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.

scholarly journals Natural Variation in Translational Activities of the 5′ Nontranslated RNAs of Hepatitis C Virus Genotypes 1a and 1b: Evidence for a Long-Range RNA-RNA Interaction outside of the Internal Ribosomal Entry Site

1999 ◽  
Vol 73 (6) ◽  
pp. 4941-4951 ◽  
Author(s):  
Masao Honda ◽  
Rene Rijnbrand ◽  
Geoff Abell ◽  
Desok Kim ◽  
Stanley M. Lemon
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Mutational Analysis ◽  
Inhibitory Effect ◽  
Translation Efficiency ◽  
Entry Site ◽  
Translational Activity ◽  
Genotype 1B ◽  
Rna Interaction

ABSTRACT The 5′ nontranslated RNA (5′NTR) of a genotype 1b hepatitis C virus (HCV-N) directs cap-independent translation of the HCV-N polyprotein with about twofold less efficiency than the 5′NTR of a genotype 1a virus under physiologic conditions (Hutchinson strain, or HCV-H) (M. Honda et al., Virology 222:31–42, 1996). Here, we show by mutational analysis that substitution of the AG dinucleotide sequence at nucleotides (nt) 34 and 35 of HCV-N with GA (present in HCV-H) restores the translational activity to that of the HCV-H 5′NTR both in vitro and in vivo. These nucleotides are located upstream of the minimal essential internal ribosome entry site (IRES), as a 6-nt deletion spanning nt 32 to 37 also increased the translational activity of the HCV-N 5′NTR to that of HCV-H. Thus, the upstream AG dinucleotide sequence has an inhibitory effect on IRES-directed translation. Surprisingly, however, this inhibitory effect was observed only when the translated, downstream RNA sequence contained nt 408 to 929 of HCV (capsid-coding RNA). Further analysis of RNA transcripts containing frameshift mutations demonstrated that the nucleotide sequence of the transcript, and not the amino acid sequence of the expressed capsid protein, determines this difference in translation efficiency. The difference between the translational activities of the HCV-N and HCV-H transcripts was increased when translation was carried out in reticulocyte lysates containing high K+ concentrations, with a sevenfold difference evident at 130 to 150 mM K+. These results suggest that there is an RNA-RNA interaction involving 5′NTR and capsid-coding sequences flanking the IRES and that this is responsible for the reduced IRES activity of the genotype 1b virus, HCV-N.

scholarly journals Development and characterization of a transient-replication assay for the genotype 2a hepatitis C virus subgenomic replicon

2005 ◽  
Vol 86 (11) ◽  
pp. 3075-3080 ◽  
Author(s):  
Paul Targett-Adams ◽  
John McLauchlan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Osteosarcoma Cell ◽  
Dependent Manner ◽  
Entry Site ◽  
Subgenomic Replicon ◽  
Replication Assay ◽  
Cell Extracts ◽  
Genotype 1B

Dicistronic, subgenomic hepatitis C virus (HCV) replicons were constructed containing sequences from JFH1, a genotype 2a strain, that also incorporated the firefly luciferase gene under the control of the HCV internal ribosome entry site element. Luciferase activity in Huh-7 cell extracts containing in vitro-transcribed subgenomic JFH1 RNA was monitored over a 72 h period to examine early stages of HCV replication in the absence of any selective pressure. Enzyme activities produced by the replicon were almost 200-fold greater than those generated from corresponding genotype 1b replicons and correlated with an accumulation of NS5A protein and replicon RNA. Transient replication was sensitive to IFN treatment in a dose-dependent manner and, in addition to Huh-7 cells, the U2OS human osteosarcoma cell line supported efficient replication of the JFH1 replicon. Thus, this system based on JFH1 sequences offers improvements over prior genotype 1b replicons for quantitative measurement of viral RNA replication.

scholarly journals Hepatitis C virus depends on E-cadherin as an entry factor and regulates its expression in epithelial-to-mesenchymal transition

2016 ◽  
Vol 113 (27) ◽  
pp. 7620-7625 ◽  
Author(s):  
Qisheng Li ◽  
Catherine Sodroski ◽  
Brianna Lowey ◽  
Cameron J. Schweitzer ◽  
Helen Cha ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Cell Surface ◽  
Viral Entry ◽  
Epithelial To Mesenchymal Transition ◽  
Hcv Infection ◽  
Hcv Rna ◽  
Entry Site ◽  
Mesenchymal Transition ◽  
E Cadherin

Hepatitis C virus (HCV) enters the host cell through interactions with a cascade of cellular factors. Although significant progress has been made in understanding HCV entry, the precise mechanisms by which HCV exploits the receptor complex and host machinery to enter the cell remain unclear. This intricate process of viral entry likely depends on additional yet-to-be-defined cellular molecules. Recently, by applying integrative functional genomics approaches, we identified and interrogated distinct sets of host dependencies in the complete HCV life cycle. Viral entry assays using HCV pseudoparticles (HCVpps) of various genotypes uncovered multiple previously unappreciated host factors, including E-cadherin, that mediate HCV entry. E-cadherin silencing significantly inhibited HCV infection in Huh7.5.1 cells, HepG2/miR122/CD81 cells, and primary human hepatocytes at a postbinding entry step. Knockdown of E-cadherin, however, had no effect on HCV RNA replication or internal ribosomal entry site (IRES)-mediated translation. In addition, an E-cadherin monoclonal antibody effectively blocked HCV entry and infection in hepatocytes. Mechanistic studies demonstrated that E-cadherin is closely associated with claudin-1 (CLDN1) and occludin (OCLN) on the cell membrane. Depletion of E-cadherin drastically diminished the cell-surface distribution of these two tight junction proteins in various hepatic cell lines, indicating that E-cadherin plays an important regulatory role in CLDN1/OCLN localization on the cell surface. Furthermore, loss of E-cadherin expression in hepatocytes is associated with HCV-induced epithelial-to-mesenchymal transition (EMT), providing an important link between HCV infection and liver cancer. Our data indicate that a dynamic interplay among E-cadherin, tight junctions, and EMT exists and mediates an important function in HCV entry.

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 Hepatitis C Virus 3′X Region Interacts with Human Ribosomal Proteins

2001 ◽  
Vol 75 (3) ◽  
pp. 1348-1358 ◽  
Author(s):  
Jonny Wood ◽  
Robert M. Frederickson ◽  
Stanley Fields ◽  
Arvind H. Patel
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Fusion Protein ◽  
Ribosomal Proteins ◽  
Infectious Clone ◽  
Cdna Libraries ◽  
Entry Site ◽  
Reporter Protein ◽  
Barr Virus

ABSTRACT To identify proteins that can bind the 3′ untranslated region (UTR) of hepatitis C virus (HCV) we screened human cDNA libraries using theSaccharomyces cerevisiae three-hybrid system. Screening with an RNA sequence derived from the 3′-terminal 98 nucleotides (3′X region) of an infectious clone of HCV (H77c) yielded clones of human ribosomal proteins L22, L3, S3, and mL3, a mitochondrial homologue of L3. We performed preliminary characterization of the binding between the 3′X region and these proteins by a three-hybrid mating assay using mutant 3′X sequences. We have further characterized the interaction between 3′X and L22, since this protein is known to be associated with two small Epstein-Barr virus (EBV)-encoded RNA species (EBERs) which are abundantly produced in cells latently infected with EBV. The EBERs, which have similar predicted secondary structure to the HCV 3′X, assemble into ribonucleoprotein particles that include L22 and La protein. To confirm that L22 binds HCV 3′X we performed in vitro binding assays using recombinant L22 (expressed as a glutathioneS-transferase [GST] fusion protein) together with a 3′X riboprobe. The 3′X region binds to the GST-L22 fusion protein (but not to GST alone), and this interaction is subject to competition with unlabeled 3′X RNA. To establish the functional role played by L22 in internal ribosome entry site (IRES)-mediated translation of HCV sequences we performed translational analysis in HuH-7 cells using monocistronic and bicistronic reporter constructs. The relative amount of core-chloramphenicol acetyltransferase reporter protein translated under the control of the HCV IRES was stimulated in the presence of L22 and La when these proteins were supplied in trans.

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.

scholarly journals Hepatitis C Virus p7 and NS2 Proteins Are Essential for Production of Infectious Virus

2007 ◽  
Vol 81 (16) ◽  
pp. 8374-8383 ◽  
Author(s):  
Christopher T. Jones ◽  
Catherine L. Murray ◽  
Dawnnica K. Eastman ◽  
Jodie Tassello ◽  
Charles M. Rice
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Infectious Virus ◽  
Early Stage ◽  
Virus Production ◽  
Health Concern ◽  
Cell Culture Systems ◽  
Catalytic Active Site

ABSTRACT Hepatitis C virus (HCV) infection is a global health concern affecting an estimated 3% of the world's population. Recently, cell culture systems have been established, allowing recapitulation of the complete virus life cycle for the first time. Since the HCV proteins p7 and NS2 are not predicted to be major components of the virion, nor are they required for RNA replication, we investigated whether they might have other roles in the viral life cycle. Here we utilize the recently described infectious J6/JFH chimera to establish that the p7 and NS2 proteins are essential for HCV infectivity. Furthermore, unprocessed forms of p7 and NS2 were not required for this activity. Mutation of two conserved basic residues, previously shown to be important for the ion channel activity of p7 in vitro, drastically impaired infectious virus production. The protease domain of NS2 was required for infectivity, whereas its catalytic active site was dispensable. We conclude that p7 and NS2 function at an early stage of virion morphogenesis, prior to the assembly of infectious virus.

scholarly journals Inhibition of Hepatitis C Virus (HCV) RNA Polymerase by DNA Aptamers: Mechanism of Inhibition of In Vitro RNA Synthesis and Effect on HCV-Infected Cells

2008 ◽  
Vol 52 (6) ◽  
pp. 2097-2110 ◽  
Author(s):  
Pantxika Bellecave ◽  
Christian Cazenave ◽  
Julie Rumi ◽  
Cathy Staedel ◽  
Ophélie Cosnefroy ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Selection Procedure ◽  
Inhibitory Effect ◽  
Hcv Rna ◽  
Dna Aptamers ◽  
Huh7 Cells

ABSTRACT We describe here the further characterization of two DNA aptamers that specifically bind to hepatitis C virus (HCV) RNA polymerase (NS5B) and inhibit its polymerase activity in vitro. Although they were obtained from the same selection procedure and contain an 11-nucleotide consensus sequence, our results indicate that aptamers 27v and 127v use different mechanisms to inhibit HCV polymerase. While aptamer 27v was able to compete with the RNA template for binding to the enzyme and blocked both the initiation and the elongation of RNA synthesis, aptamer 127v competed poorly and exclusively inhibited initiation and postinitiation events. These results illustrate the power of the selective evolution of ligands by exponential enrichment in vitro selection procedure approach to select specific short DNA aptamers able to inhibit HCV NS5B by different mechanisms. We also determined that, in addition to an in vitro inhibitory effect on RNA synthesis, aptamer 27v was able to interfere with the multiplication of HCV JFH1 in Huh7 cells. The efficient cellular entry of these short DNAs and the inhibitory effect observed on human cells infected with HCV indicate that aptamers are useful tools for the study of HCV RNA synthesis, and their use should become a very attractive and alternative approach to therapy for HCV infection.

scholarly journals La Autoantigen Is Necessary for Optimal Function of the Poliovirus and Hepatitis C Virus Internal Ribosome Entry Site In Vivo and In Vitro

2004 ◽  
Vol 24 (15) ◽  
pp. 6861-6870 ◽  
Author(s):  
Mauro Costa-Mattioli ◽  
Yuri Svitkin ◽  
Nahum Sonenberg
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Internal Ribosome Entry Site ◽  
Ribosomal Subunit ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Entry Site ◽  
Internal Ribosome Entry

ABSTRACT Translation of poliovirus and hepatitis C virus (HCV) RNAs is initiated by recruitment of 40S ribosomes to an internal ribosome entry site (IRES) in the mRNA 5′ untranslated region. Translation initiation of these RNAs is stimulated by noncanonical initiation factors called IRES trans-activating factors (ITAFs). The La autoantigen is such an ITAF, but functional evidence for the role of La in poliovirus and HCV translation in vivo is lacking. Here, by two methods using small interfering RNA and a dominant-negative mutant of La, we demonstrate that depletion of La causes a dramatic reduction in poliovirus IRES function in vivo. We also show that 40S ribosomal subunit binding to HCV and poliovirus IRESs in vitro is inhibited by a dominant-negative form of La. These results provide strong evidence for a function of the La autoantigen in IRES-dependent translation and define the step of translation which is stimulated by La.

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