The hepatitis C virus minigenome: A new cellular model for studying viral replication

Dumas, Estelle; Masante, Cyril; Astier-Gin, T.; Lapaillerie, Delphine; Ventura, Michel

doi:10.1016/j.jviromet.2007.01.005

Cited by 8 publications

(15 citation statements)

References 31 publications

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“…The stronger the selective pressure, the larger the number of fluorescent cells, produced as a consequence of minigenome replication. Replication of the RNA minigenome in Huh7/NS3-5B cells was also demonstrated by the presence of minus-strand RNA 96 h after transfection of the corresponding plus strand (Dumas et al, 2007).…”

Section: Translation Activity From Genotypes 1 Andmentioning

confidence: 93%

“…All plasmid clones described were verified by sequencing. (iii) The minigenome pGEM-T/5UTR-H2AE-3UTR construct, consisting of a gene encoding a polyprotein composed of hygromycin phosphotransferase (Hygro R ), the foot-and-mouth disease virus 2A protein and the enhanced green fluorescent protein (EGFP), flanked by the HCV 59 and 39 UTRs, was described previously (Dumas et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

“…The Huh7/rep5.1 cell line supporting the subgenomic HCV replicon rep5.1 was maintained under G418 selection pressure. The Huh7/NS3-5B cell line, constitutively expressing the non-structural (NS) proteins from HCV strain J4L6, and cured Huh7 cells were described previously (Dumas et al, 2007).…”

Section: Methodsmentioning

confidence: 99%

“…Since no HCV genotype 3 isolate has currently been found to replicate in cell culture, we used an ex vivo system aiming at (i) constitutively producing the HCV replication complex thanks to integration of the HCV NS3-5B coding region into the genome of Huh7 cells (Huh7/NS3-5B) and (ii) studying the replication events using an RNA minigenome (Fig. 3a) (Dumas et al, 2007). The 5UTR-H2AE-3UTR minigenome was used to analyse the replicative activity of the HCV replication complex in Huh7 cells.…”

Section: Translation Activity From Genotypes 1 Andmentioning

confidence: 99%

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Seven nucleotide changes characteristic of the hepatitis C virus genotype 3 5′ untranslated region: correlation with reduced in vitro replication

Masante

Mahias

Lourenco

et al. 2008

Journal of General Virology

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Seven nucleotide changes characteristic of the hepatitis C virus genotype 3 59 untranslated region: correlation with reduced in vitro replication Computer analysis of 158 hepatitis C virus (HCV) 59 untranslated region (59 UTR) sequences from the six genotypes showed that the 59 UTR from genotype 3 displays seven specific non-contiguous nucleotide changes, at positions 8, 13, 14, 70, 97, 203 and 224. The purpose of this study was to investigate the impact of these changes on translation and replication activities. Indeed, these modifications could alter both the internal ribosome entry site (IRES) present in the 59 UTR of the plus-strand RNA and the 39 end of the minus strand involved in the initiation of plus-strand RNA synthesis. We found that the genotype 3-specific nucleotide changes do not modify the in vitro or ex vivo translation activity of the corresponding IRES, in comparison with that of genotype 1. In contrast, in vitro replication from the minus-strand RNA is eight times less efficient for genotype 3 than for genotype 1 RNA, suggesting the involvement of some nucleotide changes in the reduction of RNA synthesis. Nucleotides 13, 14 and 224 were found to be responsible for this effect. Moreover, a reduced replicative activity was confirmed ex vivo for genotype 3, but to a lesser extent than that observed in vitro, using an RNA minigenome. INTRODUCTIONHepatitis C virus (HCV) affects nearly 200 million people worldwide. Five to seven per cent of patients die as a consequence of liver disease. Hepatic steatosis is a common feature of liver biopsy specimens from patients with chronic hepatitis C, and its presence is associated with fibrotic progression (Rubbia-Brandt et al., 2000). Numerous factors including gender, age of infection, alcohol consumption, exposure to other hepatotoxins and perturbation of lipid metabolism have been identified as determinants of pathogenesis. Viral factors may also be critical determinants of steatosis in chronic hepatitis C, particularly HCV genotype (Rubbia-Brandt et al., 2004). Genetic variability of the RNA genome has made it possible to distinguish six HCV genotypes and over 70 subtypes (Simmonds et al., 2005). Each of these genotypes displays particular features such as resistance to interferon/ribavirin treatments. Thus, genotype 1-infected patients respond less efficiently to therapy than those infected with genotype 2 and 3 viruses. Conversely, patients with HCV genotype 3 infection and chronic hepatitis C are more likely to be subjected to a liver steatosis than those infected with HCV genotype 1 (Lonardo et al., 2006). Viral determinants of this differential progression are as yet poorly understood. It has been suspected that sequence variations in the envelope E1 and E2 glycoproteins might be involved in differences in pathogenesis between genotypes 1 and 3 (Shaw et al., 2003). More recently, in vitro models suggested involvement of the core protein as a viral factor associated with lipid accumulation in genotype 3 infection (Abid et al., 2005;Hourioux et al., 20...

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Section: Translation Activity From Genotypes 1 Andmentioning

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Translation Activity From Genotypes 1 Andmentioning

confidence: 99%

See 2 more Smart Citations

Seven nucleotide changes characteristic of the hepatitis C virus genotype 3 5′ untranslated region: correlation with reduced in vitro replication

Masante

Mahias

Lourenco

et al. 2008

Journal of General Virology

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“…These cells (Huh7-QR) were obtained from the Huh7/Rep5.1 cell line cured for the Rep5.1 replicon by treatment with 150 U ⅐ ml Ϫ1 alpha interferon. After 3 weeks in the presence of this drug and the absence of G418, no replicon RNA could be detected (13).…”

mentioning

confidence: 94%

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

Bellecave¹,

Cazenave²,

Rumi³

et al. 2008

Antimicrob Agents Chemother

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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.Hepatitis C virus (HCV) infection causes serious liver diseases, such as chronic hepatitis, which can evolve into cirrhosis and hepatocellular carcinoma (20,39). The most effective therapy, a combination of pegylated interferon and ribavirin, is efficient in only 50% of patients (32). Therefore, new treatments based on specific and well-tolerated compounds need to be developed.HCV RNA replication is catalyzed by the viral polymerase NS5B. This RNA-dependent RNA polymerase (RdRp) synthesizes a negative-strand RNA that serves as a template for the synthesis of new positive RNA strands. Viral RNA synthesis can be divided into two major steps: (i) initiation, which corresponds to the formation of a 2-or 3-nucleotide (nt) product and which occurs by a de novo mechanism in vitro as well as in the cellular replicon system that mimics some steps of the in vivo viral cycle (9, 31, 49), and (ii) elongation, which yields a full copy of the template. The transition between these two steps, in which 3-nt to 5-nt products are synthesized, may involve conformational changes of the viral polymerase that are not yet understood (14). The structure of the enzyme, as determined by X-ray crystallography, revealed, like for many other RNA and DNA polymerases, a right-handed-like structure with finger, palm, and thumb domains (1,7,28). It has been shown that the NS5B protein may adopt a closed and active conformation. The contact between the two loops extending from the fingers and the thumb links these two domains and closes the back of the enzyme to form a tunnel that constitutes the entry site for ribonucleotides. This suggests that a concerted movement of the thumb and fingertips occurs during the polymerization steps. The...

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Mutations of the SL2 dimerization sequence of the hepatitis C genome abrogate viral replication

Masante

Jaubert

Palau

et al. 2015

Cell. Mol. Life Sci.

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Stem-loop SL2 is a self-interacting palindromic sequence that has been identified within the hepatitis C virus genome (HCV). While, RNA dimerization of the HCV genome has been observed in vitro with short RNA sequences, the role of a putative RNA dimerization during viral replication has not been elucidated. To determine the effect of genomic dimerization on viral replication, we introduced mutations into SL2 predicted to disrupt genomic dimerization. Using surface plasmon resonance, we show that mutations within the SL2 bulge impact dimerization in vitro. Transfection of Huh7 cells with luciferase-encoding full-length genomes containing SL2 mutations abolishes viral replication. Luciferase expression indicates that viral translation is not or slightly affected and that the viral RNA is properly encapsidated. However, RT-qPCR analysis demonstrates that viral RNA synthesis is drastically decreased. In vitro synthesis experiments using the viral recombinant polymerase show that modifications of intra-molecular interactions have no effect on RNA synthesis, while impairing inter-molecular interactions decreases polymerase activity. This confirms that dimeric templates are preferentially replicated by the viral polymerase. Altogether, these results indicate that the dimerization of the HCV genomic RNA is a crucial step for the viral life cycle especially for RNA replication. RNA dimerization could explain the existence of HCV recombinants in cell culture and patients reported recently in other studies.

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The hepatitis C virus minigenome: A new cellular model for studying viral replication

Cited by 8 publications

References 31 publications

Seven nucleotide changes characteristic of the hepatitis C virus genotype 3 5′ untranslated region: correlation with reduced in vitro replication

Seven nucleotide changes characteristic of the hepatitis C virus genotype 3 5′ untranslated region: correlation with reduced in vitro replication

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

Mutations of the SL2 dimerization sequence of the hepatitis C genome abrogate viral replication

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