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

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

Hepatitis delta virus and hepatocellular carcinoma: an update

International audienc

Cytoplasmic localization of core in HCV-infected cells by immunofluorescence without LMB treatment.

<p>(A) <i>Silencing of the proteasome activator PA28γ</i>. Huh 7.5 cells were transfected with an siRNA targeting PA28γ or a control non targeting siRNA 18 h before infection with JFH1. For analysis of the expression of PA28γ by immunofluorescence, cells were stained with rabbit anti-PA28γ antibody, followed by Alexa Fluor 488-conjugated anti-rabbit IgG. Staining for HCV core was carried out 48 h after infection, with the monoclonal anti-core antibody ACAP-27, followed by Alexa Fluor 568-tagged anti-mouse IgG (in red). (a) Non treated HCV (JFH1)-infected Huh 7.5 cells; (b) HCV-infected cells transfected with control, non-targeting siRNA before infection; (c) cells with PA28γ knockdown due to transfection with a specific PA28γ-targeting siRNA. (B) Expression of core in Huh7.5 cells after silencing of the PA28γ proteasome activator. JFH1-infected cells were stained with rabbit anti-lamin B antibody and Alexa Fluor 488-conjugated anti-rabbit IgG as a secondary antibody, to outline the cell nuclei, and with ACAP27 anti-core antibody followed by Alexa Fluor 568-conjugated anti-mouse IgG, for subcellular localization of HCV core. (d) JFH1-infected Huh 7.5 cells without PA28γ silencing, (corresponding to the image shown in (a) panel A); (e) HCV-infected cells transfected with control, non-targeting siRNA before infection (corresponding to the image shown in (b) panel A); and (f) cells with PA28γ knockdown with a PA28γ-specific siRNA before infection with HCV (corresponding to (c) in panel A). Staining of the nuclear membrane with anti-lamin B (green) and with anti-core antibody (red), as described above. No nuclear staining of core was detected, in either siRNA-silenced cells or in cells transfected with a control si-RNA.</p

Identification of a functional, “non-classical” NES” in the core protein.

<p>(A) CLUSTAL W (1.81) software was used for multiple sequence alignment analysis, leading to the identification of a “non classical” NES sequence in domain II of the core protein. The potential NES signal aa(109–133) in core was compared with known viral NES sequences. Underlined regions correspond to hydrophobic amino-acid residues, and letters in bold typeface identify the conserved LXL motifs. The frame delineates a region of the export sequence containing amino-acid residues L(119), I(123) and L(126), which were replaced by alanine residues (the corresponding immunofluorescence analyses are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0025854#pone-0025854-g006" target="_blank">Figure 6</a>). (B) The amino-acid sequences of the fragment corresponding to the putative NES aa(109–133) in HCV core proteins are well conserved in different HCV genotypes. The consensus sequences are shown, for each virus genotype, and were obtained by the alignment of 1245 sequences corresponding to the putative NES for HCV type 1A, 2078 sequences for HCV type 1B, and 95, 264, 60, 12 and 121 sequences for HCV types 2, 3, 4, 5 and 6, respectively. Sequences were obtained from the Los Alamos Data Bank (National Institutes of Health). (C) Schematic diagram of the plasmids used to investigate the functionality of the putative export sequence of core, aa(109–133). The SV40 NLS was used as a nuclear reporter, and the NES of the HIV Rev protein was used as a control export signal. The sequences shown were fused to either EGFP or m-Cherry, to allow the visualization of proteins in transfected cells. (D) Subcellular distribution of the proteins encoded by the plasmids depicted above. Huh7 cells grown on coverslips were transfected with the appropriate plasmids; 40 h after transfection, the cells were fixed in 4% PFA and examined by fluorescence microscopy. Panels a-c represent proteins labeled with EGFP, d-f the equivalent proteins labeled with m-Cherry. Proteins containing only the SV40 NLS were present mostly in the cell nuclei (a, d); proteins containing the control SV40 NLS and Rev NES were found in both the nucleus and the cytoplasm (b, e). The core sequence containing a putative NES, aa(109–133), is functional, because it was exported from the nucleus to the cytoplasm (c, f), like HIV Rev NES (b,e). Staining of nuclei with DAPI. (E) Graphical representation of nonparametric one-way ANOVA of the ratios of fluorescence between the nucleus and cytoplasm for the three plasmids. Cytoplasmic fluorescence is significantly higher for both EGFP-NLSSV40-NESRev and EGFP-NLSSV40-core aa(109–133).</p

Nuclear export of core NES is mediated by CRM-1.

<p>Huh7 cells were transfected with a plasmid encoding EGFP-tagged NLS SV40 (a, b), EGFP-tagged NLS SV40 and HIV Rev NES (c, d) or the plasmid encoding EGFP-tagged NLS SV40 and putative core NES, aa(109-133) (e, f). Cells transfected with the EGFP-tagged control plasmid pEGFPC1 are shown (g, h). Twenty-two h after transfection, the cells were treated for 4 h with 10 ng/ml LMB (b, d, f, h; +LMB). The cells were subsequently washed, fixed and analyzed by immunofluorescence microscopy. The bar represents 10 µm.</p