In vivo detection of hepatitis C virus (HCV) RNA in the brain in a case of encephalitis: evidence for HCV neuroinvasion

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.

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Novel Robust Hepatitis C Virus Mouse Efficacy Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00413-06 ◽

2006 ◽

Vol 50 (10) ◽

pp. 3260-3268 ◽

Cited By ~ 22

Author(s):

Qing Zhu ◽

Yoko Oei ◽

Dirk B. Mendel ◽

Evelyn N. Garrett ◽

Montesa B. Patawaran ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Protease Inhibitor ◽

Small Animal ◽

Rna Replication ◽

Treatment Withdrawal ◽

Whole Body ◽

Hcv Rna ◽

Whole Body Imaging

ABSTRACT The lack of a robust small-animal model for hepatitis C virus (HCV) has hindered the discovery and development of novel drug treatments for HCV infections. We developed a reproducible and easily accessible xenograft mouse efficacy model in which HCV RNA replication is accurately monitored in vivo by real-time, noninvasive whole-body imaging of gamma-irradiated SCID mice implanted with a mouse-adapted luciferase replicon-containing Huh-7 cell line (T7-11). The model was validated by demonstrating that both a small-molecule NS3/4A protease inhibitor (BILN 2061) and human alpha interferon (IFN-α) decreased HCV RNA replication and that treatment withdrawal resulted in a rebound in replication, which paralleled clinical outcomes in humans. We further showed that protease inhibitor and IFN-α combination therapy was more effective in reducing HCV RNA replication than treatment with each compound alone and supports testing in humans. This robust mouse efficacy model provides a powerful tool for rapid evaluation of potential anti-HCV compounds in vivo as part of aggressive drug discovery efforts.

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Expression of hepatitis C virus proteins in epithelial intestinal cells in vivo

Journal of General Virology ◽

10.1099/vir.0.80071-0 ◽

2004 ◽

Vol 85 (9) ◽

pp. 2515-2523 ◽

Cited By ~ 44

Author(s):

Séverine Deforges ◽

Alexey Evlashev ◽

Magali Perret ◽

Mireille Sodoyer ◽

Stéphane Pouzol ◽

...

Keyword(s):

Small Intestine ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Core Protein ◽

Lipoprotein Metabolism ◽

Hcv Rna ◽

Hybrid Particles ◽

Hepatitis C Infection ◽

Hcv Antibody

Previous work on hepatitis C virus (HCV) led to the discovery of a new form of virus particle associating virus and lipoprotein elements. These hybrid particles (LVP for lipo-viro-particles) are enriched in triglycerides and contain at least apolipoprotein B (apoB), HCV RNA and core protein. These findings suggest that LVP synthesis could occur in liver and intestine, the two main organs specialized in the production of apoB-containing lipoprotein. To identify the site of LVP production, the genetic diversity and phylogenetic relationship of HCV quasispecies from purified LVP, whole serum and liver biopsies from chronically infected patients were studied. HCV quasispecies from LVP and liver differed significantly, suggesting that LVP were not predominantly synthesized in the liver but might also originate in the intestine. The authors therefore searched for the presence of HCV in the small intestine. Paraffin-embedded intestinal biopsies from 10 chronically HCV-infected patients and from 12 HCV RNA-negative controls (10 anti-HCV antibody-negative and two anti-HCV antibody-positive patients) were tested for HCV protein expression. HCV NS3 and NS5A proteins were stained in small intestine epithelial cells in four of the 10 chronically infected patients, and not in controls. Cells expressing HCV proteins were apoB-producing enterocytes but not mucus-secreting cells. These data indicate that the small intestine can be infected by HCV, and identify this organ as a potential reservoir and replication site. This further emphasizes the interaction between lipoprotein metabolism and HCV, and offers new insights into hepatitis C infection and pathophysiology.

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A role for B cells to transmit hepatitis C virus infection

10.21203/rs.3.rs-41273/v1 ◽

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.

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A 7-Deaza-Adenosine Analog Is a Potent and Selective Inhibitor of Hepatitis C Virus Replication with Excellent Pharmacokinetic Properties

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.10.3944-3953.2004 ◽

2004 ◽

Vol 48 (10) ◽

pp. 3944-3953 ◽

Cited By ~ 165

Author(s):

David B. Olsen ◽

Anne B. Eldrup ◽

Linda Bartholomew ◽

Balkrishen Bhat ◽

Michele R. Bosserman ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Treatment Options ◽

Lethal Dose ◽

Current Treatment ◽

Hcv Infection ◽

Hcv Rna ◽

Inhibitory Potency ◽

Pharmacokinetic Properties

ABSTRACT Improved treatments for chronic hepatitis C virus (HCV) infection are needed due to the suboptimal response rates and deleterious side effects associated with current treatment options. The triphosphates of 2′-C-methyl-adenosine and 2′-C-methyl-guanosine were previously shown to be potent inhibitors of the HCV RNA-dependent RNA polymerase (RdRp) that is responsible for the replication of viral RNA in cells. Here we demonstrate that the inclusion of a 7-deaza modification in a series of purine nucleoside triphosphates results in an increase in inhibitory potency against the HCV RdRp and improved pharmacokinetic properties. Notably, incorporation of the 7-deaza modification into 2′-C-methyl-adenosine results in an inhibitor with a 20-fold-increased potency as the 5′-triphosphate in HCV RdRp assays while maintaining the inhibitory potency of the nucleoside in the bicistronic HCV replicon and with reduced cellular toxicity. In contrast, while 7-deaza-2′-C-methyl-GTP also displays enhanced inhibitory potency in enzyme assays, due to poor cellular penetration and/or metabolism, the nucleoside does not inhibit replication of a bicistronic HCV replicon in cell culture. 7-Deaza-2′-C-methyl-adenosine displays promising in vivo pharmacokinetics in three animal species, as well as an acute oral lethal dose in excess of 2,000 mg/kg of body weight in mice. Taken together, these data demonstrate that 7-deaza-2′-C-methyl-adenosine is an attractive candidate for further investigation as a potential treatment for HCV infection.

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

Journal of Virology ◽

10.1128/jvi.02334-06 ◽

2007 ◽

Vol 81 (9) ◽

pp. 4405-4411 ◽

Cited By ~ 85

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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The C-Terminal Transmembrane Domain of Hepatitis C Virus (HCV) RNA Polymerase Is Essential for HCV Replication In Vivo

Journal of Virology ◽

10.1128/jvi.78.7.3797-3802.2004 ◽

2004 ◽

Vol 78 (7) ◽

pp. 3797-3802 ◽

Cited By ~ 27

Author(s):

Ki Jeong Lee ◽

Jinah Choi ◽

Jing-hsiung Ou ◽

Michael M. C. Lai

Keyword(s):

Amino Acids ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Rna Polymerase ◽

Rna Synthesis ◽

Transmembrane Domain ◽

Hcv Rna ◽

Membrane Association ◽

Subgenomic Replicon

ABSTRACT Hepatitis C virus (HCV) RNA replication is dependent on the enzymatic activities of the viral RNA-dependent RNA polymerase NS5B, which is a membrane-anchored protein. Recombinant NS5B lacking the C-terminal transmembrane domain (21 amino acids) is enzymatically active. To address the role of this domain in HCV replication in vivo, we introduced a series of mutations into the NS5B of an HCV subgenomic replicon and examined the replication capabilities of the resultant mutants by a colony formation assay. Replicons lacking the transmembrane domain did not yield any colonies. Furthermore, when Huh-7 cells harboring the HCV subgenomic replicon were treated with a synthetic peptide consisting of the NS5B transmembrane domain fused to the antennapedia peptide, the membrane association of NS5B was completely disrupted. Correspondingly, the HCV RNA titer was reduced by approximately 50%. A scrambled peptide used as a control did not have any effects. These findings suggest that the membrane association of NS5B facilitates HCV RNA synthesis. However, a related transmembrane domain derived from bovine viral diarrhea virus could not replace the HCV NS5B transmembrane segment. This finding suggests that the C-terminal 21 amino acids not only have a membrane-anchoring function but also may perform additional functions for RNA synthesis in vivo.

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Alpha Interferon Induces Distinct Translational Control Programs To Suppress Hepatitis C Virus RNA Replication

Journal of Virology ◽

10.1128/jvi.77.7.3898-3912.2003 ◽

2003 ◽

Vol 77 (7) ◽

pp. 3898-3912 ◽

Cited By ~ 171

Author(s):

Chunfu Wang ◽

Jill Pflugheber ◽

Rhea Sumpter ◽

Donald L. Sodora ◽

Daniel Hui ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Translational Control ◽

Rna Replication ◽

Viral Rna ◽

Hcv Rna ◽

Control Programs ◽

Eif2α Phosphorylation ◽

Ifn Treatment

ABSTRACT Hepatitis C virus (HCV) infection is treated with interferon (IFN)-based therapy. The mechanisms by which IFN suppresses HCV replication are not known, and only limited efficacy is achieved with therapy because the virus directs mechanisms to resist the host IFN response. In the present study we characterized the effects of IFN action upon the replication of two distinct quasispecies of an HCV replicon whose encoded NS5A protein exhibited differential abilities to bind and inhibit protein kinase R (PKR). Metabolic labeling experiments revealed that IFN had little overall effect upon HCV protein stability or polyprotein processing but specifically blocked translation of the HCV RNA, such that the replication of both viral quasispecies was suppressed by IFN treatment of the Huh7 host cells. However, within cells expressing an NS5A variant that inhibited PKR, we observed a reduced level of eukaryotic initiation factor 2 alpha subunit (eIF2α) phosphorylation and a concomitant increase in HCV protein synthetic rates, enhancement of viral RNA replication, and a partial rescue of viral internal ribosome entry site (IRES) function from IFN suppression. Assessment of the ribosome distribution of the HCV replicon RNA demonstrated that the NS5A-mediated block in eIF2α phosphorylation resulted in enhanced recruitment of the HCV RNA into polyribosome complexes in vivo but only partially rescued the RNA from polyribosome dissociation induced by IFN treatment. Examination of cellular proteins associated with HCV-translation complexes in IFN-treated cells identified the P56 protein as an eIF3-associated factor that fractionated with the initiator ribosome-HCV RNA complex. Importantly, we found that P56 could independently suppress HCV IRES function both in vitro and in vivo, but a mutant P56 that was unable to bind eIF3 had no suppressive action. We conclude that IFN blocks HCV replication through translational control programs involving PKR and P56 to, respectively, target eIF2- and eIF3-dependent steps in the viral RNA translation initiation process.

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

Journal of Virology ◽

10.1128/jvi.77.7.4149-4159.2003 ◽

2003 ◽

Vol 77 (7) ◽

pp. 4149-4159 ◽

Cited By ~ 69

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.

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

Journal of Virology ◽

10.1128/jvi.02612-08 ◽

2009 ◽

Vol 83 (13) ◽

pp. 6383-6390 ◽

Cited By ~ 42

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.

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