An infectious and selectable full‐length replicon system with hepatitis C virus JFH‐1 strain

Date, Tomoko; Miyamoto, Michiko; Kato, Takanobu; Morikawa, Kenichi; Murayama, Asako; Akazawa, Daisuke; Tanabe, Junichi; Sone, Saburo; Mizokami, Masashi; Wakita, Takaji

doi:10.1111/j.1872-034x.2007.00056.x

Cited by 22 publications

(17 citation statements)

References 32 publications

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“…VSV glycoprotein-mediated HCV replicon colony formation assay. HCV replicon cells stably expressing the Neo-labeled JFH1 genome lacking the regions encoding E1 and E2 were produced by transfecting in vitro transcripts of pFGR-JFH1-delE into Huh7 cells as described previously (11). The VSV-G expression plasmids or the empty vector was transfected into the established replicon cells using Lipofectamine 2000 (Invitrogen).…”

Section: Cells and Virusesmentioning

confidence: 99%

“…Plasmids pUC-JFH1-delE and pFGR-JFH1-delE, which contain an in-frame deletion in the regions of JFH1 encoding E1 and E2, have been described previously (11,53). Plasmid pcDNA3-JFH1-E1/E2, expressing JFH1 envelope glycoproteins, was constructed by PCR amplification of the JFH1 E1 and E2 regions (amino acid residues 171 to 750) and insertion of the PCR product into pcDNA3.1.…”

Section: Cells and Virusesmentioning

confidence: 99%

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Production of Hepatitis C Virus Lacking the Envelope-Encoding Genes for Single-Cycle Infection by Providing Homologous Envelope Proteins or Vesicular Stomatitis Virus Glycoproteins in trans

Yan

et al. 2011

J Virol

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Hepatitis C virus (HCV) infection is a major worldwide health problem. The envelope glycoproteins are the major components of viral particles. Here we developed a trans-complementation system that allows the production of infectious HCV particles in whose genome the regions encoding envelope proteins are deleted (HCV⌬E). The lack of envelope proteins could be efficiently complemented by the expression of homologous envelope proteins in trans. HCV⌬E production could be enhanced significantly by previously described adaptive mutations in NS3 and NS5A. Moreover, HCV⌬E could be propagated and passaged in packaging cells stably expressing HCV envelope proteins, resulting in only single-round infection in wild-type cells. Interestingly, we found that vesicular stomatitis virus (VSV) glycoproteins could efficiently rescue the production of HCV lacking endogenous envelope proteins, which no longer required apolipoprotein E for virus production. VSV glycoprotein-mediated viral entry could allow for the bypass of the natural HCV entry process and the delivery of HCV replicon RNA into HCV receptor-deficient cells. Our development provides a new tool for the production of single-cycle infectious HCV particles, which should be useful for studying individual steps of the HCV life cycle and may also provide a new strategy for HCV vaccine development.Hepatitis C virus (HCV) is a major etiological agent of severe liver diseases, including liver cirrhosis and hepatocellular carcinoma, with an estimated 170 million people infected worldwide (2). No vaccine is available to prevent HCV infection, and the sole therapeutic treatment available, based on interferon, does not always lead to cure and is often associated with significant side effects (12). HCV is an enveloped plusstrand RNA virus belonging to the family Flaviviridae. The 9.6-kb viral genome encodes a single polyprotein that is co-or posttranslationally cleaved into structural (core, E1, and E2) and nonstructural (p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) proteins (4). The structural proteins encapsidate the viral genome into infectious particles and mediate the entry of the virus into permissive cells; the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B are the viral components of the membrane-bound replication complexes that catalyze genomic RNA replication (18).HCV envelope glycoproteins E1 and E2 are processed by cellular signal peptidases in the endoplasmic reticulum (ER), are highly glycosylated in the amino-terminal ectodomains, and are anchored to the membrane by the carboxyl-terminal transmembrane domains to form a stable noncovalent heterodimer complex. This oligomer is thought to be the prebudding form of the functional HCV glycoproteins and is essential for interaction with the receptor during HCV entry (52). According to the current model for HCV assembly and secretion, HCV core particles containing the genome assemble in the lipid droplets and acquire the viral envelope by budding into the ER (43), during which time the E1 and E2 proteins are inserted i...

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Section: Cells and Virusesmentioning

confidence: 99%

Section: Cells and Virusesmentioning

confidence: 99%

Production of Hepatitis C Virus Lacking the Envelope-Encoding Genes for Single-Cycle Infection by Providing Homologous Envelope Proteins or Vesicular Stomatitis Virus Glycoproteins in trans

Yan

et al. 2011

J Virol

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“…Specifically, we conducted co-immunoprecipitation experiments in human hepatoma Huh7 cells, which overexpress core and NS5A (Figure 2A, top panel) and in Huh7 subgenomic JFH1 replicon cells (SGR-JFH1) [59] transfected with core (Figure 2A, bottom panel). N-terminally-3xFlag-tagged core protein was immunoprecipitated with an anti-Flag antibody and resulting immunocomplexes were examined for NS5A content by Western blotting.…”

Section: Resultsmentioning

confidence: 99%

“…Huh7 cells carrying a subgenomic JFH1 replicon (SGR-JFH1) were maintained in the same medium as mentioned above and supplemented with 300 μg/ml G418 (BioPioneer) [59]. Huh7.5.1 and SGR-JFH1 cells were kindly provided by Francis V. Chisari.…”

Section: Methodsmentioning

confidence: 99%

HCV Core Residues Critical for Infectivity Are Also Involved in Core-NS5A Complex Formation

et al. 2014

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Hepatitis C virus (HCV) infection is a major cause of liver disease. The molecular machinery of HCV assembly and particle release remains obscure. A better understanding of the assembly events might reveal new potential antiviral strategies. It was suggested that the nonstructural protein 5A (NS5A), an attractive recent drug target, participates in the production of infectious particles as a result of its interaction with the HCV core protein. However, prior to the present study, the NS5A-binding site in the viral core remained unknown. We found that the D1 domain of core contains the NS5A-binding site with the strongest interacting capacity in the basic P38-K74 cluster. We also demonstrated that the N-terminal basic residues of core at positions 50, 51, 59 and 62 were required for NS5A binding. Analysis of all substitution combinations of R50A, K51A, R59A, and R62A, in the context of the HCVcc system, showed that single, double, triple, and quadruple mutants were fully competent for viral RNA replication, but deficient in secretion of viral particles. Furthermore, we found that the extracellular and intracellular infectivity of all the mutants was abolished, suggesting a defect in the formation of infectious particles. Importantly, we showed that the interaction between the single and quadruple core mutants and NS5A was impaired in cells expressing full-length HCV genome. Interestingly, mutations of the four basic residues of core did not alter the association of core or NS5A with lipid droplets. This study showed for the first time that basic residues in the D1 domain of core that are critical for the formation of infectious extracellular and intracellular particles also play a role in core-NS5A interactions.

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“…The constructs were linearized and then used for in vitro transcription as described above. Huh7 cell-derived cell lines containing the HCV SGR or HCV FGR were established by transfection of in vitro -transcribed HCV subgenomic or HCV full-genomic RNA, followed by selection with 500 μg/mL G418 sulfate as previously described (Date et al, 2007). The selected cell lines were maintained in complete DMEM containing 500 μg/mL G418 sulfate.…”

Section: Methodsmentioning

confidence: 99%

GRIM-19 Restricts HCV Replication by Attenuating Intracellular Lipid Accumulation

et al. 2017

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Gene-associated with retinoid-interferon-induced mortality 19 (GRIM-19) targets multiple signaling pathways involved in cell death and growth. However, the role of GRIM-19 in the pathogenesis of hepatitis virus infections remains unexplored. Here, we investigated the restrictive effects of GRIM-19 on the replication of hepatitis C virus (HCV). We found that GRIM-19 protein levels were reduced in HCV-infected Huh7 cells and Huh7 cells harboring HCV replicons. Moreover, ectopically expressed GRIM-19 caused a reduction in both intracellular viral RNA levels and secreted viruses in HCVcc-infected cell cultures. The restrictive effect on HCV replication was restored by treatment with siRNA against GRIM-19. Interestingly, GRIM-19 overexpression did not alter the level of phosphorylated STAT3 or its subcellular distribution. Strikingly, forced expression of GRIM-19 attenuated an increase in intracellular lipid droplets after oleic acid (OA) treatment or HCVcc infection. GRIM-19 overexpression abrogated fatty acid-induced upregulation of sterol regulatory element-binding transcription factor-1 (SREBP-1c), resulting in attenuated expression of its target genes such as fatty acid synthase (FAS) and acetyl CoA carboxylase (ACC). Treatment with OA or overexpression of SREBP-1c in GRIM-19-expressing, HCVcc-infected cells restored HCV replication. Our results suggest that GRIM-19 interferes with HCV replication by attenuating intracellular lipid accumulation and therefore is an anti-viral host factor that could be a promising target for HCV treatment.

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An infectious and selectable full‐length replicon system with hepatitis C virus JFH‐1 strain

Abstract: Selectable infection was established using HCV replicon containing full-length genotype 2a JFH-1 cDNA. This system might be useful for HCV research.

Cited by 22 publications

References 32 publications

Production of Hepatitis C Virus Lacking the Envelope-Encoding Genes for Single-Cycle Infection by Providing Homologous Envelope Proteins or Vesicular Stomatitis Virus Glycoproteins in trans

Production of Hepatitis C Virus Lacking the Envelope-Encoding Genes for Single-Cycle Infection by Providing Homologous Envelope Proteins or Vesicular Stomatitis Virus Glycoproteins in trans

HCV Core Residues Critical for Infectivity Are Also Involved in Core-NS5A Complex Formation

GRIM-19 Restricts HCV Replication by Attenuating Intracellular Lipid Accumulation

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