A Gaussia luciferase cell-based system to assess the infection of cell culture- and serum-derived hepatitis C virus

Robust replication of hepatitis C virus (HCV) in cell culture occurs only with the JFH-1 (genotype 2a) recombinant genome. The aim of this study was to develop a system for HCV infection quantification analysis and apply it for the selection of patient sera that may contain cell culture infectious viruses, particularly of the most clinically important genotype 1. Initially, a hepatoma cell line (designated Huh-7.5/EG(4A/4B)GLuc) was generated that stably expressed the enhanced green fluorescent protein (EGFP) fused in-frame to the secreted Gaussia luciferase via a recognition sequence of the viral NS3/4A protease. Upon HCV infection, NS3/4A cleaved at its signal and the Gaussia was secreted to the culture medium, thus facilitating the infection quantification. The Huh-7.5/EG(4A/4B)GLuc cell line provided a rapid and highly sensitive quantification of HCV infection in cell culture using JFH-1-derived viruses. Furthermore, the Huh-7.5/EG(4A/4B)GLuc cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target distinct stages of the HCV life cycle; the Ź-factor of this assay ranged from 0.72 to 0.75. Additionally, eighty-six sera derived from HCV genotype 1b infected liver transplant recipients were screened for their in vitro infection and replication potential. Approximately 12% of the sera contained in vitro replication-competent viruses, as deduced by the Gaussia signal, real time quantitative PCR, immunofluorescence and capsid protein secretion. We conclude that the Huh-7.5/EG(4A/4B)GLuc cell line is an excellent system not only for the screening of in vitro replication-competent serum-derived viruses, but also for the subsequent cloning of recombinant isolates. Additionally, it can be utilized for high-throughput screening of antiviral compounds

Interplay between Basic Residues of Hepatitis C Virus Glycoprotein E2 with Vi ral Receptors Neutralizing Antibodies and Lipoproteins.

Positively-charged amino acids are located at specific positions in the envelope glycoprotein E2 of the hepatitis C virus (HCV): two histidines (H) and four arginines (R) in two conserved WHY and one RGERCDLEDRDR motifs, respectively. Additionally, the E2 hypervariable region 1 (HVR1) is rich in basic amino acids. To investigate the role(s) of these residues in HCV entry, we subjected to comparative infection and sedimentation analysis cell culture-produced (HCVcc, genotype 2a) wild type virus, a panel of alanine single-site mutants and a HVR1-deletion variant. Initially, we analyzed the effects of these mutations on E2-heparan sulfate (HS) interactions. The positive milieu of the HVR1, formulated by its basic amino acids (key residues the conserved H386 and R408), and the two highly conserved basic residues H488 and R648 contributed to E2-HS interactions. Mutations in these residues did not alter the HCVcc-CD81 entry, but they modified the HCVcc-scavenger receptor class B type I (SR-BI) dependent entry and the neutralization by anti-E2 or patients IgG. Finally, separation by density gradients revealed that mutant viruses abolished partially or completely the infectivity of low density particles, which are believed to be associated with lipoproteins. This study shows that there exists a complex interplay between the basic amino acids located in HVR1 and other conserved E2 motifs with the HS, the SR-BI, and neutralizing antibodies and suggests that HCV-associated lipoproteins are implicated in these interactions

Hepatitis C virus intrinsic molecular determinants may contribute to the development of cholestatic hepatitis after liver transplantation

Cholestatic hepatitis C (CHC) is a severe form of hepatitis C virus (HCV) infection recurrence that leads to high graft loss rates early after liver transplantation (LT). To investigate the pathogenic mechanisms of CHC, we analysed HCV quasispecies in CHC patients compared to a control group (mild hepatitis C recurrence) by deep pyrosequencing. At the time of LT, NS5B quasispecies complexity was similar between the two groups but, after LT, it decreased more sharply in CHC patients than in the control group. Interestingly, the major variant before LT propagated efficiently and remained as the dominant sequence after LT in 62% of CHC patients versus 11% of controls (P=0.031). Sequence analysis of the complete nonstructural region in a limited number of patients revealed a potential 12 aa signature specific to the CHC group. These data suggest that intrinsic molecular determinants in the circulating HCV quasispecies may provide a fitness advantage, contributing to the development of CHC

Cell Culture Replication of a Genotype 1b Hepatitis C Virus Isolate Cloned from a Patient Who Underwent Liver Transplantation

The introduction of the genotype 2a isolate JFH1 was a major breakthrough in the field of hepatitis C virus (HCV), allowing researchers to study the complete life cycle of the virus in cell culture. However, fully competent culture systems encompassing the most therapeutically relevant HCV genotypes are still lacking, especially for the highly drug-resistant genotype 1b. For most isolated HCV clones, efficient replication in cultured hepatoma cells requires the introduction of replication-enhancing mutations. However, such mutations may interfere with viral assembly, as occurs in the case of the genotype 1b isolate Con1. In this study, we show that a clinical serum carrying a genotype 1b virus with an exceptionally high viral load was able to infect Huh7.5 cells. Similar to previous reports, inoculation of Huh7.5 cells by natural virus is very inefficient compared to infection by cell culture HCV. A consensus sequence of a new genotype 1b HCV isolate was cloned from the clinical serum (designated Barcelona HCV1), and then subjected to replication studies. This virus replicated poorly in a transient fashion in Huh7.5 cells after electroporation with in vitro transcribed RNA. Nonetheless, approximately 3 weeks post electroporation and thereafter, core protein-positive cells were detected by immunofluorescence. Surprisingly, small amounts of core protein were also measurable in the supernatant of electroporated cells, suggesting that HCV particles might be assembled and released. Our findings not only enhance the current method of cloning in vitro HCV replication-competent isolates, but also offer valuable insights for the realization of fully competent culture systems for HCV

Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations

With the advent of subgenomic hepatitis C virus (HCV) replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs), previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A), but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I) as well as NS5A (S2204R), whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes

Scavenger receptor class B type I is a key host factor for hepatitis C virus infection required for an entry step closely linked to CD81.

International audienceHepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. Scavenger receptor class B type I (SR-BI) has been shown to bind HCV envelope glycoprotein E2, participate in entry of HCV pseudotype particles, and modulate HCV infection. However, the functional role of SR-BI for productive HCV infection remains unclear. In this study, we investigated the role of SR-BI as an entry factor for infection of human hepatoma cells using cell culture-derived HCV (HCVcc). Anti-SR-BI antibodies directed against epitopes of the human SR-BI extracellular loop specifically inhibited HCVcc infection in a dose-dependent manner. Down-regulation of SR-BI expression by SR-BI-specific short interfering RNAs (siRNAs) markedly reduced the susceptibility of human hepatoma cells to HCVcc infection. Kinetic studies demonstrated that SR-BI acts predominately after binding of HCV at an entry step occurring at a similar time point as CD81-HCV interaction. Although the addition of high-density lipoprotein (HDL) enhanced the efficiency of HCVcc infection, anti-SR-BI antibodies and SR-BI-specific siRNA efficiently inhibited HCV infection independent of lipoprotein. Conclusion: Our data suggest that SR-BI (i) represents a key host factor for HCV entry, (ii) is implicated in the same HCV entry pathway as CD81, and (iii) targets an entry step closely linked to HCV-CD81 interaction

In vivo και ex vivo μελέτη της ενεργοπίησης-απόπτωσης, που προκαλείται από την V3 περιοχή της γλυκππρωτείνης gp120 του ιού HIV-1, σε ενεργοποιημένα CD4+T λεμφοκύτταρα

Ο ιός της επίκτητης ανοσολογικής ανεπάρκειας (Human Immunodeficiency Virus, HIV) δημιουργεί στον άνθρωπο κατά τη μόλυνση μια σταδιακή μείωση στον αριθμό των CD4+ T λεμφοκυττάρων, η οποία έχει ως αποτέλεσμα την κατάσταση της ανοσοανεπάρκειας και αυξημένη ευαισθησία σε ευκαιριακές μολύνσεις και ογκογενέσεις, δηλαδή το AIDS( Acquired Immune Deficiency Syndrome). Η παρατηρούμενη μείωση του αριθμού των λεμφοκυττάρων οφείλεται σε διαδικασίες παθογονικότητας του ιού αλλά πλέον στις μέρες μας έχει αποδειχθεί ότι η πρωταρχική βάση για την αυξημένη μείωση του συνολικού αριθμού των Τ λεμφοκυττάρων αποτελεί η ενισχυμένη απόπτωση των CD4+ λεμφοκυττάρων και στα τελικά στάδια της νόσου και των CD8+ Τ λεμφοκύτταρων. Ιδιαίτερο βάρος έχει πάρει η μελέτη της απόπτωσης που προκαλείται από την gp120. H γλυκοπρωτεΐνη gp120 αποτελεί τμήμα του καψιδίου και είναι υπεύθυνη για τη σύντηξη με τον υποδοχέα CD4 και με βασικούς υποδοχείς χυμοκινών για την είσοδο του ιικού σωματιδίου στα κύτταρα-στόχους. Η πρόσδεση της gp120 με τον υποδοχέα CD4 και με υποδοχείς χυμοκινών (CCR5, CXCR4) ενεργοποιεί κατά περίπτωση όλους τους μηχανισμούς απόπτωσης: Fas/FasL, TNF/TNFRII, TRAIL, κασπάσες και απελευθέρωση του κυτοχρώματος c από τα μιτοχόνδρια. Το αντικείμενο αυτής της διατριβής αποτέλεσε η μελέτη του φαινομένου ενεργοποίσης-απόπτωσης που προκαλείται από την περιοχή V3 της gp120 σε διεγερμένα Τ βοηθητικά λεμφοκύτταρα τα οποία αναγνωρίζουν ένα αντιγόνο. Για την επίτευξη του παραπάνω στόχου πραγματοποιήθηκε ανάλυση της έκφρασης επιφανειακών πρωτεϊνών με κυτταρομετρία ροής (FACS καθώς και χρήση ενός ποντικίσιου πειραματικού μοντέλου για την in vivo μελέτη της απόπτωσης. Τα αποτελέσματα έδειξαν ότι παρόλο που ο πολλαπλασιασμός των T λεμφοκυττάρων που διεγείρονται με lipoV3/TT (το λιπόσωμα χρησιμοποιήθηκε ως φορέας της περιοχής V3) διαφέρει χρονικά από τον πολλαπλασιασμό που παρατηρείται μετά από διέγερση με το διαλυτό ΤΤ, η αλλαγή των επιφανειακών μαρτύρων ενεργοποίησης (CD45RO), απόπτωσης (CD95) και υποδοχέων χυμοκινών (CCR5, CXCR4) είναι ποσοτικά και ποιοτικά ίδια για όλους τους επιφανειακούς μάρτυρες σε σχέση με την αλλαγή κατά την αναμνηστική απάντηση στο διαλυτό ΤΤ. Ένεση των λιποσωμάτων lipoV3/TT στα ποντίκια δεν φάνηκε να επηρεάζει κάτω από αυτές τις συνθήκες συγκεκριμένους Τ κυτταρικούς πληθυσμούς και έτσι το πείραμα πρέπει να επαναληφθεί. Απώτερος στόχος ήταν η εξακρίβωση του πλήρους αποπτωτικού μηχανισμού που ενεργοποιείται κατά τη σύνδεση V3/CCR5 και η χρήση της πληροφορίας αυτής για το σχεδιασμό νέων θεραπευτικών προσεγγίσεων για τους ασθενείς που εμφανίζουν συμπτώματα από την μόλυνση με τον HIV που στο σύνολο τους καλούνται AIDS

The Level of CD81 Cell Surface Expression Is a Key Determinant for Productive Entry of Hepatitis C Virus into Host Cells

Recently a cell culture model supporting the complete life cycle of the hepatitis C virus (HCV) was developed. Searching for host cell determinants involved in the HCV replication cycle, we evaluated the efficiency of virus propagation in different Huh-7-derived cell clones. We found that Huh-7.5 cells and Huh7-Lunet cells, two former replicon cell clones that had been generated by removal of an HCV replicon by inhibitor treatment, supported comparable levels of RNA replication and particle production, whereas virus spread was severely impaired in the latter cells. Analysis of cell surface expression of CD81 and scavenger receptor class B type I (SR-BI), two molecules previously implicated in HCV entry, revealed similar expression levels for SR-BI, while CD81 surface expression was much higher on Huh-7.5 cells than on Huh7-Lunet cells. Ectopic expression of CD81 in Huh7-Lunet cells conferred permissiveness for HCV infection to a level comparable to that for Huh-7.5 cells. Modulation of CD81 cell surface density in Huh-7.5 cells by RNA interference indicated that a certain amount of this molecule (∼7 × 10(4) molecules per cell) is required for productive infection with a low dose of HCV. Consistent with this, we show that susceptibility to HCV infection depends on a critical quantity of CD81 molecules. While infection is restricted in cells expressing very small amounts of CD81, susceptibility rapidly rises within a narrow range of CD81 levels, reaching a plateau where higher expression does not further increase the efficiency of infection. Together these data indicate that a high density of cell surface-exposed CD81 is a key determinant for productive HCV entry into host cells

Characterization of the Early Steps of Hepatitis C Virus Infection by Using Luciferase Reporter Viruses

The lack of an efficient system to produce hepatitis C virus (HCV) particles has impeded the analysis of the HCV life cycle. Recently, we along with others demonstrated that transfection of Huh7 hepatoma cells with a novel HCV isolate (JFH1) yields infectious viruses. To facilitate studies of HCV replication, we generated JFH1-based bicistronic luciferase reporter virus genomes. We found that RNA replication of the reporter construct was only slightly attenuated and that virus titers produced were only three- to fivefold lower compared to the parental virus, making these reporter viruses an ideal tool for quantitative analyses of HCV infections. To expand the scope of the system, we created two chimeric JFH1 luciferase reporter viruses with structural proteins from the Con1 (genotype 1b) and J6CF (genotype 2a) strains. Using these and the authentic JFH1 reporter viruses, we analyzed the early steps of the HCV life cycle. Our data show that the mode of virus entry is conserved between these isolates and involves CD81 as a key receptor for pH-dependent virus entry. Competition studies and time course experiments suggest that interactions of HCV with cell surface-resident glycosaminoglycans aid in efficient infection of Huh7 cells and that CD81 acts during a postattachment step. The reporter viruses described here should be instrumental for investigating the viral life cycle and for the development of HCV inhibitors