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 Combination of a Hepatitis C Virus NS3-NS4A Protease Inhibitor and Alpha Interferon Synergistically Inhibits Viral RNA Replication and Facilitates Viral RNA Clearance in Replicon Cells

2004 ◽  
Vol 48 (12) ◽  
pp. 4784-4792 ◽  
Author(s):  
Kai Lin ◽  
Ann D. Kwong ◽  
Chao Lin
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Adverse Effects ◽  
Protease Inhibitor ◽  
Standard Of Care ◽  
Rna Replication ◽  
Viral Rna ◽  
Alpha Interferon ◽  
Hcv Rna

ABSTRACT The present standard of care for hepatitis C virus (HCV) infection is pegylated alpha interferon (IFN-α) in combination with ribavirin. However, specific antivirals such as HCV NS3-NS4A protease inhibitors are now in clinical development, and these agents can potentially be used in combination with the present treatments. Therefore, it is important to investigate the potential benefits or adverse effects of these new combinations by using available in vitro HCV culture systems first. In the present study we demonstrate that the combination of a specific HCV NS3-NS4A protease inhibitor and IFN-α synergistically inhibits HCV RNA replication in replicon cells, with little or no increase in cytotoxicity. Furthermore, the benefit of the combination was sustained over time, such that a greater than 3-log reduction in HCV RNA levels was achieved following 9 days of treatment. The viral RNA appeared to be cleared from the replicon cells after 14 days of treatment, and no viral RNA rebound was observed upon withdrawal of the inhibitors. In each case, the antiviral effects obtained with higher concentrations of either the protease inhibitor alone or IFN-α alone can be achieved by a combination of both agents at lower concentrations, which may potentially reduce the risk of possible adverse effects associated with high doses of either agent.

scholarly journals mTORC1 Restricts Hepatitis C Virus Replication Through ULK1-mediated Suppression of miR-122 and Facilitates Post-replication Events

2018 ◽  
Author(s):  
Manish Kumar Johri ◽  
Hiren Vasantrai Lashkari ◽  
Dhiviya Vedagiri ◽  
Divya Gupta ◽  
Krishnan Harinivas Harshan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Infection ◽  
Virus Replication ◽  
Viral Rna ◽  
Hcv Rna ◽  
Significant Drop ◽  
Mechanistic Target Of Rapamycin ◽  
Mtorc1 Activation ◽  
Rna Levels

ABSTRACTMechanistic target of rapamycin (mTOR) is an important kinase that assimilates several upstream signals including viral infection and facilitates appropriate response by the cell through two unique complexes mTORC1 and mTORC2. Here, we demonstrate that mTORC1 is activated early during HCV infection as antiviral response. Pharmacological inhibition of mTORC1 promoted HCV replication as suggested by elevated levels of HCV (+) and (-) RNA strands. This was accompanied by significant drop in extracellular HCV RNA levels indicating defective post-replication stages. The increase in viral RNA levels failed to augment intracellular infectious virion levels, suggesting that mTORC1 inhibition is detrimental to post-replication steps. Lower infectivity of the supernatant confirmed this observation. Depletion of Raptor and ULK1 accurately reproduced these results suggesting that mTORC1 imparted these effects on HCV through mTORC1-ULK1 arm. Interestingly, ULK1 depletion resulted in increased levels of miR-122, a critical host factor for HCV replication, thus revealing a new mechanism of regulation by ULK1. The binary effect of mTORC1 on HCV replication and egress suggests that mTORC1-ULK1 could be critical in replication: egress balance. Interestingly we discover that ULK1 depletion did not interfere with autophagy in Huh7.5 cells and hence the effects on HCV replication and post-replication events are not resultant of involvement of autophagy. Our studies demonstrate an overall ULK1 mediated anti-HCV function of mTORC1 and identifies an ULK1-independent autophagy that allows HCV replication in spite of mTORC1 activation.

scholarly journals Poly(I:C) and Lipopolysaccharide Innate Sensing Functions of Circulating Human Myeloid Dendritic Cells Are Affected In Vivo in Hepatitis C Virus-Infected Patients

2007 ◽  
Vol 81 (11) ◽  
pp. 5537-5546 ◽  
Author(s):  
Ian Gaël Rodrigue-Gervais ◽  
Loubna Jouan ◽  
Geneviève Beaulé ◽  
Dominike Sauvé ◽  
Julie Bruneau ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Poly I:C ◽  
Hcv Rna ◽  
Genomic Rna ◽  
Tnf Α ◽  
Wide Range ◽  
Rna Levels

ABSTRACT The role of peripheral dendritic cells (DCs) in hepatitis C virus (HCV) infection is unclear. To determine if persistent infection exerts an inhibitory pressure on HCV-specific innate responses, we analyzed DC function in blood through quantification of cell-associated HCV RNA levels in conjunction with multiparametric flow cytometry analysis of pathogen recognition receptor-induced cytokine expression. Independently of the serum viral load, fluorescence-activated cell sorter-purified total DCs had a wide range of cell-associated HCV genomic RNA copy numbers (mean log10, 5.0 per 106 cells; range, 4.3 to 5.8). Here we report that for viremic patients with high viral loads in their total DCs, the myeloid DC (MDC) subset displayed impaired expression of interleukin-12 (IL-12) and tumor necrosis factor alpha (TNF-α) but normal IL-6 or chemokine CCL3 expression in response to poly(I:C) and lipopolysaccharide (LPS). IL-6-expressing cells from this subgroup of viremic patients demonstrated a significant increase (sixfold more) in TNF-α− IL-12− cell frequency compared to healthy donors (mean, 38.8% versus 6.5%; P < 0.0001), indicating a functional defect in a subpopulation of cytokine-producing MDCs (∼6% of MDCs). Attenuation of poly(I:C) and LPS innate sensing was HCV RNA density dependent and did not correlate with viremia or deficits in circulating MDC frequencies in HCV-infected patients. Monocytes from these patients were functionally intact, responding normally on a per-cell basis following stimulation, independent of cell-associated HCV RNA levels. Taken together, these data indicate that detection of HCV genomic RNA in DCs and loss of function in the danger signal responsiveness of a small proportion of DCs in vivo are interrelated rather than independent phenomena.

scholarly journals Preclinical Profile and Clinical Efficacy of a Novel Hepatitis C Virus NS5A Inhibitor, EDP-239

2016 ◽  
Vol 60 (10) ◽  
pp. 6207-6215 ◽  
Author(s):  
Christopher M. Owens ◽  
Bradley B. Brasher ◽  
Alex Polemeropoulos ◽  
Michael H. J. Rhodin ◽  
Nicole McAllister ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Nonstructural Protein ◽  
Viral Rna ◽  
Controlled Clinical Trial ◽  
Genotype 1A ◽  
Pharmacokinetic Properties ◽  
Direct Acting

ABSTRACTEDP-239, a novel hepatitis C virus (HCV) inhibitor targeting nonstructural protein 5A (NS5A), has been investigatedin vitroandin vivo. EDP-239 is a potent, selective inhibitor with potency at picomolar to nanomolar concentrations against HCV genotypes 1 through 6. In the presence of human serum, the potency of EDP-239 was reduced by less than 4-fold. EDP-239 is additive to synergistic with other direct-acting antivirals (DAAs) or host-targeted antivirals (HTAs) in blocking HCV replication and suppresses the selection of resistancein vitro. Furthermore, EDP-239 retains potency against known DAA- or HTA-resistant variants, with half-maximal effective concentrations (EC50s) equivalent to those for the wild type. In a phase I, single-ascending-dose, placebo-controlled clinical trial, EDP-239 demonstrated excellent pharmacokinetic properties that supported once daily dosing. A single 100-mg dose of EDP-239 resulted in reductions in HCV genotype 1a viral RNA of >3 log10IU/ml within the first 48 h after dosing and reductions in genotype 1b viral RNA of >4-log10IU/ml within 96 h. (This study has been registered at ClinicalTrials.gov under identifier NCT01856426.)

scholarly journals Modulation of alpha interferon anti-hepatitis C virus activity by ISG15

2009 ◽  
Vol 90 (12) ◽  
pp. 2929-2939 ◽  
Author(s):  
Pong Kian Chua ◽  
Matthew F. McCown ◽  
Sonal Rajyaguru ◽  
Simran Kular ◽  
Ram Varma ◽  
...  
Keyword(s):  
Gene Expression ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Specific Effect ◽  
Alpha Interferon ◽  
Hcv Rna ◽  
Moderate Increase ◽  
Knock Down ◽  
Inducible Genes

ISG15 has recently been reported to possess antiviral properties against viruses, both in vivo and in vitro. Knock-down of ISG15 gene expression by small interfering RNA followed by alpha interferon (IFN-α) treatment in Huh-7 cells resulted in an increased phenotypic sensitivity to IFN-α, as determined by measuring hepatitis C virus (HCV) RNA replication inhibition in stably transfected HCV replicon cells and in cells infected with genotype 1a HCVcc (infectious HCV). This IFN-α-specific effect, which was not observed with IFN-γ, correlated with an increase in expression of the IFN-α-inducible genes IFI6, IFITM3, OAS1 and MX1, whereas the expression of the non-IFN-α-inducible genes PTBP-1 and JAK1 remained unchanged. It has previously been reported that, unlike ISG15 knock-down, increased sensitivity to IFN-α after knock-down of USP18 occurs through the prolonged phosphorylation of STAT-1. Combination knock-down of ISG15 and USP18 resulted in a moderate increase in IFN-α-inducible gene expression compared with single ISG15 or USP18 knock-down. Furthermore, the phenotype of increased gene expression after ISG15 knock-down and IFN-α treatment was also observed in non-hepatic cell lines A549 and HeLa. Taken together, these results reveal a novel function for ISG15 in the regulation of the IFN-α pathway and its antiviral effect.

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 VX-950, a Novel Hepatitis C Virus (HCV) NS3-4A Protease Inhibitor, Exhibits Potent Antiviral Activities in HCV Replicon Cells

2006 ◽  
Vol 50 (5) ◽  
pp. 1813-1822 ◽  
Author(s):  
Kai Lin ◽  
Robert B. Perni ◽  
Ann D. Kwong ◽  
Chao Lin
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Agents ◽  
Alpha Interferon ◽  
Hcv Rna ◽  
Resistance Mutations ◽  
In Vitro Characterization ◽  
Antiviral Activities

ABSTRACT The NS3-4A serine protease of hepatitis C virus (HCV) is essential for viral replication and therefore has been one of the most attractive targets for developing specific antiviral agents against HCV. VX-950, a highly selective, reversible, and potent peptidomimetic inhibitor of the HCV NS3-4A protease, is currently in clinical development for the treatment of hepatitis C. In this report, we describe the in vitro characterization of anti-HCV activities of VX-950 in subgenomic HCV replicon cells. Incubation with VX-950 resulted in a time- and dose-dependent reduction of HCV RNA and proteins in replicon cells. Moreover, following a 2-week incubation with VX-950, a reduction in HCV RNA levels of 4.7 log10 was observed, and this reduction resulted in elimination of HCV RNA from replicon cells, since there was no rebound in replicon RNA after withdrawal of the inhibitor. The combination of VX-950 and alpha interferon was additive to moderately synergistic in reducing HCV RNA in replicon cells with no significant increase in cytotoxicity. The benefit of the combination was sustained over time: a 4-log10 reduction in HCV RNA level was achieved following a 9-day incubation with VX-950 and alpha interferon at lower concentrations than when either VX-950 or alpha interferon was used alone. The combination of VX-950 and alpha interferon also suppressed the emergence of in vitro resistance mutations against VX-950 in replicon cells.

scholarly journals Relationships between Hepatitis C Virus Replication and CXCL-8 Production In Vitro

2006 ◽  
Vol 80 (16) ◽  
pp. 7885-7893 ◽  
Author(s):  
Bon Chang A. Koo ◽  
Paula McPoland ◽  
Jessica P. Wagoner ◽  
Olivia J. Kane ◽  
Volker Lohmann ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Expression ◽  
Hcv Rna ◽  
Protein Levels ◽  
Huh7 Cells ◽  
Mrna And Protein Expression ◽  
Chronic Hcv ◽  
Rna Levels

ABSTRACT The chemokine CXCL-8 (interleukin-8) is induced by many viruses, including hepatitis C virus (HCV). In the current study, we examined CXCL-8 levels in the context of acute and chronic HCV replication in vitro. Two different small interfering RNAs were used to silence CXCL-8 mRNA and protein expression in Huh7 and BB7 replicon cells. HCV RNA synthesis in BB7 cells was inhibited by CXCL-8 knockdown. Furthermore, antibody neutralization of endogenous CXCL-8 activity inhibited HCV replication, while addition of recombinant human CXCL-8 stimulated NS5A protein expression. Moreover, CXCL-8 protein levels correlated positively with HCV RNA levels in four independent subgenomic and genomic replicon lines (R = 0.41, P = 0.0013). However, CXCL-8 mRNA levels correlated inversely with CXCL-8 protein and HCV RNA levels in all replicon lines and in Huh7 cells. Transient replication assays with strongly permissive and weakly permissive Huh7 cells and three independent subgenomic replicons with various replicative capacities revealed that CXCL-8 protein levels were higher in weakly than in strongly permissive cells. The JFH-1 subgenomic replicon, which replicated to high levels in both strongly and weakly permissive Huh7 cells, induced CXCL-8 protein to high levels in both cell types. The data indicate that in the replicon system, CXCL-8 protein levels are positively associated with chronic HCV replication and that CXCL-8 removal inhibits HCV replication. During acute HCV replication, CXCL-8 production may be inhibitory to viruses with low replicative capacity. The data underscore the complex regulation of CXCL-8 mRNA and protein expression and further suggest that in addition to contributing to HCV pathology via proinflammatory actions, CXCL-8 may have opposing antiviral and proviral effects depending on the level of HCV replication, the cellular context, and whether the infection is acute or chronic.

scholarly journals New Antiviral Pathway That Mediates Hepatitis C Virus Replicon Interferon Sensitivity through ADAR1

2005 ◽  
Vol 79 (10) ◽  
pp. 6291-6298 ◽  
Author(s):  
Deborah R. Taylor ◽  
Montserrat Puig ◽  
Miriam E. R. Darnell ◽  
Kathleen Mihalik ◽  
Stephen M. Feinstone
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Therapeutic Strategy ◽  
Viral Infections ◽  
Viral Rna ◽  
Hcv Rna ◽  
Double Stranded Rna ◽  
Translation Inhibition ◽  
Antiviral Role

ABSTRACT While many clinical hepatitis C virus (HCV) infections are resistant to alpha interferon (IFN-α) therapy, subgenomic in vitro self-replicating HCV RNAs (HCV replicons) are characterized by marked IFN-α sensitivity. IFN-α treatment of replicon-containing cells results in a rapid loss of viral RNA via translation inhibition through double-stranded RNA-activated protein kinase (PKR) and also through a new pathway involving RNA editing by an adenosine deaminase that acts on double-stranded RNA (ADAR1). More than 200 genes are induced by IFN-α, and yet only a few are attributed with an antiviral role. We show that inhibition of both PKR and ADAR1 by the addition of adenovirus-associated RNA stimulates replicon expression and reduces the amount of inosine recovered from RNA in replicon cells. Small inhibitory RNA, specific for ADAR1, stimulated the replicon 40-fold, indicating that ADAR1 has a role in limiting replication of the viral RNA. This is the first report of ADAR's involvement in a potent antiviral pathway and its action to specifically eliminate HCV RNA through adenosine to inosine editing. These results may explain successful HCV replicon clearance by IFN-α in vitro and may provide a promising new therapeutic strategy for HCV as well as other viral infections.

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.

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