Isolation and Characterization of an Huh.7.5.1-Derived Cell Clone Highly Permissive to Hepatitis C Virus

Shirasago, Yoshitaka; Sekizuka, Tsuyoshi; Saito, Kyoko; Suzuki, Tetsuro; Wakita, Takaji; Hanada, Kentaro; Kuroda, Makoto; Ryo, Akihide; Fukasawa, Masayoshi

doi:10.7883/yoken.jjid.2014.231

Cited by 25 publications

(36 citation statements)

References 43 publications

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“…9) We have isolated various hepatic cell mutants resistant to HCV infection, among which CD81-defective 751r cells and CLDN1-defective S7-A cells have been established as clones that are non-permissive to HCV infection. 10,11) Using genetic approaches with these mutant cells, we have confirmed that CD81 and CLDN1 are essential for HCV entry into hepatic cells, which is consistent with other studies. 6,7,12) There is as yet no conclusive evidence for the necessity of OCLN for HCV infection of hepatic cells, although OCLN has been shown by RNA interference (RNAi) experiments to be importantly involved in the infection.…”

supporting

confidence: 89%

“…Cell Culture In this study, several human hepatic cell lines derived from Huh7.5.1 cells 19) were used: Huh7.5.1-8 cells, which are highly permissive to HCV 10) ; CD81-defective Huh7.5.1-5 cells, 10) renamed as 751r cells; and CLDN1-defective S7-A cells.…”

Section: Methodsmentioning

confidence: 99%

“…[21][22][23] Detection of HCV infection was performed using quantitative reverse transcription-PCR (qRT-PCR) and immunohistochemistry as previously described. 10,11) HCV Pseudoparticle Infection HCV pseudoparticles (HCVpp) were generated as previously described. 24) Briefly, a Gag-Pol packaging construct (Gag-Pol 5349), a transfer vector construct (Luc 126), and an envelope glycoprotein (E1 and E2)-expressing vector [H77, genotype 1a (GenBank accession number JX472009.1); TH, genotype 1b (GenBank accession number AB985268.1); J6, genotype 2a (GenBank accession number AB047639); JFH1, genotype 2a (GenBank accession number AB047639.1); or VSV-G (GenBank accession number M27165)] were transfected into HEK 293T cells.…”

Section: Construction Of Retroviral Expression Vectorsmentioning

confidence: 99%

“…10) After culture for 4 h, the medium was changed to fresh medium, and cells were further cultured for 1-4 d. Cells were lysed, and the luciferase activity of the lysates was measured.…”

Section: Construction Of Retroviral Expression Vectorsmentioning

confidence: 99%

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Occludin-Knockout Human Hepatic Huh7.5.1-8-Derived Cells Are Completely Resistant to Hepatitis C Virus Infection

Shirasago

Shimizu

Tanida

et al. 2016

Biological & Pharmaceutical Bulletin

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It is well known that occludin (OCLN) is involved in hepatitis C virus (HCV) entry into hepatocytes, but there has been no conclusive evidence that OCLN is essential for HCV infection. In this study, we first established an OCLN-knockout cell line derived from human hepatic Huh7.5.1-8 cells using the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 system, in which two independent targeting plasmids expressing single-guide RNAs were used. One established cell clone, named OKH-4, had the OCLN gene truncated in the N-terminal region, and a complete defect of the OCLN protein was shown using immunoblot analysis. Infection of OKH-4 cells with various genotypes of HCV was abolished, and exogenous expression of the OCLN protein in OKH-4 cells completely reversed permissiveness to HCV infection. In addition, using a co-culture system of HCV-infected Huh7. 5.1-8 cells with OKH-4 cells, we showed that OCLN is also critical for cell-to-cell HCV transmission. Thus, we concluded that OCLN is essential for HCV infection of human hepatic cells. Further experiments using HCV genomic RNA-transfected OKH-4 cells or HCV subgenomic replicon-harboring OKH-4 cells suggested that OCLN is mainly involvedin the entry step of the HCV life cycle. It was also demonstrated that the second extracellular loop of OCLN, especially the two cysteine residues, is critical for HCV infection of hepatic cells. OKH-4 cells may be a useful tool for understanding not only the entire mechanism of HCV entry, but also the biological functions of OCLN.

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supporting

confidence: 89%

Section: Methodsmentioning

confidence: 99%

Section: Construction Of Retroviral Expression Vectorsmentioning

confidence: 99%

“…10) After culture for 4 h, the medium was changed to fresh medium, and cells were further cultured for 1-4 d. Cells were lysed, and the luciferase activity of the lysates was measured.…”

Section: Construction Of Retroviral Expression Vectorsmentioning

confidence: 99%

See 2 more Smart Citations

Occludin-Knockout Human Hepatic Huh7.5.1-8-Derived Cells Are Completely Resistant to Hepatitis C Virus Infection

Shirasago

Shimizu

Tanida

et al. 2016

Biological & Pharmaceutical Bulletin

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“…1). We examined the impact of SQS inhibition on HCV production by using an HCV cell culture system with human hepatoma Huh-7.5.1-derived cells and an HCV genotype 2a isolate, JFH-1 (35)(36)(37). We present data showing that SQS-mediated cholesterol biosynthesis is important for viral production, and we propose that SQS is a potential anti-HCV target.…”

mentioning

confidence: 99%

Targeting Cellular Squalene Synthase, an Enzyme Essential for Cholesterol Biosynthesis, Is a Potential Antiviral Strategy against Hepatitis C Virus

Saito

Shirasago

Suzuki

et al. 2015

J Virol

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Hepatitis C virus (HCV) exploits host membrane cholesterol and its metabolism for progeny virus production. Here, we examined the impact of targeting cellular squalene synthase (SQS), the first committed enzyme for cholesterol biosynthesis, on HCV production. By using the HCV JFH-1 strain and human hepatoma Huh-7.5.1-derived cells, we found that the SQS inhibitors YM-53601 and zaragozic acid A decreased viral RNA, protein, and progeny production in HCV-infected cells without affecting cell viability. Similarly, small interfering RNA (siRNA)-mediated knockdown of SQS led to significantly reduced HCV production, confirming the enzyme as an antiviral target. A metabolic labeling study demonstrated that YM-53601 suppressed the biosynthesis of cholesterol and cholesteryl esters at antiviral concentrations. Unlike YM-53601, the cholesterol esterification inhibitor Sandoz 58-035 did not exhibit an antiviral effect, suggesting that biosynthesis of cholesterol is more important than that of cholesteryl esters for HCV production. YM-53601 inhibited transient replication of a JFH-1 subgenomic replicon and entry of JFH-1 pseudoparticles, suggesting that at least suppression of viral RNA replication and entry contributes to the antiviral effect of the drug. Collectively, our findings highlight the importance of the cholesterol biosynthetic pathway in HCV production and implicate SQS as a potential target for antiviral strategies against HCV. IMPORTANCEHepatitis C virus (HCV) is known to be closely associated with host cholesterol and its metabolism throughout the viral life cycle. However, the impact of targeting cholesterol biosynthetic enzymes on HCV production is not fully understood. We found that squalene synthase, the first committed enzyme for cholesterol biosynthesis, is important for HCV production, and we propose this enzyme as a potential anti-HCV target. We provide evidence that synthesis of free cholesterol is more important than that of esterified cholesterol for HCV production, highlighting a marked free cholesterol dependency of HCV production. Our findings also offer a new insight into a role of the intracellular cholesterol pool that is coupled to its biosynthesis in the HCV life cycle.

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Occludin‐binding single‐chain variable fragment and antigen‐binding fragment antibodies prevent hepatitis C virus infection

et al. 2020

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Occludin (OCLN) is a tetraspan membrane component of epithelial tight junctions and a known receptor for hepatitis C virus (HCV). Previously, we established functional monoclonal antibodies (mAbs) that bind to each extracellular loop of OCLN and showed their ability to prevent in vitro and in vivo HCV infection. In this study, we converted these mAbs to corresponding monovalent antigen‐binding fragments (Fabs) and single‐chain variable fragment (scFv) antibodies. These Fab fragments and scFv antibodies demonstrate similar binding specificity and affinity to parental anti‐OCLN mAbs. Moreover, Fab fragments and scFv antibodies inhibit in vitro HCV infection. The small functional monovalent OCLN‐binding probes reported in our study have high potential as drug candidates and tools for biological and pharmaceutical studies of OCLN.

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Isolation and Characterization of an Huh.7.5.1-Derived Cell Clone Highly Permissive to Hepatitis C Virus

Cited by 25 publications

References 43 publications

Occludin-Knockout Human Hepatic Huh7.5.1-8-Derived Cells Are Completely Resistant to Hepatitis C Virus Infection

Occludin-Knockout Human Hepatic Huh7.5.1-8-Derived Cells Are Completely Resistant to Hepatitis C Virus Infection

Targeting Cellular Squalene Synthase, an Enzyme Essential for Cholesterol Biosynthesis, Is a Potential Antiviral Strategy against Hepatitis C Virus

Occludin‐binding single‐chain variable fragment and antigen‐binding fragment antibodies prevent hepatitis C virus infection

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