Genetic Analysis of Hepatitis C Virus with Defective Genome and Its Infectivity in Vitro

Sugiyama, Kazuo; Suzuki, Kenji; Nakazawa, Takahide; Funami, Kenji; Hishiki, Takayuki; Ogawa, K.; Saito, Satoru; Shimotohno, K; Suzuki, Takeshi; Shimizu, Yuko; Tobita, Reiri; Hijikata, Makoto; Takaku, Hiroshi; Shimotohno, Kunitada

doi:10.1128/jvi.02674-08

Cited by 31 publications

(37 citation statements)

References 16 publications

(11 reference statements)

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“…S1B), a finding consistent with previous reports (4,6,15). Downregulated virus release and reduced production of infectious virus were also observed when the infectious chimeric HCV genome, TNS2J1 (30), which contains the HCV-1b-derived structural region and the JFH1-derived nonstructural region, was examined (see Fig. S1B, right).…”

Section: Resultssupporting

confidence: 78%

“…RNA was extracted from 10-times-concentrated HCVcc for real-time reverse transcription-PCR (RT-PCR). Quantitative real-time RT-PCR analysis of the 5Ј untranslated region of the HCV genome was performed as described previously (30). The forward and reverse primers were 5Ј-CCCTC CCGGGAGAGCCATAGTG-3Ј and 5Ј-GTCTCGCGGGGGCACGCCCAAA T-3Ј, respectively.…”

Section: Methodsmentioning

confidence: 99%

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Infectivity of Hepatitis C Virus Is Influenced by Association with Apolipoprotein E Isoforms

Hishiki¹,

Shimizu²,

Tobita

et al. 2010

J Virol

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117

113

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Hepatitis C virus (HCV) is a causative agent of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV in circulating blood associates with lipoproteins such as very low density lipoprotein (VLDL) and low-density lipoprotein (LDL). Although these associations suggest that lipoproteins are important for HCV infectivity, the roles of lipoproteins in HCV production and infectivity are not fully understood. To clarify the roles of lipoprotein in the HCV life cycle, we analyzed the effect of apolipoprotein E (ApoE), a component of lipoprotein, on virus production and infectivity. The production of infectious HCV was significantly reduced by the knockdown of ApoE. When an ApoE mutant that fails to be secreted into the culture medium was used, the amount of infectious HCV in the culture medium was dramatically reduced; the infectious HCV accumulated inside these cells, suggesting that infectious HCV must associate with ApoE prior to virus release. We performed rescue experiments in which ApoE isoforms were ectopically expressed in cells depleted of endogenous ApoE. The ectopic expression of the ApoE2 isoform, which has low affinity for the LDL receptor (LDLR), resulted in poor recovery of infectious HCV, whereas the expression of other isoforms, ApoE3 and ApoE4, rescued the production of infectious virus, raising it to an almost normal level. Furthermore, we found that the infectivity of HCV required both the LDLR and scavenger receptor class B, member I (SR-BI), ligands for ApoE. These findings indicate that ApoE is an essential apolipoprotein for HCV infectivity.

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Section: Resultssupporting

confidence: 78%

Section: Methodsmentioning

confidence: 99%

Infectivity of Hepatitis C Virus Is Influenced by Association with Apolipoprotein E Isoforms

Hishiki¹,

Shimizu²,

Tobita

et al. 2010

J Virol

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117

113

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“…Experiments to elucidate the role of mannose trimming of N-glycans in the HCV life cycle are currently under way. It has recently been demonstrated that subgenomic replicons or defective genomes of HCV that have the potential of translation and self-replication can be encapsidated into infectious viruslike particles by trans-complementation of the viral structural proteins (1,17,32,41,44). In these studies, the viral RNAs were generally generated by in vitro transcription from linearized corresponding plasmids, followed by electroporation into the cells.…”

Section: Discussionmentioning

confidence: 99%

Production of Infectious Hepatitis C Virus by Using RNA Polymerase I-Mediated Transcription

Masaki

Suzuki

Saeed

et al. 2010

J Virol

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In this study, we used an RNA polymerase I (Pol I) transcription system for development of a reverse genetics protocol to produce hepatitis C virus (HCV), which is an uncapped positive-strand RNA virus. Transfection with a plasmid harboring HCV JFH-1 full-length cDNA flanked by a Pol I promoter and Pol I terminator yielded an unspliced RNA with no additional sequences at either end, resulting in efficient RNA replication within the cytoplasm and subsequent production of infectious virions. Using this technology, we developed a simple replicon trans-packaging system, in which transient transfection of two plasmids enables examination of viral genome replication and virion assembly as two separate steps. In addition, we established a stable cell line that constitutively produces HCV with a low mutation frequency of the viral genome. The effects of inhibitors of N-linked glycosylation on HCV production were evaluated using this cell line, and the results suggest that certain step(s), such as virion assembly, intracellular trafficking, and secretion, are potentially up-and downregulated according to modifications of HCV envelope protein glycans. This Pol I-based HCV expression system will be beneficial for a high-throughput antiviral screening and vaccine discovery programs.Over 170 million people worldwide have been infected with hepatitis C virus (HCV) (22,33,37), and persistence of HCV infection is one of the leading causes of liver diseases, such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma (16,25,38). The HCV genome is an uncapped 9.6-kb positivestrand RNA sequence consisting of a 5Ј untranslated region (UTR), an open reading frame encoding at least 10 viral proteins (Core, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B), and a 3ЈUTR (46). The structural proteins (Core, E1, and E2) reside in the N-terminal region.The best available treatment for HCV infection, which is pegylated alpha interferon (IFN-␣) combined with ribavirin, is effective in only about half of patients and is often difficult to tolerate (25). To date, a prophylactic or therapeutic vaccine is not available. There is an urgent need to develop more effective and better tolerated therapies for HCV infection. Recently, a robust system for HCV production and infection in cultured cells has been developed. The discovery that some HCV isolates can replicate in cell cultures and release infectious particles has allowed the complete viral life cycle to be studied (23,49,53). The most robust system for HCV production involves transfection of Huh-7 cells with genomic HCV RNA of the JFH-1 strain by electroporation. However, using this RNA transfection system, the amount of secreted infectious viruses often fluctuate and mutations emerge in HCV genome with multiple passages for an extended period of time (54), which limits its usefulness for antiviral screening and vaccine development.DNA-based expression systems for HCV replication and virion production have also been examined (5, 15, 21). With DNA-based expression systems, transcriptio...

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“…[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.…”

Section: Construction Of Retroviral Expression Vectorsmentioning

confidence: 99%

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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Genetic Analysis of Hepatitis C Virus with Defective Genome and Its Infectivity in Vitro

Cited by 31 publications

References 16 publications

Infectivity of Hepatitis C Virus Is Influenced by Association with Apolipoprotein E Isoforms

Infectivity of Hepatitis C Virus Is Influenced by Association with Apolipoprotein E Isoforms

Production of Infectious Hepatitis C Virus by Using RNA Polymerase I-Mediated Transcription

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

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