-8, 2012 Oct.
Article de Anglais | MEDLINE | ID: mdl-23017246

RÉSUMÉ

Interferons (IFNs) have long been used as an immunomodulatory therapy for a large array of acute and chronic viral infections. However, IFN therapies have been plagued by severe side effects. The discovery of pathogen recognition receptors (PRR) rejuvenated the interest for immunomodulatory therapies. The successes obtained with Toll-like receptor (TLR) agonists in activating immune cells and as adjuvant for prophylactic vaccines against different viruses paved the way to targeted immunomodulatory therapy. Better characterization of pathogen-induced immune disorders and newly discovered regulators of innate immunity have now the potential to specifically withdraw prevailing subversion mechanisms and to transform antiviral treatments by introducing panviral therapeutics with less adverse effects than IFN therapies.


Sujet(s)
Antiviraux/pharmacologie , Immunité innée/effets des médicaments et des substances chimiques , Maladies virales/traitement médicamenteux , Maladies virales/immunologie , Virus/effets des médicaments et des substances chimiques , Animaux , Évaluation préclinique de médicament , Humains , Interférons/effets indésirables , Interférons/usage thérapeutique , Récepteurs de type Toll , Maladies virales/virologie , Virus/génétique , Virus/immunologie
11.
J Virol ; 85(21): 11022-37, 2011 Nov.
Article de Anglais | MEDLINE | ID: mdl-21849455

RÉSUMÉ

The hepatitis C virus (HCV) NS3/4A protein has several essential roles in the virus life cycle, most probably through dynamic interactions with host factors. To discover cellular cofactors that are co-opted by HCV for its replication, we elucidated the NS3/4A interactome using mass spectrometry and identified Y-box-binding protein 1 (YB-1) as an interacting partner of NS3/4A protein and HCV genomic RNA. Importantly, silencing YB-1 expression decreased viral RNA replication and severely impaired the propagation of the infectious HCV molecular clone JFH-1. Immunofluorescence studies further revealed a drastic HCV-dependent redistribution of YB-1 to the surface of the lipid droplets, an important organelle for HCV assembly. Core and NS3 protein-dependent polyprotein maturation were shown to be required for YB-1 relocalization. Unexpectedly, YB-1 knockdown cells showed the increased production of viral infectious particles while HCV RNA replication was impaired. Our data support that HCV hijacks YB-1-containing ribonucleoparticles and that YB-1-NS3/4A-HCV RNA complexes regulate the equilibrium between HCV RNA replication and viral particle production.


Sujet(s)
Protéines de transport/métabolisme , Hepacivirus/physiologie , Interactions hôte-pathogène , ARN viral/métabolisme , Protéines virales non structurales/métabolisme , Réplication virale , Protéine-1 de liaison à la boîte Y/métabolisme , Extinction de l'expression des gènes , Protéines et peptides de signalisation intracellulaire , Spectrométrie de masse , Microscopie de fluorescence , Protéine-1 de liaison à la boîte Y/génétique
12.
Viruses ; 2(8): 1752-1765, 2010 Aug.
Article de Anglais | MEDLINE | ID: mdl-21994705

RÉSUMÉ

Hepatitis C virus (HCV) infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease) that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection.

13.
J Virol ; 83(3): 1299-311, 2009 Feb.
Article de Anglais | MEDLINE | ID: mdl-19036819

RÉSUMÉ

The mitochondrial antiviral signaling (MAVS) protein plays a central role in innate antiviral immunity. Upon recognition of a virus, intracellular receptors of the RIG-I-like helicase family interact with MAVS to trigger a signaling cascade. In this study, we investigate the requirement of the MAVS structure for enabling its signaling by structure-function analyses and resonance energy transfer approaches in live cells. We now report the essential role of the MAVS oligomer in signal transduction and map the transmembrane domain as the main determinant of dimerization. A combination of mutagenesis and computational methods identified a cluster of residues making favorable van der Waals interactions at the MAVS dimer interface. We also correlated the activation of IRF3 and NF-kappaB with MAVS oligomerization rather than its mitochondrial localization. Finally, we demonstrated that MAVS oligomerization is disrupted upon expression of HCV NS3/4A protease, suggesting a mechanism for the loss of antiviral signaling. Altogether, our data suggest that the MAVS oligomer is essential in the formation of a multiprotein membrane-associated signaling complex and enables downstream activation of IRF3 and NF-kappaB in antiviral innate immunity.


Sujet(s)
Protéines de transport/métabolisme , Immunité innée , Transduction du signal , Protéines virales non structurales/métabolisme , Protéines virales/métabolisme , Séquence nucléotidique , Protéines de transport/génétique , Lignée cellulaire , Amorces ADN , Dimérisation , Transfert d'énergie par résonance de fluorescence , Humains , Immunoprécipitation , Protéines et peptides de signalisation intracellulaire , Mutagenèse , Protéines virales non structurales/génétique , Protéines virales/génétique
14.
Eur J Immunol ; 38(5): 1225-30, 2008 May.
Article de Anglais | MEDLINE | ID: mdl-18389477

RÉSUMÉ

IL-10 is a potent anti-inflammatory cytokine interfering with antigen presentation by inducing the intracellular sequestration of MHC class II (MHC-II) molecules. Here we studied the contribution of membrane-associated RING-CH (MARCH) ubiquitin ligase family members to the IL-10-induced down-regulation of MHC-II molecules. We found that MARCH1 and MARCH8 proteins are the most potent family members for the down-regulation of MHC-II surface expression in transfected cells, but only MARCH1 mRNA expression is strongly induced by IL-10 in human primary monocytes. We detected mono- and poly-ubiquitinated forms of MHC-II molecules both in IL-10-treated monocytes and in cells transfected with MARCH1. We also show direct interaction between MHC-II and MARCH1 molecules in co-immunoprecipitation assays. Finally, we found that siRNA-mediated knockdown of MARCH1 reverses IL-10-induced MHC-II down-regulation in primary monocytes. Thus, the immunosuppressive effect of IL-10 on antigen presentation is mediated through induced expression of MARCH1.


Sujet(s)
Antigènes HLA-D/métabolisme , Interleukine-10/physiologie , Monocytes/métabolisme , Ubiquitin-protein ligases/physiologie , Antigène CD86/métabolisme , Régulation négative , Expression des gènes/effets des médicaments et des substances chimiques , Antigènes HLA-DR/métabolisme , Cellules HeLa , Humains , Interféron gamma/pharmacologie , Interleukine-10/pharmacologie , Protéines membranaires/métabolisme , Monocytes/effets des médicaments et des substances chimiques , Protéines nucléaires/génétique , Liaison aux protéines , Petit ARN interférent/génétique , Transactivateurs/génétique , Ubiquitin-protein ligases/génétique , Ubiquitination/immunologie
15.
Inorg Chem ; 46(21): 8652-61, 2007 Oct 15.
Article de Anglais | MEDLINE | ID: mdl-17850141

RÉSUMÉ

Five new antimony(III) complexes with the heterocyclic thiones 2-mercapto-benzimidazole (MBZIM), 5-ethoxy-2-mercapto-benzimidazole (EtMBZIM), and 2-mercapto-thiazolidine (MTZD) of formulas {[SbCl(2)(MBZIM)4]+.Cl-.2H(2)O. (CH(3)OH)} (1), {[SbCl(2)(MBZIM)4]+.Cl-.3H(2)O.(CH3CN)} (2), [SbCl(3)(MBZIM)2] (3), [SbCl(3)(EtMBZIM)(2)] (4), and [SbCl(3)(MTZD)2] (5) have been synthesized and characterized by elemental analysis, FT-IR, far-FT-IR, differential thermal analysis-thermogravimetry, X-ray diffraction, and conductivity measurements. Complex {[SbCl2(tHPMT)(2)]+Cl-}, (tHPMT = 2-mercapto-3,4,5,6-tetrahydro-pyrimidine), already known, was also prepared, and its X-ray crystal structure was solved. It is shown that the complex is better described as {[SbCl3(tHPMT)(2)]} (6). Crystal structures of all other complexes (1-5) have also been determined by X-ray diffraction at ambient conditions. The crystal structure of the hydrated ligand, EtMBZIM.H2O is also reported. Compound [C(28)H(24)Cl(2)N(8)S(4)Sb.2H(2)O.Cl.(CH(3)OH)] (1) crystallizes in space group P2(1), with a = 7.7398(8) A, b = 16.724(3) A, c = 13.717(2) A, beta = 98.632(11) degrees, and Z = 2. Complex [C(28)H(24)Cl(2)N(8)S(4)S(b).Cl.3H(2)O.(CH(3)CN)] (2) corresponds to space group P2(1), with a = 7.8216(8) A, b = 16.7426(17) A, c = 13.9375(16) A, beta = 99.218(10) degrees , and Z = 2. In both 1 and 2 complexes, four sulfur atoms from thione ligands and two chloride ions form an octahedral (Oh) cationic [SbS(4)Cl(2)]+ complex ion, where chlorides lie at axial positions. A third chloride counteranion neutralizes it. Complexes 1 and 2 are the first examples of antimony(III) compounds with positively charged Oh geometries. Compound [C(14)H(12)Cl(3)N(4)S(2)S(b)] (3) crystallizes in space group P, with a = 7.3034(5) A, b = 11.2277(7) A, c = 12.0172(8) A, alpha = 76.772(5) degrees, beta = 77.101(6) degrees, gamma = 87.450(5) degrees, and Z = 2. Complex [C(18)H(20)Cl(3)N(4)O(2)S(2)S(b)] (4) crystallizes in space group P1, with a = 8.6682(6) A, b = 10.6005(7) A, c = 13.0177(9) A, alpha = 84.181(6) degrees, beta = 79.358(6) degrees, gamma = 84.882(6) degrees, and Z = 2, while complex [C(6)H(10)Cl(3)N(2)S(4)S(b)] (5) in space group P2(1)/c shows a = 8.3659(10) A, b = 14.8323(19) A, c = 12.0218(13) A, beta = 99.660(12) degrees, and Z = 4 and complex [C(8)H(16)Cl(3)N(4)S(2)S(b)] (6) in space group P1 shows a = 7.4975(6) A, b = 10.3220(7) A, c = 12.1094(11) A, alpha = 71.411(7) degrees, beta = 84.244(7) degrees, gamma = 73.588(6) degrees, and Z = 2. Crystals of complexes 3-6 grown from acetonitrile solutions adopt a square-pyramidal (SP) geometry, with two sulfur atoms from thione ligands and three chloride anions around Sb(III). The equatorial plane is formed by two sulfur and two chloride atoms in complexes 3-5, in a cis-S, cis-Cl arrangement in 3 and 5 and a trans-S, trans-Cl arrangement in 4. Finally, in the case of 6, the equatorial plane is formed by three chloride ions and one sulfur from the thione ligand while the second sulfur atom takes an axial position leading to a unique SP conformation. The complexes showed a moderate cytostatic activity against tumor cell lines.


Sujet(s)
Antimoine/composition chimique , Chimie/méthodes , Solvants/composition chimique , Thiones/analyse , Thiones/composition chimique , Animaux , Anisotropie , Lignée cellulaire tumorale , Cristallographie aux rayons X/méthodes , Électrochimie/méthodes , Cellules HeLa , Humains , Concentration inhibitrice 50 , Souris , Modèles chimiques , Modèles théoriques , Spectrophotométrie IR/méthodes , Diffraction des rayons X
16.
Inorg Chem ; 46(4): 1187-95, 2007 Feb 19.
Article de Anglais | MEDLINE | ID: mdl-17291115

RÉSUMÉ

Organotin(IV) complexes with the formulas [(C6H5)3Sn(mbzt)] (1), [(C6H5)3Sn(cmbzt)] (3), and [(C6H5)2Sn(cmbzt)2] (4) (Hmbzt = 2-mercaptobenzothiazole and Hcmbzt = 5-chloro-2-mercaptobenzothiazole) have been synthesized and characterized by elemental analysis; FT-IR, Raman, 1H, 13C, and 119Sn NMR, and Mössbauer spectroscopic techniques; and X-ray crystallography at various temperatures. The crystal structures of complexes 1, 3, and 4 were determined by X-ray diffraction at room temperature [295(1) or 293(2) K]. The complexes [(C6H5)3Sn(mbzo)] (2) and [(n-C4H9)2Sn(cmbzt)2] (5) (Hmbzo = 2-mercaptobenzoxazole) were synthesized by new improved methods, and their structures were determined at low temperature [100(1) K] and compared to those solved at room temperature. Comparison with {(CH3)2Sn(cmbzt)2]} (6), already reported, was also attempted. The influence of temperature on the geometry of the complexes is discussed. In the cases of complexes 1-3, three carbon atoms from phenyl groups and one sulfur atom and one nitrogen atom from thione ligands form a tetrahedrally distorted trigonal-bipyramidal geometry around the five-coordinate tin(IV) ion. In complexes 4-6, two carbon atoms from aryl groups and two sulfur atoms and two nitrogen atoms from thione ligands form a distorted tetrahedral geometry, tending toward octahedral, around the six-coordinate tin(IV) ions, with trans-C2, cis-N2, and cis-S2 configurations. Although the C-Sn and S-Sn bond distances are found to be constant in compounds 1-6, their N-Sn bond lengths vary significantly (from 2.635 to 3.078 A), with the longer distances found in the cases of five-coordinate complexes 1-3.


Sujet(s)
Benzothiazoles/composition chimique , Composés organiques de l'étain/synthèse chimique , Antifongiques/composition chimique , Benzène/composition chimique , Carbone/composition chimique , Chlore/composition chimique , Cristallographie aux rayons X , Ligands , Azote/composition chimique , Analyse spectrale , Stéréoisomérie , Soufre/composition chimique , Température , Thioamides/composition chimique
17.
Chemistry ; 12(26): 6888-97, 2006 Sep 06.
Article de Anglais | MEDLINE | ID: mdl-16773663

RÉSUMÉ

Four selenium analogues of the antithyroid drug 6-n-propyl-2-thiouracil (PTU), of formulae RSeU, (R = methyl (Me) (1), ethyl (Et) (2), n-propyl (nPr) (3), and isopropyl (iPr) 4), have been synthesized. Reaction of 1-4 with diiodine in a 1:1 molar ratio in dichloromethane results in the formation of [(RSeU)I(2)] (R = methyl (5), ethyl (6), n-propyl (7) and isopropyl (8)). All compounds have been characterized by elemental analysis, FT-Raman, FT-IR, UV/Vis, (1)H-, (13)C-, (77)Se-1D and -2D NMR spectroscopy, and ESI-MS spectrometric techniques. Recrystallization of 4 from dichloromethane afforded (4CH(2)Cl(2)). Crystals of [(nPrSeU)I(2)] (7), a charge-transfer complex, were obtained from chloroform solutions, while crystallization of 6 and 7 from acetone afforded the diselenides [N-(6-Et-4-pyrimidone)(6-EtSeU)(2)] (92 H(2)O) and [N-(6-nPr-4-pyrimidone)(6-nPrSeU)(2)] (10) as oxidation products. Recrystallization of 7 from methanol/acetonitrile solutions led to deselenation with the formation of 6-n-propyl-2-uracil (nPrU) (11). [(nPrSeU)I(2)] (7) was found to be a charge-transfer complex with a Se--I bond. These results are discussed in relation to the mechanism of action of antithyroid drugs.


Sujet(s)
Antithyroïdiens/synthèse chimique , Iode/composition chimique , Composés organiques du sélénium/synthèse chimique , Propylthiouracile/analogues et dérivés , Antithyroïdiens/composition chimique , Cristallographie aux rayons X , Spectroscopie par résonance magnétique , Structure moléculaire , Composés organiques du sélénium/composition chimique , Composés organiques du sélénium/pharmacologie , Propylthiouracile/composition chimique , Spectrophotométrie UV
18.
Nucleic Acids Res ; 33(5): 1474-86, 2005.
Article de Anglais | MEDLINE | ID: mdl-15755749

RÉSUMÉ

The hepatitis C virus (HCV) genome contains an internal ribosome entry site (IRES) followed by a large open reading frame coding for a polyprotein that is cleaved into 10 proteins. An additional HCV protein, the F protein, was recently suggested to result from a +1 frameshift by a minority of ribosomes that initiated translation at the HCV AUG initiator codon of the polyprotein. In the present study, we reassessed the mechanism accounting for the synthesis of the F protein by measuring the expression in cultured cells of a luciferase reporter gene with an insertion encompassing the IRES plus the beginning of the HCV-coding region preceding the luciferase-coding sequence. The insertion was such that luciferase expression was either in the +1 reading frame relative to the HCV AUG initiator codon, mimicking the expression of the F protein, or in-frame with this AUG, mimicking the expression of the polyprotein. Introduction of a stop codon at various positions in-frame with the AUG initiator codon and substitution of this AUG with UAC inhibited luciferase expression in the 0 reading frame but not in the +1 reading frame, ruling out that the synthesis of the F protein results from a +1 frameshift. Introduction of a stop codon at various positions in the +1 reading frame identified the codon overlapping codon 26 of the polyprotein in the +1 reading frame as the translation start site for the F protein. This codon 26(+1) is either GUG or GCG in the viral variants. Expression of the F protein strongly increased when codon 26(+1) was replaced with AUG, or when its context was mutated into an optimal Kozak context, but was severely decreased in the presence of low concentrations of edeine. These observations are consistent with a Met-tRNA(i)-dependent initiation of translation at a non-AUG codon for the synthesis of the F protein.


Sujet(s)
Codon d'initiation , Hepacivirus/génétique , Initiation de la traduction , Protéines du core viral/génétique , Séquence nucléotidique , Lignée cellulaire , Édéine/pharmacologie , Décalage ribosomique , Humains , Données de séquences moléculaires , Inhibiteurs de la synthèse d'acide nucléique/pharmacologie , Initiation de la traduction/effets des médicaments et des substances chimiques , Polyprotéines/biosynthèse , Polyprotéines/génétique , ARN viral/composition chimique , Cadres de lecture , Séquences régulatrices de l'acide ribonucléique , Protéines du core viral/biosynthèse
19.
RNA ; 9(10): 1246-53, 2003 Oct.
Article de Anglais | MEDLINE | ID: mdl-13130138

RÉSUMÉ

The synthesis of the Gag-Pol polyprotein, the precursor of the enzymes of the human immunodeficiency virus type 1 (HIV-1), requires a programmed -1 ribosomal frameshift. This frameshift has been investigated so far only for subtype B of HIV-1 group M. In this subtype, the frameshift stimulatory signal was found to be a two-stem helix, in which a three-purine bulge interrupts the two stems. In this study, using a luciferase reporter system, we compare, for the first time, the frameshift efficiency of all the subtypes of group M. Mutants of subtype B, including a natural variant were also investigated. Our results with mutants of subtype B confirm that the bulge and the lower stem of the frameshift stimulatory signal contribute to the frameshift in addition to the upper stem-loop considered previously as the sole participant. Our results also show that the frameshift stimulatory signal of all of the other subtypes of group M can be folded into the same structure as in subtype B, despite sequence variations. Moreover, the frameshift efficiency of these subtypes, when assessed in cultured cells, falls within a narrow window (the maximal deviation from the mean value calculated from the experimental values of all the subtypes being approximately 35%), although the predicted thermodynamic stability of the frameshift stimulatory signal differs between the subtypes (from -17.2 kcal/mole to -26.2 kcal/mole). The fact that the frameshift efficiencies fall within a narrow range for all of the subtypes of HIV-1 group M stresses the potential of the frameshift event as an antiviral target.


Sujet(s)
Décalage ribosomique , Protéines de fusion gag-pol/génétique , Régulation de l'expression des gènes viraux , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , ARN viral/génétique , Réplication virale , Séquence nucléotidique , Séquence conservée , Gènes rapporteurs , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/isolement et purification , Humains , Luciferases/analyse , Luciferases/génétique , Données de séquences moléculaires , Mutagenèse dirigée , Mutation , Conformation d'acide nucléique , Biosynthèse des protéines , ARN viral/composition chimique , Similitude de séquences d'acides nucléiques , Transcription génétique
20.
J Mol Biol ; 331(3): 571-83, 2003 Aug 15.
Article de Anglais | MEDLINE | ID: mdl-12899829

RÉSUMÉ

Human immunodeficiency virus type 1 (HIV-1) requires a programmed -1 ribosomal frameshift to produce Gag-Pol, the precursor of its enzymatic activities. This frameshift occurs at a slippery sequence on the viral messenger RNA and is stimulated by a specific structure, downstream of the shift site. While in group M, the most abundant HIV-1 group, the frameshift stimulatory signal is an extended bulged stem-loop, we show here, using a combination of mutagenesis and probing studies, that it is a pseudoknot in group O. The mutagenesis and probing studies coupled to an in silico analysis show that group O pseudoknot is a hairpin-type pseudoknot with two coaxially stacked stems of eight base-pairs (stem 1 and stem 2), connected by single-stranded loops of 2nt (loop 1) and 20nt (loop 2). Mutations impairing formation of stem 1 or stem 2 of the pseudoknot reduce frameshift efficiency, whereas compensatory changes that allow re-formation of these stems restore the frameshift efficiency to near wild-type level. The difference between the frameshift stimulatory signal of group O and group M supports the hypothesis that these groups originate from a different monkey to human transmission.


Sujet(s)
Décalage ribosomique/génétique , VIH-1 (Virus de l'Immunodéficience Humaine de type 1)/génétique , Conformation d'acide nucléique , ARN viral/composition chimique , ARN viral/génétique , Séquences régulatrices de l'acide ribonucléique/génétique , Séquence nucléotidique , Lignée cellulaire , Simulation numérique , Humains , Modèles moléculaires , Données de séquences moléculaires , Analyse de séquence d'ADN
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