scholarly journals Production of Infectious Hepatitis C Virus of Various Genotypes in Cell Cultures

2007 ◽  
Vol 81 (9) ◽  
pp. 4405-4411 ◽  
Author(s):  
Takanobu Kato ◽  
Takuya Matsumura ◽  
Theo Heller ◽  
Satoru Saito ◽  
Ronda K. Sapp ◽  
...  
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Culture Medium ◽  
Expression System ◽  
Core Antigen ◽  
Hcv Rna ◽  
Transfected Cells

ABSTRACT A unique hepatitis C virus (HCV) strain JFH-1 has been shown to replicate efficiently in cell culture with production of infectious HCV. We previously developed a DNA expression system containing HCV cDNA flanked by two self-cleaving ribozymes to generate HCV particles in cell culture. In this study, we produced HCV particles of various genotypes, including 1a (H77), 1b (CG1b), and 2a (J6 and JFH-1), in the HCV-ribozyme system. The constructs also contain the secreted alkaline phosphatase gene to control for transfection efficiency and the effects of culture conditions. After transfection into the Huh7-derived cell line Huh7.5.1, continuous HCV replication and secretion were confirmed by the detection of HCV RNA and core antigen in the culture medium. HCV replication levels of strains H77, CG1b, and J6 were comparable, whereas the JFH-1 strain replicates at a substantially higher level than the other strains. To evaluate the infectivity in vitro, the culture medium of JFH-1-transfected cells was inoculated into naive Huh7.5.1 cells. HCV proteins were detected by immunofluorescence 3 days after inoculation. To evaluate the infectivity in vivo, the culture medium from HCV genotype 1b-transfected cells was inoculated into a chimpanzee and caused a typical course of HCV infection. The HCV 1b propagated in vitro and in vivo had sequences identical to those of the HCV genomic cDNA used for cell culture transfection. The development of culture systems for production of various HCV genotypes provides a valuable tool not only to study the replication and pathogenesis of HCV but also to screen for antivirals.

scholarly journals Efficient Infectious Cell Culture Systems of the Hepatitis C Virus (HCV) Prototype Strains HCV-1 and H77

2014 ◽  
Vol 89 (1) ◽  
pp. 811-823 ◽  
Author(s):  
Yi-Ping Li ◽  
Santseharay Ramirez ◽  
Lotte Mikkelsen ◽  
Jens Bukh
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Adaptive Mutations ◽  
Culture Systems ◽  
Infectious Clones ◽  
Cell Culture Systems ◽  
A Genome

ABSTRACTThe first discovered and sequenced hepatitis C virus (HCV) genome and the firstin vivoinfectious HCV clones originated from the HCV prototype strains HCV-1 and H77, respectively, both widely used in research of this important human pathogen. In the present study, we developed efficient infectious cell culture systems for these genotype 1a strains by using the HCV-1/SF9_A and H77Cin vivoinfectious clones. We initially adapted a genome with the HCV-1 5′UTR-NS5A (where UTR stands for untranslated region) and the JFH1 NS5B-3′UTR (5-5A recombinant), including the genotype 2a-derived mutations F1464L/A1672S/D2979G (LSG), to grow efficiently in Huh7.5 cells, thus identifying the E2 mutation S399F. The combination of LSG/S399F and reported TNcc(1a)-adaptive mutations A1226G/Q1773H/N1927T/Y2981F/F2994S promoted adaptation of the full-length HCV-1 clone. An HCV-1 recombinant with 17 mutations (HCV1cc) replicated efficiently in Huh7.5 cells and produced supernatant infectivity titers of 104.0focus-forming units (FFU)/ml. Eight of these mutations were identified from passaged HCV-1 viruses, and the A970T/I1312V/C2419R/A2919T mutations were essential for infectious particle production. Using CD81-deficient Huh7 cells, we further demonstrated the importance of A970T/I1312V/A2919T or A970T/C2419R/A2919T for virus assembly and that the I1312V/C2419R combination played a major role in virus release. Using a similar approach, we found that NS5B mutation F2994R, identified here from culture-adapted full-length TN viruses and a common NS3 helicase mutation (S1368P) derived from viable H77C and HCV-1 5-5A recombinants, initiated replication and culture adaptation of H77C containing LSG and TNcc(1a)-adaptive mutations. An H77C recombinant harboring 19 mutations (H77Ccc) replicated and spread efficiently after transfection and subsequent infection of naive Huh7.5 cells, reaching titers of 103.5and 104.4FFU/ml, respectively.IMPORTANCEHepatitis C virus (HCV) was discovered in 1989 with the cloning of the prototype strain HCV-1 genome. In 1997, two molecular clones of H77, the other HCV prototype strain, were shown to be infectious in chimpanzees, but notin vitro. HCV research was hampered by a lack of infectious cell culture systems, which became available only in 2005 with the discovery of JFH1 (genotype 2a), a genome that could establish infection in Huh7.5 cells. Recently, we developedin vitroinfectious clones for genotype 1a (TN), 2a (J6), and 2b (J8, DH8, and DH10) strains by identifying key adaptive mutations. Globally, genotype 1 is the most prevalent. Studies using HCV-1 and H77 prototype sequences have generated important knowledge on HCV. Thus, thein vitroinfectious clones developed here for these 1a strains will be of particular value in advancing HCV research. Moreover, our findings open new avenues for the culture adaptation of HCV isolates of different genotypes.

scholarly journals A role for B cells to transmit hepatitis C virus infection

2020 ◽  
Author(s):  
Isabelle Desombere ◽  
Freya Van Houtte ◽  
Ali Farhoudi ◽  
Lieven Verhoye ◽  
Caroline Buysschaert ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
B Cells ◽  
Neutralizing Antibody ◽  
Hcv Rna ◽  
Serum Samples ◽  
Cell Culture Systems ◽  
Hcv Transmission

Abstract Hepatitis C virus (HCV) is highly variable and transmits through infected blood to establish a chronic liver infection in the majority of patients. Our knowledge of the infectivity of clinical HCV strains is hampered by the lack of in vitro cell culture systems that support efficient viral replication. We previously reported that laboratory strains of HCV associated with non-permissive B cells could trans-infect hepatocytes and thereby evade host neutralizing antibody responses, suggesting a role for B cells in HCV transmission. To evaluate this hypothesis, we assessed the ability of B cells and sera from recent (<2 years) or chronic (≥ 2 years) infections to infect humanized liver chimeric mice. HCV was efficiently transmitted by B cells from chronically infected patients whereas the sera were non-infectious. In contrast, we noted that B cells from recently infected patients failed to transmit HCV to the mice, whereas all serum samples were infectious. Only patients with circulating anti-glycoprotein antibodies harbored genomic HCV-RNA in B cells. Taken together, our studies provide direct in vivo evidence for HCV transmission by B cells and these findings may have clinical implications for prophylactic and therapeutic antibody-based vaccine design.

scholarly journals Modeling hepatitis C virus kinetics during liver transplantation highlights the role of the liver in virus clearance

2020 ◽  
Author(s):  
Louis Shekhtman ◽  
Miquel Navasa ◽  
Natasha Sansone ◽  
Gonzalo Crespo ◽  
Gitanjali Subramanya ◽  
...  
Keyword(s):  
Cell Culture ◽  
Liver Transplantation ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Clearance Rate ◽  
Culture Medium ◽  
Viral Clearance ◽  
Huh7 Cells

AbstractWhile the liver, specifically hepatocytes, are widely accepted as the main source for hepatitis C virus (HCV) production, the role of the liver/hepatocytes in the clearance of circulating HCV remains largely unknown. Here we evaluated the function of the liver/hepatocytes in clearing virus from the circulation by investigating viral clearance during liver transplantation and from culture medium in vitro. Frequent HCV kinetic data during liver transplantation were recorded from 5 individuals throughout the anhepatic (AH) phase and for 4 hours after reperfusion (RP), along with recordings of fluid balances. Using mathematical modeling, the serum viral clearance rate, c, was estimated. Analogously, we monitored the clearance rate of HCV at 37°C from culture medium in vitro in the absence and presence of chronically infected Huh7 human hepatoma cells. During the AH phase, in 3 transplant cases viral levels remained at pre-AH levels, while in the other 2 cases HCV declined (half-life, t1/2~1h). Immediately post-RP, virus declined in a biphasic manner in Cases 1-4 consisting of an extremely rapid (median t1/2=5min) decline followed by a slower decline (HCV t1/2=67min). In Case 5, HCV remained at the same level post-RP as at the end of AH. Declines in virus level were not explained by adjusting for dilution from IV fluid and blood products. Consistent with what was observed in the majority of patients in the anhepatic phase, the t1/2 of HCV in cell culture was much longer in the absence of chronically HCV-infected Huh7 cells. Therefore, kinetic and modeling results from both in vivo liver transplantation cases and in vitro cell culture studies suggest that the liver plays a major role in clearing HCV from the circulation.

scholarly journals Comparative Analysis of Translation Efficiencies of Hepatitis C Virus 5′ Untranslated Regions among Intraindividual Quasispecies Present in Chronic Infection: Opposite Behaviors Depending on Cell Type

2000 ◽  
Vol 74 (22) ◽  
pp. 10827-10833 ◽  
Author(s):  
Julien Laporte ◽  
Isabelle Malet ◽  
Thibault Andrieu ◽  
Vincent Thibault ◽  
Jean-Jacques Toulme ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Expression System ◽  
Luciferase Expression ◽  
Hcv Rna ◽  
Translation Efficiency ◽  
Cell Type ◽  
Entry Site ◽  
Conserved Sequence

ABSTRACT Hepatitis C virus (HCV) RNA translation initiation is dependent on the presence of an internal ribosome entry site (IRES) that is found mostly in its 5′ untranslated region (5′ UTR). While exhibiting the most highly conserved sequence within the genome, the 5′ UTR accumulates small differences, which may be of biological and clinical importance. In this study, using a bicistronic dual luciferase expression system, we have examined the sequence of 5′ UTRs from quasispecies characterized in the serum of a patient chronically infected with HCV genotype 1a and its corresponding translational activity. Sequence heterogeneity between IRES elements led to important changes in their translation efficiency both in vitro and in different cell cultures lines, implying that interactions of RNA with related transacting factors may vary according to cell type. These data suggest that variants occasionally carried by the serum prior to reinfection could be selected toward different compartments of the same infected organism, thus favoring the hypothesis of HCV multiple tropism.

scholarly journals Interaction with a Ubiquitin-Like Protein Enhances the Ubiquitination and Degradation of Hepatitis C Virus RNA-Dependent RNA Polymerase

2003 ◽  
Vol 77 (7) ◽  
pp. 4149-4159 ◽  
Author(s):  
Lu Gao ◽  
Hong Tu ◽  
Stephanie T. Shi ◽  
Ki-Jeong Lee ◽  
Miyuki Asanaka ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Hcv Rna ◽  
Rna Dependent Rna Polymerase ◽  
Subgenomic Replicon ◽  
C Terminus ◽  
Huh7 Cells

ABSTRACT To identify potential cellular regulators of hepatitis C virus (HCV) RNA-dependent RNA polymerase (NS5B), we searched for cellular proteins interacting with NS5B protein by yeast two-hybrid screening of a human hepatocyte cDNA library. We identified a ubiquitin-like protein, hPLIC1 (for human homolog 1 of protein linking intergrin-associated protein and cytoskeleton), which is expressed in the liver (M. F. Kleijnen, A. H. Shih, P. Zhou, S. Kumar, R. E. Soccio, N. L. Kedersha, G. Gill, and P. M. Howley, Mol. Cell 6: 409-419, 2000). In vitro binding assays and in vivo coimmunoprecipitation studies confirmed the interaction between hPLIC1 and NS5B, which occurred through the ubiquitin-associated domain at the C terminus of the hPLIC1 protein. As hPLICs have been shown to physically associate with two E3 ubiquitin protein ligases as well as proteasomes (Kleijnen et al., Mol. Cell 6: 409-419, 2000), we investigated whether the stability and posttranslational modification of NS5B were affected by hPLIC1. A pulse-chase labeling experiment revealed that overexpression of hPLIC1, but not the mutant lacking the NS5B-binding domain, significantly shortened the half-life of NS5B and enhanced the polyubiquitination of NS5B. Furthermore, in Huh7 cells that express an HCV subgenomic replicon, the amounts of both NS5B and the replicon RNA were reduced by overexpression of hPLIC1. Thus, hPLIC1 may be a regulator of HCV RNA replication through interaction with NS5B.

scholarly journals Modeling Subgenomic Hepatitis C Virus RNA Kinetics during Treatment with Alpha Interferon

2009 ◽  
Vol 83 (13) ◽  
pp. 6383-6390 ◽  
Author(s):  
Harel Dahari ◽  
Bruno Sainz ◽  
Alan S. Perelson ◽  
Susan L. Uprichard
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Rna ◽  
Alpha Interferon ◽  
Hcv Rna ◽  
Second Phase ◽  
Inhibition Kinetics ◽  
Rna Levels

ABSTRACT Although replicons have been used to demonstrate hepatitis C virus (HCV) inhibition by alpha interferon (IFN-α), the detailed inhibition kinetics required to mathematically model HCV RNA decline have been lacking. Therefore, we measured genotype 1b subgenomic replicon (sg1b) RNA levels under various IFN-α concentrations to assess the inhibition kinetics of intracellular HCV RNA. During nine days of IFN-α treatment, sg1b RNA decreased in a biphasic, dose-dependent manner. Using frequent measurements to dissect these phases during IFN-α treatments of 100 and 250 U/ml revealed that the first-phase sg1b RNA decline began ∼12 h posttreatment, continued for 2 to 4 days, and then exhibited a distinct flat or slower second phase. Based on these data, we developed a mathematical model of IFN-α-induced intracellular sg1b RNA decline, and we show that the mechanism(s) mediating IFN-α inhibition of HCV acts primarily by reducing sg1b RNA amplification, with an additional effect on HCV RNA stability/degradation detectable at a dose of 250 U/ml IFN-α. While the extremely slow or flat second phase of viral RNA inhibition observed in vitro, in which there is little or no cell death, supports the in vivo modeling prediction that the more profound second-phase decline observed in IFN-α-treated patients reflects immune-mediated death/loss of productively infected cells, the second-phase decline in viral RNA with a dose of 250 U/ml IFN-α suggests that a further inhibition of intracellular HCV RNA levels may contribute as well. As such, dissection of HCV IFN-α inhibition kinetics in vitro has brought us closer to understanding the mechanism(s) by which IFN-α may be inhibiting HCV in vivo.

scholarly journals Biochemical Characterization of the Active Anti-Hepatitis C Virus Metabolites of 2,6-Diaminopurine Ribonucleoside Prodrug Compared to Sofosbuvir and BMS-986094

2016 ◽  
Vol 60 (8) ◽  
pp. 4659-4669 ◽  
Author(s):  
Maryam Ehteshami ◽  
Sijia Tao ◽  
Tugba Ozturk ◽  
Longhu Zhou ◽  
Jong Hyun Cho ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Biochemical Characterization ◽  
Nonstructural Protein ◽  
Nucleoside Triphosphate ◽  
Hcv Rna ◽  
Mitochondrial Rna ◽  
Active Metabolites

ABSTRACTRibonucleoside analog inhibitors (rNAI) target the hepatitis C virus (HCV) RNA-dependent RNA polymerase nonstructural protein 5B (NS5B) and cause RNA chain termination. Here, we expand our studies on β-d-2′-C-methyl-2,6-diaminopurine-ribonucleotide (DAPN) phosphoramidate prodrug 1 (PD1) as a novel investigational inhibitor of HCV. DAPN-PD1 is metabolized intracellularly into two distinct bioactive nucleoside triphosphate (TP) analogs. The first metabolite, 2′-C-methyl-GTP, is a well-characterized inhibitor of NS5B polymerase, whereas the second metabolite, 2′-C-methyl-DAPN-TP, behaves as an adenosine base analog.In vitroassays suggest that both metabolites are inhibitors of NS5B-mediated RNA polymerization. Additional factors, such as rNAI-TP incorporation efficiencies, intracellular rNAI-TP levels, and competition with natural ribonucleotides, were examined in order to further characterize the potential role of each nucleotide metabolitein vivo. Finally, we found that although both 2′-C-methyl-GTP and 2′-C-methyl-DAPN-TP were weak substrates for human mitochondrial RNA (mtRNA) polymerase (POLRMT)in vitro, DAPN-PD1 did not cause off-target inhibition of mtRNA transcription in Huh-7 cells. In contrast, administration of BMS-986094, which also generates 2′-C-methyl-GTP and previously has been associated with toxicity in humans, caused detectable inhibition of mtRNA transcription. Metabolism of BMS-986094 in Huh-7 cells leads to 87-fold higher levels of intracellular 2′-C-methyl-GTP than DAPN-PD1. Collectively, our data characterize DAPN-PD1 as a novel and potent antiviral agent that combines the delivery of two active metabolites.

scholarly journals The Cyclophilin Inhibitor SCY-635 Disrupts Hepatitis C Virus NS5A-Cyclophilin A Complexes

2012 ◽  
Vol 56 (7) ◽  
pp. 3888-3897 ◽  
Author(s):  
Sam Hopkins ◽  
Michael Bobardt ◽  
Udayan Chatterji ◽  
Jose A. Garcia-Rivera ◽  
Precious Lim ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Replication ◽  
Cyclophilin A ◽  
Hcv Rna ◽  
Dependent Manner ◽  
Mutant Proteins ◽  
Cypa Binding

ABSTRACTThe nonimmunosuppressive cyclophilin (Cyp) inhibitor SCY-635 blocks hepatitis C virus (HCV) replication bothin vitroandin vivoand represents a novel potent anti-HCV agent. However, its mechanism of action remains to be fully elucidated. A growing body of evidence suggests that cyclophilin A (CypA) is absolutely necessary for HCV replication and that the HCV nonstructural 5A (NS5A) protein serves as a main viral ligand for CypA. In this study, we examined the effect of SCY-635 on HCV replication. Specifically, we asked whether SCY-635 blocks HCV replication by targeting CypA-NS5A interactions. We also investigated the possibility that HCV can escape SCY-635 selection pressure and whether this resistance influences either CypA-NS5A interactions or the dependence of HCV on CypA. We found not only that SCY-635 efficiently inhibits HCV replication, but it is sufficient alone to clear HCV replicon-containing cells. We found that SCY-635 prevents CypA-NS5A interactions in a dose-dependent manner. SCY-635 prevents the contact between CypA and NS5A derived from genotypes 1 to 3. Together, these data suggest that NS5A-CypA interactions control HCV replication and that SCY-635 blocks viral replication by preventing the formation of these complexes. We also found that NS5A mutant proteins found in SCY-635-resistant HCV replicons behave similarly to wild-type NS5A in terms of both CypA binding and SCY-635-mediated dissociation and inhibition of CypA binding. However, the NS5A mutations found in SCY-635-resistant HCV replicons rescued viral replication in CypA-knockdown cells, suggesting that the NS5A mutations, which arosein vitrounder SCY-635 selection, do not alter the binding affinity of CypA for NS5A. These specific mutations in NS5A eliminate the dependence of HCV RNA replication on the expression of host CypA

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