scholarly journals Effect of Cell Polarization on Hepatitis C Virus Entry

2007 ◽  
Vol 82 (1) ◽  
pp. 461-470 ◽  
Author(s):  
Christopher J. Mee ◽  
Joe Grove ◽  
Helen J. Harris ◽  
Ke Hu ◽  
Peter Balfe ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Scavenger Receptor ◽  
Cell Polarization ◽  
Receptor Expression ◽  
Cell Junction ◽  
Apical Surface ◽  
Viral Receptor ◽  
Lateral Cell

ABSTRACT The primary reservoir for hepatitis C virus (HCV) replication in vivo is believed to be hepatocytes within the liver. Three host cell molecules have been reported to be important entry factors for receptors for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI), and the tight-junction (TJ) protein claudin 1 (CLDN1). The recent discovery of a TJ protein as a critical coreceptor highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. The colorectal adenocarcinoma Caco-2 cell line forms polarized monolayers containing functional TJs and was found to express the CD81, SR-BI, and CLDN1 proteins. Viral receptor expression levels increased upon polarization, and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins ZO-1 and occludin 1 were unchanged upon polarization. HCV infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry was neutralized by anti-E2 monoclonal antibodies, demonstrating glycoprotein-dependent entry. HCV pseudoparticle infection and recombinant HCV E1E2 glycoprotein interaction with polarized Caco-2 cells occurred predominantly at the apical surface. Disruption of TJs significantly increased HCV entry. These data support a model where TJs provide a physical barrier for viral access to receptors expressed on lateral and basolateral cellular domains.

scholarly journals Scavenger Receptor Class B Type I and Hepatitis C Virus Infection of Primary Tupaia Hepatocytes

2005 ◽  
Vol 79 (9) ◽  
pp. 5774-5785 ◽  
Author(s):  
Heidi Barth ◽  
Raffaele Cerino ◽  
Mirko Arcuri ◽  
Marco Hoffmann ◽  
Peter Schürmann ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Surface ◽  
Scavenger Receptor ◽  
Hcv Infection ◽  
Type I ◽  
Scavenger Receptor Class ◽  
Class B

ABSTRACT Hepatitis C virus (HCV) is a major cause of chronic hepatitis worldwide. The study of early steps during HCV infection has been hampered by the lack of suitable in vitro or in vivo models. Primary Tupaia hepatocytes (PTH) have been shown to be susceptible to HCV infection in vitro and in vivo. Human scavenger receptor class B type I (SR-BI) represents an HCV receptor candidate mediating the cellular binding of E2 glycoprotein to HepG2 hepatoma cells. However, the function of SR-BI for viral infection of hepatocytes is unknown. In this study, we used PTH to assess the functional role of SR-BI as a putative HCV receptor. Sequence analysis of cloned tupaia SR-BI revealed a high homology between tupaia and human SR-BI. Transfection of CHO cells with human or tupaia SR-BI but not mouse SR-BI cDNA resulted in cellular E2 binding, suggesting that E2-binding domains between human and tupaia SR-BI are highly conserved. Preincubation of PTH with anti-SR-BI antibodies resulted in marked inhibition of E2 or HCV-like particle binding. However, anti-SR-BI antibodies were not able to block HCV infection of PTH. In conclusion, our results demonstrate that SR-BI represents an important cell surface molecule for the binding of the HCV envelope to hepatocytes and suggest that other or additional cell surface molecules are required for the initiation of HCV infection. Furthermore, the structural and functional similarities between human and tupaia SR-BI indicate that PTH represent a useful model system to characterize the molecular interaction of the HCV envelope and SR-BI on primary hepatocytes.

Funktion von CEACAM-1 im Rahmen der Therapie der Hepatitis C-Virus Infektion mit Interferon-alpha in vivo und in vitro

2006 ◽  
Vol 44 (08) ◽  
Author(s):  
P Hilgard ◽  
R Bröring ◽  
M Trippler ◽  
S Viazov ◽  
G Gerken ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Interferon Alpha

scholarly journals Functional and Transcriptional Adaptations of Blood Monocytes Recruited to the Cystic Fibrosis Airway Microenvironment In Vitro

2021 ◽  
Vol 22 (5) ◽  
pp. 2530
Author(s):  
Bijean D. Ford ◽  
Diego Moncada Giraldo ◽  
Camilla Margaroli ◽  
Vincent D. Giacalone ◽  
Milton R. Brown ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Bactericidal Activity ◽  
Scavenger Receptor ◽  
Receptor Expression ◽  
Blood Monocytes ◽  
Bacterial Killing ◽  
Blood Neutrophils ◽  
Vitro Model

Cystic fibrosis (CF) lung disease is dominated by the recruitment of myeloid cells (neutrophils and monocytes) from the blood which fail to clear the lung of colonizing microbes. In prior in vitro studies, we showed that blood neutrophils migrated through the well-differentiated lung epithelium into the CF airway fluid supernatant (ASN) mimic the dysfunction of CF airway neutrophils in vivo, including decreased bactericidal activity despite an increased metabolism. Here, we hypothesized that, in a similar manner to neutrophils, blood monocytes undergo significant adaptations upon recruitment to CFASN. To test this hypothesis, primary human blood monocytes were transmigrated in our in vitro model into the ASN from healthy control (HC) or CF subjects to mimic in vivo recruitment to normal or CF airways, respectively. Surface phenotype, metabolic and bacterial killing activities, and transcriptomic profile by RNA sequencing were quantified post-transmigration. Unlike neutrophils, monocytes were not metabolically activated, nor did they show broad differences in activation and scavenger receptor expression upon recruitment to the CFASN compared to HCASN. However, monocytes recruited to CFASN showed decreased bactericidal activity. RNASeq analysis showed strong effects of transmigration on monocyte RNA profile, with differences between CFASN and HCASN conditions, notably in immune signaling, including lower expression in the former of the antimicrobial factor ISG15, defensin-like chemokine CXCL11, and nitric oxide-producing enzyme NOS3. While monocytes undergo qualitatively different adaptations from those seen in neutrophils upon recruitment to the CF airway microenvironment, their bactericidal activity is also dysregulated, which could explain why they also fail to protect CF airways from infection.

scholarly journals Control of PKR Protein Kinase by Hepatitis C Virus Nonstructural 5A Protein: Molecular Mechanisms of Kinase Regulation

1998 ◽  
Vol 18 (9) ◽  
pp. 5208-5218 ◽  
Author(s):  
Michael Gale ◽  
Collin M. Blakely ◽  
Bart Kwieciszewski ◽  
Seng-Lai Tan ◽  
Michelle Dossett ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Kinase ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Functional Assay ◽  
Binding Region ◽  
Dimerization Domain ◽  
Antiviral Effects

ABSTRACT The PKR protein kinase is a critical component of the cellular antiviral and antiproliferative responses induced by interferons. Recent evidence indicates that the nonstructural 5A (NS5A) protein of hepatitis C virus (HCV) can repress PKR function in vivo, possibly allowing HCV to escape the antiviral effects of interferon. NS5A presents a unique tool by which to study the molecular mechanisms of PKR regulation in that mutations within a region of NS5A, termed the interferon sensitivity-determining region (ISDR), are associated with sensitivity of HCV to the antiviral effects of interferon. In this study, we investigated the mechanisms of NS5A-mediated PKR regulation and the effect of ISDR mutations on this regulatory process. We observed that the NS5A ISDR, though necessary, was not sufficient for PKR interactions; we found that an additional 26 amino acids (aa) carboxyl to the ISDR were required for NS5A-PKR complex formation. Conversely, we localized NS5A binding to within PKR aa 244 to 296, recently recognized as a PKR dimerization domain. Consistent with this observation, we found that NS5A from interferon-resistant HCV genotype 1b disrupted kinase dimerization in vivo. NS5A-mediated disruption of PKR dimerization resulted in repression of PKR function and inhibition of PKR-mediated eIF-2α phosphorylation. Introduction of multiple ISDR mutations abrogated the ability of NS5A to bind to PKR in mammalian cells and to inhibit PKR in a yeast functional assay. These results indicate that mutations within the PKR-binding region of NS5A, including those within the ISDR, can disrupt the NS5A-PKR interaction, possibly rendering HCV sensitive to the antiviral effects of interferon. We propose a model of PKR regulation by NS5A which may have implications for therapeutic strategies against HCV.

scholarly journals Mass Balance, Metabolite Profile, andIn Vitro-In VivoComparison of Clearance Pathways of Deleobuvir, a Hepatitis C Virus Polymerase Inhibitor

2014 ◽  
Vol 59 (1) ◽  
pp. 25-37 ◽  
Author(s):  
Lin-Zhi Chen ◽  
John P. Sabo ◽  
Elsy Philip ◽  
Lois Rowland ◽  
Yan Mao ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mass Balance ◽  
Metabolite Profile ◽  
Fecal Samples ◽  
Acyl Glucuronide ◽  
Polymerase Inhibitor ◽  
Main Components

ABSTRACTThe pharmacokinetics, mass balance, and metabolism of deleobuvir, a hepatitis C virus (HCV) polymerase inhibitor, were assessed in healthy subjects following a single oral dose of 800 mg of [14C]deleobuvir (100 μCi). The overall recovery of radioactivity was 95.2%, with 95.1% recovered from feces. Deleobuvir had moderate to high clearance, and the half-life of deleobuvir and radioactivity in plasma were ∼3 h, indicating that there were no metabolites with half-lives significantly longer than that of the parent. The most frequently reported adverse events (in 6 of 12 subjects) were gastrointestinal disorders. Two major metabolites of deleobuvir were identified in plasma: an acyl glucuronide and an alkene reduction metabolite formed in the gastrointestinal (GI) tract by gut bacteria (CD 6168), representing ∼20% and 15% of the total drug-related material, respectively. Deleobuvir and CD 6168 were the main components in the fecal samples, each representing ∼30 to 35% of the dose. The majority of the remaining radioactivity found in the fecal samples (∼21% of the dose) was accounted for by three metabolites in which deleobuvir underwent both alkene reduction and monohydroxylation. In fresh human hepatocytes that form biliary canaliculi in sandwich cultures, the biliary excretion for these excretory metabolites was markedly higher than that for deleobuvir and CD 6168, implying that rapid biliary elimination upon hepatic formation may underlie the absence of these metabolites in circulation. The lowin vitroclearance was not predictive of the observedin vivoclearance, likely because major deleobuvir biotransformation occurred by non-CYP450-mediated enzymes that are not well represented in hepatocyte-basedin vitromodels.

scholarly journals Characterization of Resistance to the Nonnucleoside NS5B Inhibitor Filibuvir in Hepatitis C Virus-Infected Patients

2011 ◽  
Vol 56 (3) ◽  
pp. 1331-1341 ◽  
Author(s):  
Philip J. F. Troke ◽  
Marilyn Lewis ◽  
Paul Simpson ◽  
Katrina Gore ◽  
Jennifer Hammond ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Amino Acid ◽  
Site Directed Mutagenesis ◽  
Phenotypic Resistance ◽  
Number Of Patients ◽  
Chronic Hcv ◽  
Selection Of

ABSTRACTFilibuvir (PF-00868554) is an investigational nonnucleoside inhibitor of the hepatitis C virus (HCV) nonstructural 5B (NS5B) RNA-dependent RNA polymerase currently in development for treating chronic HCV infection. The aim of this study was to characterize the selection of filibuvir-resistant variants in HCV-infected individuals receiving filibuvir as short (3- to 10-day) monotherapy. We identified amino acid M423 as the primary site of mutation arising upon filibuvir dosing. Through bulk cloning of clinical NS5B sequences into a transient-replicon system, and supported by site-directed mutagenesis of the Con1 replicon, we confirmed that mutations M423I/T/V mediate phenotypic resistance. Selection in patients of an NS5B mutation at M423 was associated with a reduced replicative capacityin vitrorelative to the pretherapy sequence; consistent with this, reversion to wild-type M423 was observed in the majority of patients following therapy cessation. Mutations at NS5B residues R422 and M426 were detected in a small number of patients at baseline or the end of therapy and also mediate reductions in filibuvir susceptibility, suggesting these are rare but clinically relevant alternative resistance pathways. Amino acid variants at position M423 in HCV NS5B polymerase are the preferred pathway for selection of viral resistance to filibuvirin vivo.

In vivo analysis at the cellular level reveals similar steatosis induction in both hepatitis C virus genotype 1 and 3 infections

2017 ◽  
Vol 25 (3) ◽  
pp. 262-271 ◽  
Author(s):  
B. Campana ◽  
D. Calabrese ◽  
M. S. Matter ◽  
L. M. Terracciano ◽  
S. F. Wieland ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cellular Level ◽  
Genotype 1 ◽  
Virus Genotype ◽  
In Vivo Analysis

Circulating and inducible IL-32α in chronic hepatitis C virus infection

2019 ◽  
Vol 2 (1) ◽  
pp. 23-30
Author(s):  
Mark Collister ◽  
Julia Rempel ◽  
Jiaqi Yang ◽  
Kelly Kaita ◽  
Zach Raizman ◽  
...  
Keyword(s):  
Chronic Hepatitis ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Chronic Hepatitis C ◽  
Mononuclear Cells ◽  
Peripheral Blood Mononuclear ◽  
Chronic Hepatitis C Virus ◽  
Chronic Hcv

Background: Interleukin 32 (IL-32) is a recently described pro-inflammatory cytokine implicated in chronic hepatitis C virus (HCV)-related inflammation and fibrosis. IL-32α is the most abundant IL-32 isoform. Methods: Circulating IL-32α levels were documented in patients with chronic HCV infections ( n = 31) and compared with individuals who spontaneously resolved HCV infection ( n = 14) and HCV-naive controls ( n = 20). In addition, peripheral blood mononuclear cells (PBMC) from the chronic HCV ( n = 12) and HCV-naive ( n = 9) cohorts were investigated for responses to HCV core and non-structural (NS)3 protein induced IL-32α production. Finally, correlations between IL-32α levels, hepatic fibrosis and subsequent responses to interferon-based therapy were documented in patients with chronic HCV. Results: Circulating IL-32α levels in patients with chronic HCV were similar to those of spontaneously resolved and HCV-naive controls. HCV protein induced IL-32α responses were similar in chronic HCV patients and HCV-naive controls. In patients with chronic HCV, serum IL-32α levels correlated with worsening METAVIR fibrosis (F) scores from F0 to F3 ( r = 0.596, P < 0.001) as did NS3 induced IL-32α responses ( r = 0.837, P < 0.05). However, these correlations were not sustained with the inclusion of IL-32α levels at F4 scores, suggesting events at F4 interfere with IL-32α synthesis or release. In chronic HCV patients who underwent treatment ( n = 28), baseline in vivo and in vitro induced IL-32α concentrations were not predictive of therapeutic outcomes. Conclusions: IL-32α activity is associated with worsening fibrosis scores in non-cirrhotic, chronic HCV patients.

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