Primary hepatocytes as targets for hepatitis C virus replication

Much of our current understanding of hepatitis C virus (HCV) replication has hailed from the use of a small number of cloned viral genomes and transformed hepatoma cell lines. Recent evidence suggests that lipoproteins play a key role in the HCV life cycle and virus particles derived from the sera of infected patients exist in association with host lipoproteins. This report will review the literature on HCV replication in primary hepatocytes and transformed cell lines, focusing largely on host factors defining particle entry

New treatments for chronic hepatitis C

Treatments for chronic hepatitis C has evolved significantly in the past 15 years. The standard of care (SOC) is peginterferon alfa-2a/-2b with ribavirin for 48 weeks or 24 weeks in patients infected with HCV genotype 1 or 2/3, respectively. The treatment duration can be individualized based on the baseline viral load and the speed of the virologic response during treatment. However, current therapies are associated with side effects, complications, and poor patient tolerability. Therefore, there is an urgent need to identify better strategies for treating this disease. An improved sustained virologic response (SVR) can be achieved with new HCV-specific inhibitors against NS3/4A and NS5B polymerases. Recent trials have found SVR rates in patients with HCV genotype 1 infection of 61~68% and 67~75% for combining the SOC with the protease inhibitors telaprevir and boceprevir, respectively. Several new HCV-specific inhibitors such as protease inhibitors and nucleoside and non-nucleoside polymerase inhibitors as well as non-HCV-specific compounds with anti-HCV activity are currently in clinical evaluation. In this review we discuss these new treatments for chronic hepatitis C

A New Model to Produce Infectious Hepatitis C Virus without the Replication Requirement

Numerous constraints significantly hamper the experimental study of hepatitis C virus (HCV). Robust replication in cell culture occurs with only a few strains, and is invariably accompanied by adaptive mutations that impair in vivo infectivity/replication. This problem complicates the production and study of authentic HCV, including the most prevalent and clinically important genotype 1 (subtypes 1a and 1b). Here we describe a novel cell culture approach to generate infectious HCV virions without the HCV replication requirement and the associated cell-adaptive mutations. The system is based on our finding that the intracellular environment generated by a West-Nile virus (WNV) subgenomic replicon rendered a mammalian cell line permissive for assembly and release of infectious HCV particles, wherein the HCV RNA with correct 5′ and 3′ termini was produced in the cytoplasm by a plasmid-driven dual bacteriophage RNA polymerase-based transcription/amplification system. The released particles preferentially contained the HCV-based RNA compared to the WNV subgenomic RNA. Several variations of this system are described with different HCV-based RNAs: (i) HCV bicistronic particles (HCVbp) containing RNA encoding the HCV structural genes upstream of a cell-adapted subgenomic replicon, (ii) HCV reporter particles (HCVrp) containing RNA encoding the bacteriophage SP6 RNA polymerase in place of HCV nonstructural genes, and (iii) HCV wild-type particles (HCVwt) containing unmodified RNA genomes of diverse genotypes (1a, strain H77; 1b, strain Con1; 2a, strain JFH-1). Infectivity was assessed based on the signals generated by the HCV RNA molecules introduced into the cytoplasm of target cells upon virus entry, i.e. HCV RNA replication and protein production for HCVbp in Huh-7.5 cells as well as for HCVwt in HepG2-CD81 cells and human liver slices, and SP6 RNA polymerase-driven firefly luciferase for HCVrp in target cells displaying candidate HCV surface receptors. HCV infectivity was inhibited by pre-incubation of the particles with anti-HCV antibodies and by a treatment of the target cells with leukocyte interferon plus ribavirin. The production of authentic infectious HCV particles of virtually any genotype without the adaptive mutations associated with in vitro HCV replication represents a new paradigm to decipher the requirements for HCV assembly, release, and entry, amenable to analyses of wild type and genetically modified viruses of the most clinically significant HCV genotypes

Molecular Determinants and Dynamics of Hepatitis C Virus Secretion

The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells

A Novel Small Molecule Inhibitor of Hepatitis C Virus Entry

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development

Generation of VSV/HCV pseudotyped particles, study of hepatitis C virus fusion with host cells

En raison de l'absence de système de culture cellulaire capable de propager efficacement le virus de l'hépatite C, peu de données sont disponibles concernant les phases précoces de l'infection. Afin d'étudier la fusion et la pénétration du VHC dans la cellule hôte, nous avons choisi de fabriquer des virus pseudotypes VSV/VHC destinés à mimer l'enveloppe du VHC dans les phases précoces de l'infection. Dans un premier temps, les glycoprotéines d'enveloppe du VHC (E1 et E2) sont modifiées pour être localisées à la membrane plasmique, site de bourgeonnement du VSV. Pour cela, les ectodomaines de E1 (jusqu'à l'acide aminé 311) et de E2 (jusqu'à l'acide aminé 661) sont fusionnés aux domaines transmembranaire et cytoplasmique de la glycoprotéine G du VSV. La protéine de fusion E1-TmG est également fusionnée à l'EGFP afin de faciliter sa détection. Les protéines de fusion EGFP-E1-TmG et E2-TmG sont produites dans la cellule grâce à deux adénovirus recombinants non réplicatifs. Pour produire les virus pseudotypes, les cellules sont tout d'abord infectées par ces deux adénovirus recombinants à 37 ʿC, puis par le VSVtsO45 à 40,5 ʿC, température à laquelle la glycoprotéine G mutée du VSV est retenue au niveau du réticulum endoplasmique. Les particules ainsi produites sont constituées d'une enveloppe contenant les glycoprotéines E1 et E2 modifiées et leur génome est celui du VSVtsO45. Par conséquent, l'infection de cellules par les pseudotypes à 33 ʿC (température permissive de la mutation thermosensible) permet la production de VSVtsO45. Dans un deuxième temps, nous nous intéressons aux mécanismes utilisés par le VHC pour pénétrer dans la cellule. Nous avons ainsi pu déterminer que la fusion des membranes virale et cellulaire se déroulait à pH acide et qu'elle avait lieu dans l'endosome. Finalement, nos résultats indiquent que les pseudotypes pénètreraient dans la cellule majoritairement par endocytose clathrine-dépendanteDue to the lack of cell culture system to propagate efficiently HCV, only few data are available concerning the early stages of HCV infection. In order to study HCV fusion and penetration into host cells, we have chosen to generate VSV/HCV pseudotyped viruses to mimic HCV envelope during the early stages of infection. First, E1 and E2 HCV glycoproteins are modified to be localized at the plasma membrane where VSV budding occurs. Thus, E1 (amino acid 311) and E2 (amino acid 661) ectodomains are fused to the transmembrane domain and the cytoplasmic tail of VSV G glycoprotein. Chimeric E1-TmG is also fused to EGFP to allow easy detection of this protein. Chimeric E1 and E2 are expressed in cells using non replicative recombinant adenoviruses. To produce pseudotypes, cells are first infected by both recombinant adenoviruses at 37 ʿC, and then super-infected by VSVtsO45 at 40.5 ʿC. At this non-permissive temperature, the mutated VSV G glycoprotein is retained in the endoplasmic reticulum. Particles resulting from this triple infection are enveloped by modified E1 and E2 but their genome is still VSVtsO45. Therefore, infection with pseudotypes at 33 ʿC (permissive temperature for the mutation) will lead to production of VSVtsO45 virions. In a second step, we are interested in mechanisms involved in HCV penetration into the host cell. Pseudotypes are used to determine optimal pH required for cellular and viral membranes fusion. The use of such a tool has allowed to investigate and to understand part of the endocytic pathway implicated in early stages of HCV infection. Our results indicate that pseudotypes enter host cells via a pH-dependant route, that fusion of viral and cellular membranes occurs in the endosome, and that clathrin is involved in this mechanism

Generation of VSV/HCV pseudotyped particles, study of hepatitis C virus fusion with host cells

En raison de l'absence de système de culture cellulaire capable de propager efficacement le virus de l'hépatite C, peu de données sont disponibles concernant les phases précoces de l'infection. Afin d'étudier la fusion et la pénétration du VHC dans la celDue to the lack of cell culture system to propagate efficiently HCV, only few data are available concerning the early stages of HCV infection. In order to study HCV fusion and penetration into host cells, we have chosen to generate VSV/HCV pseudotyped vi

Production de virus pseudotypes VSV/VHC (Etude de la fusion du VHC avec les cellules hôtes)

En raison de l'absence de système de culture cellulaire capable de propager efficacement le virus de l'hépatite C, peu de données sont disponibles concernant les phases précoces de l'infection. Afin d'étudier la fusion et la pénétration du VHC dans la cellule hôte, nous avons choisi de fabriquer des virus pseudotypes VSV/VHC destinés à mimer l'enveloppe du VHC dans les phases précoces de l'infection. Dans un premier temps, les glycoprotéines d'enveloppe du VHC (E1 et E2) sont modifiées pour être localisées à la membrane plasmique, site de bourgeonnement du VSV. Pour cela, les ectodomaines de E1 (jusqu'à l'acide aminé 311) et de E2 (jusqu'à l'acide aminé 661) sont fusionnés aux domaines transmembranaire et cytoplasmique de la glycoprotéine G du VSV. La protéine de fusion E1-TmG est également fusionnée à l'EGFP afin de faciliter sa détection. Les protéines de fusion EGFP-E1-TmG et E2-TmG sont produites dans la cellule grâce à deux adénovirus recombinants non réplicatifs. Pour produire les virus pseudotypes, les cellules sont tout d'abord infectées par ces deux adénovirus recombinants à 37 ʿC, puis par le VSVtsO45 à 40,5 ʿC, température à laquelle la glycoprotéine G mutée du VSV est retenue au niveau du réticulum endoplasmique. Les particules ainsi produites sont constituées d'une enveloppe contenant les glycoprotéines E1 et E2 modifiées et leur génome est celui du VSVtsO45. Par conséquent, l'infection de cellules par les pseudotypes à 33 ʿC (température permissive de la mutation thermosensible) permet la production de VSVtsO45. Dans un deuxième temps, nous nous intéressons aux mécanismes utilisés par le VHC pour pénétrer dans la cellule. Nous avons ainsi pu déterminer que la fusion des membranes virale et cellulaire se déroulait à pH acide et qu'elle avait lieu dans l'endosome. Finalement, nos résultats indiquent que les pseudotypes pénètreraient dans la cellule majoritairement par endocytose clathrine-dépendanteDue to the lack of cell culture system to propagate efficiently HCV, only few data are available concerning the early stages of HCV infection. In order to study HCV fusion and penetration into host cells, we have chosen to generate VSV/HCV pseudotyped viruses to mimic HCV envelope during the early stages of infection. First, E1 and E2 HCV glycoproteins are modified to be localized at the plasma membrane where VSV budding occurs. Thus, E1 (amino acid 311) and E2 (amino acid 661) ectodomains are fused to the transmembrane domain and the cytoplasmic tail of VSV G glycoprotein. Chimeric E1-TmG is also fused to EGFP to allow easy detection of this protein. Chimeric E1 and E2 are expressed in cells using non replicative recombinant adenoviruses. To produce pseudotypes, cells are first infected by both recombinant adenoviruses at 37 ʿC, and then super-infected by VSVtsO45 at 40.5 ʿC. At this non-permissive temperature, the mutated VSV G glycoprotein is retained in the endoplasmic reticulum. Particles resulting from this triple infection are enveloped by modified E1 and E2 but their genome is still VSVtsO45. Therefore, infection with pseudotypes at 33 ʿC (permissive temperature for the mutation) will lead to production of VSVtsO45 virions. In a second step, we are interested in mechanisms involved in HCV penetration into the host cell. Pseudotypes are used to determine optimal pH required for cellular and viral membranes fusion. The use of such a tool has allowed to investigate and to understand part of the endocytic pathway implicated in early stages of HCV infection. Our results indicate that pseudotypes enter host cells via a pH-dependant route, that fusion of viral and cellular membranes occurs in the endosome, and that clathrin is involved in this mechanism.STRASBOURG-Sc. et Techniques (674822102) / SudocSudocFranceF

The ubiquitin-proteasome system and skeletal muscle wasting

Abstract The ubiquitin-proteasome system (UPS) is believed to degrade the major contractile skeletal muscle proteins and plays a major role in muscle wasting. Different and multiple events in the ubiquitination, deubiquitination and proteolytic machineries are responsible for the activation of the system and subsequent muscle wasting. However, other proteolytic enzymes act upstream (possibly m-calpain, cathepsin L, and/or caspase 3) and downstream (tripeptidyl-peptidase II and aminopeptidases) of the UPS, for the complete breakdown of the myofibrillar proteins into free amino acids. Recent studies have identified a few critical proteins that seem necessary for muscle wasting {i.e. the MAFbx (muscle atrophy F-box protein, also called atrogin-1) and MuRF-1 [muscle-specific RING (really interesting new gene) finger 1] ubiquitin-protein ligases}. The characterization of their signalling pathways is leading to new pharmacological approaches that can be useful to block or partially prevent muscle wasting in human patients. 17

The ubiquitin–proteasome system and skeletal muscle wasting

International audienceThe ubiquitin-proteasome system (UPS) is believed to degrade the major contractile skeletal muscle proteins and plays a major role in muscle wasting. Different and multiple events in the ubiquitination, deubiquitination and proteolytic machineries are responsible for the activation of the system and subsequent muscle wasting. However, other proteolytic enzymes act upstream (possibly m-calpain, cathepsin L, and/or caspase 3) and downstream (tripeptidyl-peptidase II and aminopeptidases) of the UPS, for the complete breakdown of the myofibrillar proteins into free amino acids. Recent studies have identified a few critical proteins that seem necessary for muscle wasting {i.e. the MAFbx (muscle atrophy F-box protein, also called atrogin-1) and MuRF-1 [muscle-specific RING (really interesting new gene) finger 1] ubiquitin-protein ligases}. The characterization of their signalling pathways is leading to new pharmacological approaches that can be useful to block or partially prevent muscle wasting in human patients