Identification of a Functional, CRM-1-Dependent Nuclear Export Signal in Hepatitis C Virus Core Protein

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease worldwide. HCV core protein is involved in nucleocapsid formation, but it also interacts with multiple cytoplasmic and nuclear molecules and plays a crucial role in the development of liver disease and hepatocarcinogenesis. The core protein is found mostly in the cytoplasm during HCV infection, but also in the nucleus in patients with hepatocarcinoma and in core-transgenic mice. HCV core contains nuclear localization signals (NLS), but no nuclear export signal (NES) has yet been identified

Phenothiazines Inhibit Hepatitis C Virus Entry, Likely by Increasing the Fluidity of Cholesterol-Rich Membranes

Despite recent progress in the development of direct-acting antiviral agents against hepatitis C virus (HCV), more effective therapies are still urgently needed. We and others previously identified three phenothiazine compounds as potent HCV entry inhibitors. In this study, we show that phenothiazines inhibit HCV entry at the step of virus-host cell fusion, by intercalating into cholesterol-rich domains of the target membrane and increasing membrane fluidity. Perturbation of the alignment/packing of cholesterol in lipid membranes likely increases the energy barrier needed for virus-host fusion. A screening assay based on the ability of molecules to selectively increase the fluidity of cholesterol-rich membranes was subsequently developed. One compound that emerged from the library screen, topotecan, is able to very potently inhibit the fusion of liposomes with cell culture-derived HCV (HCVcc). These results yield new insights into HCV infection and provide a platform for the identification of new HCV inhibitors

Analysis of Serine Codon Conservation Reveals Diverse Phenotypic Constraints on Hepatitis C Virus Glycoprotein Evolution

Serine is encoded by two divergent codon types, UCN and AGY, which are not interchangeable by a single nucleotide substitution. Switching between codon types therefore occurs via intermediates (threonine or cysteine) or via simultaneous tandem substitutions. Hepatitis C virus (HCV) chronically infects 2 to 3% of the global population. The highly variable glycoproteins E1 and E2 decorate the surface of the viral envelope, facilitate cellular entry, and are targets for host immunity. Comparative sequence analysis of globally sampled E1E2 genes, coupled with phylogenetic analysis, reveals the signatures of multiple archaic codonswitching events at seven highly conserved serine residues. Limited detection of intermediate phenotypes indicates that associated fitness costs restrict their fixation in divergent HCV lineages. Mutational pathways underlying codon switching were probed via reverse genetics, assessing glycoprotein functionality using multiple in vitro systems. These data demonstrate selection against intermediate phenotypes can act at the structural/functional level, with some intermediates displaying impaired virion assembly and/or decreased capacity for target cell entry. These effects act in residue/isolate-specific manner. Selection against intermediates is also provided by humoral targeting, with some intermediates exhibiting increased epitope exposure and enhanced neutralization sensitivity, despite maintaining a capacity for target cell entry. Thus, purifying selection against intermediates limits their frequencies in globally sampled strains, with divergent functional constraints at the protein level restricting the fixation of deleterious mutations. Overall our study provides an experimental framework for identification of barriers limiting viral substitutional evolution and indicates that serine codon-switching represents a genomic "fossil record" of historical purifying selection against E1E2 intermediate phenotypes

Differential In Vitro Effects of Intravenous versus Oral Formulations of Silibinin on the HCV Life Cycle and Inflammation

Silymarin prevents liver disease in many experimental rodent models, and is the most popular botanical medicine consumed by patients with hepatitis C. Silibinin is a major component of silymarin, consisting of the flavonolignans silybin A and silybin B, which are insoluble in aqueous solution. A chemically modified and soluble version of silibinin, SIL, has been shown to potently reduce hepatitis C virus (HCV) RNA levels in vivo when administered intravenously. Silymarin and silibinin inhibit HCV infection in cell culture by targeting multiple steps in the virus lifecycle. We tested the hepatoprotective profiles of SIL and silibinin in assays that measure antiviral and anti-inflammatory functions. Both mixtures inhibited fusion of HCV pseudoparticles (HCVpp) with fluorescent liposomes in a dose-dependent fashion. SIL inhibited 5 clinical genotype 1b isolates of NS5B RNA dependent RNA polymerase (RdRp) activity better than silibinin, with IC50 values of 40–85 µM. The enhanced activity of SIL may have been in part due to inhibition of NS5B binding to RNA templates. However, inhibition of the RdRps by both mixtures plateaued at 43–73%, suggesting that the products are poor overall inhibitors of RdRp. Silibinin did not inhibit HCV replication in subgenomic genotype 1b or 2a replicon cell lines, but it did inhibit JFH-1 infection. In contrast, SIL inhibited 1b but not 2a subgenomic replicons and also inhibited JFH-1 infection. Both mixtures inhibited production of progeny virus particles. Silibinin but not SIL inhibited NF-κB- and IFN-B-dependent transcription in Huh7 cells. However, both mixtures inhibited T cell proliferation to similar degrees. These data underscore the differences and similarities between the intravenous and oral formulations of silibinin, which could influence the clinical effects of this mixture on patients with chronic liver diseases

Rôle de l'intégrine alpha v beta dans l'adressage des cellules tumorales à l'os

Les mécanismes responsables de l'adressage des cellules de cancer du sein au tissu osseux restent obscures. Dans cette thèse, nous avons montré, (1) in vivo l'expression d'une intégrine alpha v béta 3 par les cellules CHO ou la surexpression par les cellules du cancer du sein B02 (isolées de métastases osseuses) favorise la formation des métastases ostéolytiques chez des souris ; (2) in vitro, l'intégrine alpha v béta 3 favorise l'invasion tumorale et l'adhésion des cellules tumorales à la matrice osseuse et à la sialoprotéine osseuse (protéine spécifique de l'os). D'autre part, un antagoniste non-peptidique de l'intégrine alpha v béta 3, le HMR1404, inhibe in vivo à 90% la formation des métastases osseuses induites par les cellules exprimant l'intégrine alpha v béta 3 et in vitro inhibe l'adhésion des ces cellules. Donc, l'expression de l'intégrine alpha v béta 3 contribue à l'adressage des cellules tumorales à l'os en favorisant l'invasion et l'adhésion à l'os.LYON1-BU Santé (693882101) / SudocPARIS-BIUP (751062107) / SudocSudocFranceF

Nouveaux lipides bicaténaires polyfonctionnels (enjeux synthétiques et analytiques, applications biochimiques)

Les pathogènes comme les virus Ebola, de l hépatite C ou du VIH sont, dans la plupart des cas, mortels et représentent une menace sérieuse pour la santé humaine dans le monde. Ces virus à enveloppe sont difficiles à étudier parce qu ils doivent être manipulés dans des laboratoires de niveau de sécurité 3 ou 4. Malgré ces difficultés, la compréhension de l interaction entre les protéines d enveloppe d un virus donné et la membrane cellulaire lors de l étape d infection de la cellule cible par le virus est essentielle. Il s agit de mieux caractériser ce processus d infection et d essayer de trouver un procédé d inhibition de celui-ci à un stade précoce de l infection. Certaines de ces protéines d enveloppe, les protéines de fusion virale, sont plus particulièrement impliquées lors de l étape de fusion virale. Cette étape conduit à la délivrance du matériel génétique viral dans la cellule cible. Deux éléments structurels jouent un rôle crucial lors de l étape de fusion : le peptide de fusion et le domaine transmembranaire de la protéine de fusion virale. La cartographie de ces régions-clés portées par cette protéine est donc cruciale pour la compréhension des relations structure/fonction au cours du processus de fusion. Nous proposons donc une stratégie pour identifier les régions hydrophobes des protéines de fusion virale impliquant un photomarquage covalent d affinité hydrophobe. Cette stratégie est basée sur l utilisation de pseudo-particules virales non pathogènes ou de virus comme le virus de l hépatite B comme modèle permettant l exécution de celle-ci dans un laboratoire de niveau de sécurité 1 ou 2 moins contraignant. Cette approche repose sur l utilisation de nouvelles sondes lipidiques (2 chaînes grasses) contenant un groupement photoactivable sur une des chaînes grasses (la benzophénone) et un traceur (fluorescent : la rhodamine ou de la biotine) pour la détection et/ou la purification des adduits formés lors de la réaction de marquage.Emerging and often deadly pathogens such as HCV, HIV or Ebola viruses are a serious threat to human health worldwide. These enveloped viruses are extremely difficult to study because they must be manipulated in biosafety level 3 or 4 laboratories. In spite of these difficulties, understanding the way their envelope proteins interact with cellular membranes during infection is essential to better characterize the infection process and try to inhibit it at an early stage. These envelope proteins are involved in particular in fusion, a step of the viral infection that leads to the delivery of the viral genetic material into the cytoplasm of the target-cell. Two structural elements, common to all known fusion proteins, play a key role in fusion: the fusion peptide and the transmembrane domain. The fusion peptide is a short hydrophobic sequence present either at the N-terminus or internal to the fusion protein. Mapping these key hydrophobic regions in viral fusion proteins is therefore crucial to understand the structure/function relationships during the fusion process. We propose a strategy to delineate the hydrophobic regions of viral fusion proteins in the membrane, through hydrophobic covalent photo-affinity labeling. This method is first applied to the identification of transmembrane domains of model proteins of known structure (BmrA, bacteriorhodopsin), for validation before investigation on pseudoparticles of non pathogenic viruses or on viruses such as HBV, in order to delineate the hydrophobic regions of their fusion glycoproteins. these entities are suitable for their study in biosafety level 1 or 2 laboratories which are less restrictive. This approach relies on the use of a collection of new lipid probes (two fatty chains) containing a photoactivable group (benzophenone) and a tracer (fluorescent: rhodamine or biotin) for the detection and/or the purification of the adducts of the reaction.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

Integrin alpha(v)beta3 expression confers on tumor cells a greater propensity to metastasize to bone.

The reasons why tumor cells metastasize to bone remain obscure. There is some evidence to support the theory that integrins (acting as cell surface adhesion receptors) play a role in mediating metastasis in certain organs. Here, we report that overexpression of a functionally active integrin alpha(v)b3 in Chinese hamster ovary (CHO) tumor cells drastically increased the incidence, number, and area of bone metastases in nude mice compared with those observed in mock-transfected CHO cells (CHO dhfr+) or in CHO cells expressing a functionally inactive integrin alpha(v)b3 (CHO beta3Delta744). Moreover, a breast cancer cell line (B02) established from bone metastases caused by MDA-MB-231 cells constitutively overexpressed integrin alpha(v)b3, whereas the cell surface expression level of other integrins remained unchanged. In vivo, the extent of bone metastases in B02-bearing mice was significantly increased compared with that of MDA-MB-231-bearing mice. In vitro, B02 cells and CHO cells expressing a functionally active integrin alpha(v)b3 exhibited substantially increased invasion of and adhesion to mineralized bone, bone sialoprotein, and collagen compared with those found with MDA-MB-231, CHO dhfr+, and CHO beta3Delta744 cells, respectively. Overall, our findings suggest that integrin alpha(v)b3 expression in tumor cells accelerates the development of osteolytic lesions, presumably through increased invasion of and adhesion to bone

Hepatitis delta virus and hepatocellular carcinoma: an update

International audienc

The botrydial (BOT) and botcinic acid (BOA) biosynthetic gene clusters dispolay a bipartite genomic structure and are controlled by putatiave pathway-specific ZN(II)2CYS6 Transcription factors

The sesquiterpene Botrydial (BOT) and the polyketide botcinic acid (BOA) are non-host specific phytotoxins produced by Botrytis cinerea. In addition, their botryanes and botcinines derivatives were shown to have biological activities against bacteria and fungi. Both BOT and BOA biosynthetic gene clusters were previously identified. Inactivation of the key enzymes-encoding genes, i.e. the sesquiterpene cyclase-encoding gene Bcbot2 and the polyketide synthase-encoding gene Bcboa6, revealed that they may have a redundant role in virulence. In order to understand how BOT, BOA and their derivatives are regulated during the life cycle of B. cinerea, we investigated the genomic environment of the BOT and BOA clusters and searched for putative transcription factor (TF) encoding genes. Thanks to the recent release of the gapless genome of B05.10 strain (J. van Kan, Univ. Wageningen, NL), the BOT and BOA clusters were localized in two A+T-rich regions, one being subtelomeric (BOA). Our bioinformatics analyses revealed that the A+T/G+C-equilibrated regions that contain the Bcbot and Bcboa genes alternate with A+T-rich regions (>85%) made of relics of transposable elements that have undergone repeat-induced point (RIP) mutations. The occurrence of RIP raises questions about possible chromatin-based regulation of BOT and BOA synthesis. Several chromatin modifiers (KMT1, KMT6, HP1) are under studies to test this hypothesis. Amongst the previously identified Bcboa genes, Bcboa13 was predicted to encode a Zn(II)2Cys6 TF. In addition, the gapless version of the genome allowed us to identify a similar gene (Bcbot6) flanking the previously identified BOT cluster. Gene inactivation demonstrated that BcBot6 and BcBoa13 are the major positive regulators of BOT and BOA biosynthesis, respectively. Interestingly, the functional fusion protein BcBot6-GFP and BcBoa13-GFP localize into nuclear foci. Identification of BcBot6 as the major regulators of BOT and BOA synthesis is the first step toward a comprehensive understanding of the complete regulation network of toxin synthesis in B. cinerea. We are currently using a Yeast-One-Hybrid approach to search the upstream TF(s) that are interacting with the promoters of Bcbot6 and Bcboa13 genes