Anti-retroviral drugs do not facilitate hepatitis C virus (HCV) infection in vitro

2012 ◽  
Vol 96 (1) ◽  
pp. 51-58 ◽  
Author(s):  
Lisa Sandmann ◽  
Matthew Wilson ◽  
David Back ◽  
Heiner Wedemeyer ◽  
Michael P. Manns ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Hcv Infection

scholarly journals In vitro hepatitis C virus infection and hepatic choline metabolism

2019 ◽  
Author(s):  
Kaelan Gobeil Odai ◽  
Conor O’Dwyer ◽  
Rineke Steenbergen ◽  
Tyler A. Shaw ◽  
Tyler M. Renner ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cultured Cells ◽  
Hcv Infection ◽  
Choline Uptake ◽  
Choline Transport ◽  
Choline Metabolism ◽  
Positive Strand Rna ◽  
Uptake And Metabolism

AbstractCholine is an essential nutrient required for normal neuronal and muscular development, as well as homeostatic regulation of hepatic metabolism. In the liver, choline is incorporated into the main eukaryotic phospholipid, phosphatidylcholine (PC) and can enter one carbon metabolism via mitochondrial oxidation. Hepatitis C virus (HCV) is a hepatotropic positive-strand RNA virus that similar to other positive-strand RNA viruses can impact phospholipid metabolism. In the current study we sought to interrogate the link between choline transport and early HCV infection. Namely, we aimed to investigate how HCV modulates markers of choline metabolism following in vitro infection, while subsequently assessing how the inhibition of choline uptake and metabolism upon concurrent HCV infection may alter early viral replication. Finally, we assessed whether these parameters were consistent between cells cultured in fetal bovine serum (FBS) or human serum (HS), conditions known to differentially affect in vitro HCV infection. We observed that choline transport in FBS-cultured Huh7.5 cells is facilitated by the intermediate affinity transporter choline transporter-like family (CTL), and that CTL1 expression and the incorporation of choline into PC is diminished in 24 h infected FBS-cultured cells. Reciprocally, limiting the availability of choline for PC synthesis resulted in increased HCV replication at this early stage. In chronically HS-cultured Huh7.5 cells, there were no differences in the expression of choline transporters upon HCV infection or alterations to viral replication when choline transport was inhibited compared to control treatments. However, inhibiting choline uptake and metabolism in this system significantly impaired the production of infectious virions in HS-cultured cells. These results suggest that in addition to a known role of choline kinase, the transport of choline, potentially via CTL1, might also represent an important and regulated process during HCV infection.Abstract Figure

scholarly journals In Vitro Hepatitis C Virus Infection and Hepatic Choline Metabolism

Viruses ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 108 ◽  
Author(s):  
Kaelan Gobeil Odai ◽  
Conor O’Dwyer ◽  
Rineke Steenbergen ◽  
Tyler A. Shaw ◽  
Tyler M. Renner ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Post Infection ◽  
Cultured Cells ◽  
Hcv Infection ◽  
Choline Uptake ◽  
Choline Metabolism ◽  
Positive Strand Rna ◽  
Uptake And Metabolism

Choline is an essential nutrient required for normal neuronal and muscular development, as well as homeostatic regulation of hepatic metabolism. In the liver, choline is incorporated into the main eukaryotic phospholipid, phosphatidylcholine (PC), and can enter one-carbon metabolism via mitochondrial oxidation. Hepatitis C virus (HCV) is a hepatotropic positive-strand RNA virus that similar to other positive-strand RNA viruses and can impact phospholipid metabolism. In the current study we sought to interrogate if HCV modulates markers of choline metabolism following in vitro infection, while subsequently assessing if the inhibition of choline uptake and metabolism upon concurrent HCV infection alters viral replication and infectivity. Additionally, we assessed whether these parameters were consistent between cells cultured in fetal bovine serum (FBS) or human serum (HS), conditions known to differentially affect in vitro HCV infection. We observed that choline transport in FBS- and HS-cultured Huh7.5 cells is facilitated by the intermediate affinity transporter, choline transporter-like family (CTL). HCV infection in FBS, but not HS-cultured cells diminished CTL1 transcript and protein expression at 24 h post-infection, which was associated with lower choline uptake and lower incorporation of choline into PC. No changes in other transporters were observed and at 96 h post-infection, all differences were normalized. Reciprocally, limiting the availability of choline for PC synthesis by use of a choline uptake inhibitor resulted in increased HCV replication at this early stage (24 h post-infection) in both FBS- and HS-cultured cells. Finally, in chronic infection (96 h post-infection), inhibiting choline uptake and metabolism significantly impaired the production of infectious virions. These results suggest that in addition to a known role of choline kinase, the transport of choline, potentially via CTL1, might also represent an important and regulated process during HCV infection.

scholarly journals Preclinical Characterization of the Novel Hepatitis C Virus NS3 Protease Inhibitor GS-9451

2013 ◽  
Vol 58 (2) ◽  
pp. 647-653 ◽  
Author(s):  
Huiling Yang ◽  
Margaret Robinson ◽  
Amoreena C. Corsa ◽  
Betty Peng ◽  
Guofeng Cheng ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Antiviral Activity ◽  
Protease Inhibitor ◽  
Hcv Infection ◽  
Cross Resistance ◽  
Protease Gene ◽  
Ns5a Inhibitor ◽  
Ns3 Protease

ABSTRACTGS-9451 is a selective hepatitis C virus (HCV) NS3 protease inhibitor in development for the treatment of genotype 1 (GT1) HCV infection. Key preclinical properties of GS-9451, includingin vitroantiviral activity, selectivity, cross-resistance, and combination activity, as well as pharmacokinetic properties, were determined. In multiple GT1a and GT1b replicon cell lines, GS-9451 had mean 50% effective concentrations (EC50s) of 13 and 5.4 nM, respectively, with minimal cytotoxicity; similar potency was observed in chimeric replicons encoding the NS3 protease gene of GT1 clinical isolates. GS-9451 was less active in GT2a replicon cells (EC50= 316 nM). Additive to synergisticin vitroantiviral activity was observed when GS-9451 was combined with other agents, including alpha interferon, ribavirin, and the polymerase inhibitors GS-6620 and tegobuvir (GS-9190), as well as the NS5A inhibitor ledipasvir (GS-5885). GS-9451 retained wild-type activity against multiple classes of NS5B and NS5A inhibitor resistance mutations. GS-9451 was stable in hepatic microsomes and hepatocytes from human and three other tested species. Systemic clearance was low in dogs and monkeys but high in rats. GS-9451 showed good oral bioavailability in all three species tested. In rats, GS-9451 levels were ∼40-fold higher in liver than plasma after intravenous dosing, and elimination of GS-9451 was primarily through biliary excretion. Together, these results are consistent with the antiviral activity observed in a recent phase 1b study. The results ofin vitrocross-resistance and combination antiviral assays support the ongoing development of GS-9451 in combination with other agents for the treatment of chronic HCV infection.

Amiodarone inhibits the entry and assembly steps of hepatitis C virus life cycle

2013 ◽  
Vol 125 (9) ◽  
pp. 439-448 ◽  
Author(s):  
Yuan-Lung Cheng ◽  
Keng-Hsueh Lan ◽  
Wei-Ping Lee ◽  
Szu-Han Tseng ◽  
Li-Rong Hung ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Inhibitory Effect ◽  
Hcv Infection ◽  
Receptor Expression ◽  
Current Standard ◽  
Protein Activity ◽  
Entry Site

HCV (hepatitis C virus) infection affects an estimated 180 million people in the world's population. Adverse effects occur frequently with current standard treatment of interferon and ribavirin, while resistance of new direct anti-viral agents, NS3 protease inhibitors, is a major concern because of their single anti-HCV mechanism against the viral factor. New anti-viral agents are needed to resolve the problems. Amiodarone, an anti-arrhythmic drug, has recently been shown to inhibit HCV infection in vitro. The detailed mechanism has yet to be clarified. The aim of the present study was to elucidate the molecular mechanism of the inhibitory effect of amiodarone on HCV life cycle. The effect of amiodarone on HCV life cycle was investigated in Huh-7.5.1 cells with HCVcc (cell culture-derived HCV), HCVpp (HCV pseudoviral particles), sub-genomic replicons, IRES (internal ribosomal entry site)-mediated translation assay, and intracellular and extracellular infectivity assays. The administration of amiodarone appeared to inhibit HCV entry independent of genotypes, which was attributed to the down-regulation of CD81 receptor expression. The inhibitory effect of amiodarone also manifested in the HCV assembly step, via the suppression of MTP (microsomal triacylglycerol transfer protein) activity. Amiodarone revealed no effects on HCV replication and translation. With the host factor-targeting characteristics, amiodarone may be an attractive agent for the treatment of HCV infection.

Hepatitis C Virus Infection Involves CD34+Hematopoietic Progenitor Cells in Hepatitis C Virus Chronic Carriers

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3328-3337 ◽  
Author(s):  
Domenico Sansonno ◽  
Claudio Lotesoriere ◽  
Vito Cornacchiulo ◽  
Massimo Fanelli ◽  
Pietro Gatti ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Progenitor Cells ◽  
Hcv Infection ◽  
Hematopoietic Progenitor Cells ◽  
Hcv Rna ◽  
Rt Pcr ◽  
Hematopoietic Progenitor ◽  
Cd34 Cells

Abstract Although hepatitis C virus (HCV) mainly affects hepatocytes, infection is widespread and involves immunologically privileged sites. Whether lymphoid cells represent further targets of early HCV infection, or whether other cells in the hematopoietic microenvironment may serve as a potential virus reservoir, is still unclear. We studied whether pluripotent hematopoietic CD34+ cells support productive HCV infection and can be used to establish an in vitro infection system for HCV. Six patients were selected as part of a cohort of HCV chronic carriers who developed a neoplastic disease. Reverse transcriptase-polymerase chain reaction (RT-PCR) and branched DNA signal amplification assays were used to detect and quantitate HCV RNA in extracted nucleic acids from purified bone marrow and peripheral blood CD34+ cells. Direct in situ RT-PCR, flow cytometry analysis, and immunocytochemistry were applied to demonstrate specific viral genomic sequences and structural and nonstructural virus-related proteins in intact cells. Results indicated that both positive and negative HCV RNA strands and viral proteins were present in CD34+ cells from all HCV-positive patients and in none of the controls. Additional experiments showed that a complete viral cycle took place in CD34+ cells in vitro. Spontaneous increases in viral titers indicated that virions were produced by infected hematopoietic progenitor cells. To further define the cellular tropism, we attempted to infect CD34+ cells in vitro. We were unable to demonstrate viral uptake by cells. These findings suggest that HCV replication can occur in the early differentiation stages of hematopoietic progenitor cells, and that they may be an important source of virus production. © 1998 by The American Society of Hematology.

scholarly journals Expanding the Host Range of Hepatitis C Virus through Viral Adaptation

mBio ◽  
2016 ◽  
Vol 7 (6) ◽  
Author(s):  
Markus von Schaewen ◽  
Marcus Dorner ◽  
Kathrin Hueging ◽  
Lander Foquet ◽  
Sherif Gerges ◽  
...  
Keyword(s):  
Animal Model ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Viral Envelope ◽  
Hcv Infection ◽  
Type I ◽  
Viral Adaptation ◽  
Mouse Hepatocytes

ABSTRACTHepatitis C virus (HCV) species tropism is incompletely understood. We have previously shown that at the level of entry, human CD81 and occludin (OCLN) comprise the minimal set of human factors needed for viral uptake into murine cells. As an alternative approach to genetic humanization, species barriers can be overcome by adapting HCV to use the murine orthologues of these entry factors. We previously generated a murine tropic HCV (mtHCV or Jc1/mCD81) strain harboring three mutations within the viral envelope proteins that allowed productive entry into mouse cell lines. In this study, we aimed to characterize the ability of mtHCV to enter and infect mouse hepatocytesin vivoandin vitro. Using a highly sensitive, Cre-activatable reporter, we demonstrate that mtHCV can enter mouse hepatocytesin vivoin the absence of any human cofactors. Viral entry still relied on expression of mouse CD81 and SCARB1 and was more efficient when mouse CD81 and OCLN were overexpressed. HCV entry could be significantly reduced in the presence of anti-HCV E2 specific antibodies, suggesting that uptake of mtHCV is dependent on viral glycoproteins. Despite mtHCV’s ability to enter murine hepatocytesin vivo, we did not observe persistent infection, even in animals with severely blunted type I and III interferon signaling and impaired adaptive immune responses. Altogether, these results establish proof of concept that the barriers limiting HCV species tropism can be overcome by viral adaptation. However, additional viral adaptations will likely be needed to increase the robustness of a murine model system for hepatitis C.IMPORTANCEAt least 150 million individuals are chronically infected with HCV and are at risk of developing serious liver disease. Despite the advent of effective antiviral therapy, the frequency of chronic carriers has only marginally decreased. A major roadblock in developing a vaccine that would prevent transmission is the scarcity of animal models that are susceptible to HCV infection. It is poorly understood why HCV infects only humans and chimpanzees. To develop an animal model for hepatitis C, previous efforts focused on modifying the host environment of mice, for example, to render them more susceptible to HCV infection. Here, we attempted a complementary approach in which a laboratory-derived HCV variant was tested for its ability to infect mice. We demonstrate that this engineered HCV strain can enter mouse liver cells but does not replicate efficiently. Thus, additional adaptations are likely needed to construct a robust animal model for HCV.

scholarly journals Preclinical Characterization and Human Microdose Pharmacokinetics of ITMN-8187, a Nonmacrocyclic Inhibitor of the Hepatitis C Virus NS3 Protease

2016 ◽  
Vol 61 (1) ◽  
Author(s):  
Ravi Rajagopalan ◽  
Lin Pan ◽  
Caralee Schaefer ◽  
John Nicholas ◽  
Sharlene Lim ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Oral Absorption ◽  
Antiviral Agents ◽  
Hcv Infection ◽  
Chimeric Mouse ◽  
Elimination Kinetics ◽  
Half Maximal Effective Concentration ◽  
Chronic Hcv

Abstract The current paradigm for the treatment of chronic hepatitis C virus (HCV) infection involves combinations of agents that act directly on steps of the HCV life cycle. Here we report the preclinical characteristics of ITMN-8187, a nonmacrocyclic inhibitor of the NS3/4A HCV protease. X-ray crystallographic studies of ITMN-8187 and simeprevir binding to NS3/4A protease demonstrated good agreement between structures. Low nanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 1, 2b, 4, 5, and 6. In cell-based potency assays, half-maximal reduction of genotype 1a and 1b HCV replicon RNA was afforded by 11 and 4 nM doses of ITMN-8187, respectively. Combinations of ITMN-8187 with other directly acting antiviral agents in vitro displayed additive antiviral efficacy. A 30-mg/kg of body weight dose of ITMN-8187 administered for 4 days yielded significant viral load reductions through day 5 in a chimeric mouse model of HCV. A 3-mg/kg oral dose administered to rats, dogs, or monkeys yielded concentrations in plasma 16 h after dosing that exceeded the half-maximal effective concentration of ITMN-8187. Human microdose pharmacokinetics showed low intersubject variability and prolonged oral absorption with first-order elimination kinetics compatible with once-daily dosing. These preclinical characteristics compare favorably with those of other NS3/4A inhibitors approved for the treatment of chronic HCV infection.

scholarly journals Hepatitis C Virus Enhances the Invasiveness of Hepatocellular Carcinoma via EGFR-Mediated Invadopodia Formation and Activation

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1395 ◽  
Author(s):  
Ninio ◽  
Nissani ◽  
Meirson ◽  
Domovitz ◽  
Genna ◽  
...  
Keyword(s):  
Hepatocellular Carcinoma ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Tyrosine Phosphatase ◽  
Hcv Infection ◽  
Invasive Cancer ◽  
Cell Protein ◽  
Invadopodia Formation

Hepatocellular carcinoma (HCC) represents the fifth most common cancer worldwide and the third cause of cancer-related mortality. Hepatitis C virus (HCV) is the leading cause of chronic hepatitis, which often results in liver fibrosis, cirrhosis, and eventually HCC. HCV is the most common risk factor for HCC in western countries and leads to a more aggressive and invasive disease with poorer patient survival rates. However, the mechanism by which the virus induces the metastatic spread of HCC tumor cells through the regulation of invadopodia, the key features of invasive cancer, is still unknown. Here, the integration of transcriptome with functional kinome screen revealed that HCV infection induced invasion and invadopodia-related gene expression combined with activation of host cell tyrosine kinases, leading to invadopodia formation and maturation and consequent cell invasiveness in vitro and in vivo. The promotion of invadopodia following HCV infection was mediated by the sustained stimulation of epidermal growth factor receptor (EGFR) via the viral NS3/4A protease that inactivates the T-cell protein tyrosine phosphatase (TC-PTP), which inhibits EGFR signaling. Characterization of an invadopodia-associated gene signature in HCV-mediated HCC tumors correlated with the invasiveness of HCC and poor patient prognosis. These findings might lead to new prognostic and therapeutic strategies for virus-mediated invasive cancer.

scholarly journals Hepatitis C virus infection of primary tupaia hepatocytes leads to selection of quasispecies variants, induction of interferon-stimulated genes and NF-κB nuclear translocation

2005 ◽  
Vol 86 (11) ◽  
pp. 3065-3074 ◽  
Author(s):  
Anunciata Guitart ◽  
José-Ignacio Riezu-Boj ◽  
Edurne Elizalde ◽  
Esther Larrea ◽  
Carmen Berasain ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Semliki Forest Virus ◽  
Nuclear Translocation ◽  
Natural Infection ◽  
Hcv Infection ◽  
Interferon Response ◽  
Cell Phenotype

Systems for in vitro culture of Hepatitis C virus (HCV) are essential tools to analyse virus–cell interactions and to investigate relevant pathophysiological aspects of HCV infection. Although the HCV replicon methodology has increased our understanding of HCV biology, this system does not reproduce the natural infection. Recently, tupaia (Tupaia belangeri chinensis) hepatocytes have been utilized for in vitro culture of HCV. In the present work, primary tupaia hepatocytes infected in vitro with HCV were used to analyse the evolution of HCV quasispecies in infected cells and the ability of the virus to influence antiviral and proinflammatory responses in cells sustaining virus replication. The results confirmed the potential of tupaia hepatocytes as a model for HCV infection, although this system is limited by rapid loss of differentiated cell phenotype in culture. These findings revealed an extraordinary plasticity of HCV quasispecies, which underwent rapid evolution to tupaia-tropic variants as early as 24 h after infection. It was also shown that HCV could activate interferon-sensitive genes, albeit modestly in comparison with other viruses such as Semliki Forest virus. Importantly, HCV activated NF-κB in primary hepatocytes and upregulated NF-κB-responsive genes including the chemokines MCP-1 and CXCL2 (MIP-2). This effect may play a role in induction of the hepatic inflammatory reaction in vivo. In summary, HCV quasispecies adapt rapidly to the specific biology of the host and HCV stimulates a blunted interferon response while inducing a proinflammatory phenotype in the infected cell.

scholarly journals Initiation of Hepatitis C Virus Infection Is Dependent on Cholesterol and Cooperativity between CD81 and Scavenger Receptor B Type I

2006 ◽  
Vol 81 (1) ◽  
pp. 374-383 ◽  
Author(s):  
Sharookh B. Kapadia ◽  
Heidi Barth ◽  
Thomas Baumert ◽  
Jane A. McKeating ◽  
Francis V. Chisari
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Scavenger Receptor ◽  
Hcv Infection ◽  
Cellular Proteins ◽  
Type I ◽  
Membrane Domains ◽  
Hepatitis C Virus Infection ◽  
The Past

ABSTRACT In the past several years, a number of cellular proteins have been identified as candidate entry receptors for hepatitis C virus (HCV) by using surrogate models of HCV infection. Among these, the tetraspanin CD81 and scavenger receptor B type I (SR-BI), both of which localize to specialized plasma membrane domains enriched in cholesterol, have been suggested to be key players in HCV entry. In the current study, we used a recently developed in vitro HCV infection system to demonstrate that both CD81 and SR-BI are required for authentic HCV infection in vitro, that they function cooperatively to initiate HCV infection, and that CD81-mediated HCV entry is, in part, dependent on membrane cholesterol.

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