Production of infectious hepatitis C virus particles in three-dimensional cultures of the cell line carrying the genome-length dicistronic viral RNA of genotype 1b

Murakami, Kyoko; Ishii, Koji; Ishihara, Yousuke; Yoshizaki, Sayaka; Tanaka, Keiko; Gotoh, Yasufumi; Aizaki, Hideki; Kohara, Michinori; Yoshioka, Hiroshi; Mori, Yuichi; Manabe, Noboru; Shoji, Ikuo; Sata, Tetsutaro; Bartenschlager, Ralf; Matsuura, Yoshiharu; Miyamura, Tatsuo; Suzuki, Tetsuro

doi:10.1016/j.virol.2006.03.038

Cited by 45 publications

(37 citation statements)

References 52 publications

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“…Mitochondria were purified using Nycodenz (Nycomed Pharma, Zürich, Switzerland) according to the protocols reported by Okado-Matsumoto et al 17 For transient transfection experiments, HepG2 cells were transfected with a core-expression plasmid using TransIT-LT1 (Mirus Bio, Madison, WI). Huh7 cells harboring HCV genotype 1b full-genomic (RCYM1) 18 or subgenomic replicon (5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15), and livers of 3-month-old core-gene transgenic mice 2 were also used for the analysis.…”

Section: Methodsmentioning

confidence: 99%

Proteomics analysis of mitochondrial proteins reveals overexpression of a mitochondrial protein chaperon, prohibitin, in cells expressing hepatitis C virus core protein†

et al. 2009

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confidence: 99%

Proteomics analysis of mitochondrial proteins reveals overexpression of a mitochondrial protein chaperon, prohibitin, in cells expressing hepatitis C virus core protein†

et al. 2009

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“…4) The cells harboring the genotype 1a full-genomic replicon (RCYM1) 13) and the genotype 2a subgenomic replicon (clone 4-1) 14) have been described previously. HCV CPE Inhibition Assay Huh 7.5.1 cells were suspended in 1 : 1 Dulbecco's modified Eagle's medium : Nutrient Mixture F-12 (DMEM/F12) supplemented with 0.5% ITS-X and seeded in wells of collagen-coated 96-well plates at a density of 5000 cells per well in a volume of 125 µL.…”

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A Cell-Based, Microplate Colorimetric Screen Identifies 7,8-Benzoflavone and Green Tea Gallate Catechins as Inhibitors of the Hepatitis C Virus

Fukazawa

Suzuki

Wakita

et al. 2012

Biological & Pharmaceutical Bulletin

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We describe a cell-based, microplate colorimetric screen for anti-hepatitis C virus (HCV) drugs that exploits the HCV-JFH1 viral culture system. Antiviral activity was assessed by measuring protection against the HCV-JFH1-induced cytopathic effect (CPE) in Huh7.5.1 cells using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) viability assay. The use of serum-free medium substantially sensitized Huh7.5.1 cells to HCV-induced CPE, causing sufficient cell death to perform colorimetric assays for anti-HCV activity in 96-well plates. As a proof of concept, we carried out a pilot screen of an inhibitor library and identified cyclosporin A and tamoxifen, two compounds with reported anti-HCV activity. Using the assay, we discovered the anti-HCV properties of the plant flavonoids epigallocatechin gallate (EGCG) and 7,8-benzoflavone (α-naphthoflavone). Other gallate-type catechins and flavones also displayed anti-HCV activity, but 5,6-benzoflavone (β-naphthoflavone), flavanone, and non-gallate catechins were inactive. EGCG apparently acted mainly on HCV entry, although it may also block other steps. In contrast, 7,8-benzoflavone was presumed to inhibit later stages of the HCV life cycle. This assay is simple, reliable and cost-effective; does not require any specially engineered cell lines or viruses; and should be useful in the identification of compounds with anti-HCV activity. Key words hepatitis C virus; 7,8-benzoflavone (α-naphthoflavone); epigallocatechin gallateMore than 170 million people worldwide are chronically infected with the hepatitis C virus (HCV) and are at risk for developing liver diseases such as cirrhosis and hepatocellular carcinoma. Vaccines against HCV are not currently available; furthermore, the standard interferon/ribavirin combination therapy is not effective in approximately half of HCV-infected patients, and it has considerable side effects.1,2) Thus, there is an obvious and urgent need for new agents that can enhance or replace current HCV therapies.Screening programs using HCV replicon-based systems have successfully identified compounds that act on HCV RNA replication. However, replicon systems do not reproduce the entire HCV life cycle, and they cannot isolate inhibitors of many important steps such as viral entry, assembly, and egress. HCV cell culture infection models that recapitulate the entire viral life cycle in vitro have greatly enhanced the opportunity for HCV drug discovery. Several reports, including ours, have demonstrated that this model can overcome the limitations of the HCV replicon system and enable the discovery of compounds that target various stages of the HCV life cycle. [3][4][5][6][7][8] Using the HCV cell culture system, we previously developed a tube-capture-reverse transcription-polymerase chain reaction (RT-PCR) assay for screening HCV inhibitors and identified bisindolylmaleimides and indolocarbazoles as inhibitors of HCV replication.4) Here, we describe another screening method for the detection of anti-HCV activity. This assay measures t...

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“…Expression of the structural proteins in mammalian cells has been observed to generate virus-like particles with ultrastructural properties similar to those of HCV virions (5, 29). Packaging of these HCV-like particles into intracellular vesicles as a result of budding from the endoplasmic reticulum (ER) has also been observed (8,34). However, HCV structural proteins are observed both in the ER and in the Golgi apparatus (45).…”

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“…Plasmid pJFH1, containing full-length cDNA of the JFH-1 isolate, was used to generate HCVcc as described elsewhere (23,33,34,54). pJ6/JFH was obtained from JFH1 by replacement of the 5Ј untranslated region to the p7 region (EcoRI-BclI) of J6.…”

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Critical Role of Virion-Associated Cholesterol and Sphingolipid in Hepatitis C Virus Infection

Aizaki¹,

Morikawa²,

Fukasawa

et al. 2008

J Virol

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In this study, we establish that cholesterol and sphingolipid associated with hepatitis C virus (HCV) particles are important for virion maturation and infectivity. In a recently developed culture system enabling study of the complete life cycle of HCV, mature virions were enriched with cholesterol as assessed by the molar ratio of cholesterol to phospholipid in virion and cell membranes. Depletion of cholesterol from the virus or hydrolysis of virion-associated sphingomyelin almost completely abolished HCV infectivity. Supplementation of cholesterol-depleted virus with exogenous cholesterol enhanced infectivity to a level equivalent to that of the untreated control. Cholesterol-depleted or sphingomyelin-hydrolyzed virus had markedly defective internalization, but no influence on cell attachment was observed. Significant portions of HCV structural proteins partitioned into cellular detergent-resistant, lipid-raft-like membranes. Combined with the observation that inhibitors of the sphingolipid biosynthetic pathway block virion production, but not RNA accumulation, in a JFH-1 isolate, our findings suggest that alteration of the lipid composition of HCV particles might be a useful approach in the design of anti-HCV therapy.Hepatitis C virus (HCV) is recognized as a major cause of chronic liver disease, including chronic hepatitis, hepatic steatosis, cirrhosis, and hepatocellular carcinoma. It presently affects approximately 200 million people worldwide (26). HCV is an enveloped positive-strand RNA virus belonging to the Hepacivirus genus of the family Flaviviridae. Its genome of ϳ9.6 kb encodes a polyprotein precursor of ϳ3,000 residues, and the structural proteins (core, E1, and E2) reside in its N-terminal region.Little is known about the assembly of HCV and its virion structure, because efficient production of authentic HCV particles has only recently been achieved. Nucleocapsid assembly generally involves oligomerization of the capsid protein and encapsidation of genomic RNA. This process is thought to occur upon interaction of the core protein with viral RNA, and this core-RNA interaction may induce a change from RNA replication to packaging. As with related viruses, the mature HCV virion likely consists of a nucleocapsid and an outer envelope composed of a lipid membrane and envelope proteins. Expression of the structural proteins in mammalian cells has been observed to generate virus-like particles with ultrastructural properties similar to those of HCV virions (5, 29). Packaging of these HCV-like particles into intracellular vesicles as a result of budding from the endoplasmic reticulum (ER) has also been observed (8,34). However, HCV structural proteins are observed both in the ER and in the Golgi apparatus (45). Moreover, complex N-linked glycans have been detected on the surfaces of HCV particles isolated from patient sera, suggesting that the glycans transit through the Golgi apparatus (44). Interactions between the core and E1/E2 proteins are thought to determine viral morphology and are mediated thro...

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Production of infectious hepatitis C virus particles in three-dimensional cultures of the cell line carrying the genome-length dicistronic viral RNA of genotype 1b

Cited by 45 publications

References 52 publications

Proteomics analysis of mitochondrial proteins reveals overexpression of a mitochondrial protein chaperon, prohibitin, in cells expressing hepatitis C virus core protein†

Proteomics analysis of mitochondrial proteins reveals overexpression of a mitochondrial protein chaperon, prohibitin, in cells expressing hepatitis C virus core protein†

A Cell-Based, Microplate Colorimetric Screen Identifies 7,8-Benzoflavone and Green Tea Gallate Catechins as Inhibitors of the Hepatitis C Virus

Critical Role of Virion-Associated Cholesterol and Sphingolipid in Hepatitis C Virus Infection

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