Publications by authors named "Asako Murayama"

39 Publications

Identification of natural compounds extracted from crude drugs as novel inhibitors of hepatitis C virus.

Biochem Biophys Res Commun 2021 Aug 12;567:1-8. Epub 2021 Jun 12.

Department of Virology II, National Institute of Infectious Diseases, 162-8640, Tokyo, Japan. Electronic address:

Natural product-derived crude drugs are expected to yield an abundance of new drugs to treat infectious diseases. Hepatitis C virus (HCV) is an oncogenic virus that significantly impacts public health. In this study, we sought to identify anti-HCV compounds in extracts of natural products. A total of 110 natural compounds extracted from several herbal medicine plants were examined for antiviral activity against HCV. Using a Huh7-mCherry-NLS-IPS reporter system for HCV infection, we first performed a rapid screening for anti-HCV compounds extracted from crude drugs. The compounds threo-2,3-bis(4-hydroxy-3-methoxyphenyl)-3-butoxypropan-1-ol (#106) and medioresinol (#110), which were extracted from Crataegus cuneate, exhibited anti-HCV activity and significantly inhibited HCV production in a dose-dependent manner. Analyses using HCV pseudoparticle and subgenomic replicon systems indicated that compounds #106 and #110 specifically inhibit HCV RNA replication but not viral entry or translation. Interestingly, compound #106 also inhibited the replication and production of hepatitis A virus. Our findings suggest that C. cuneate is a new source for novel anti-hepatitis virus drug development.
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http://dx.doi.org/10.1016/j.bbrc.2021.06.022DOI Listing
August 2021

MafF Is an Antiviral Host Factor That Suppresses Transcription from Hepatitis B Virus Core Promoter.

J Virol 2021 Jul 12;95(15):e0076721. Epub 2021 Jul 12.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

Hepatitis B virus (HBV) is a stealth virus that exhibits only minimal induction of the interferon system, which is required for both innate and adaptive immune responses. However, 90% of acutely infected adults can clear the virus, suggesting the presence of additional mechanisms that facilitate viral clearance. Here, we report that Maf bZIP transcription factor F (MafF) promotes host defense against infection with HBV. Using a small interfering RNA (siRNA) library and an HBV/NanoLuc (NL) reporter virus, we screened to identify anti-HBV host factors. Our data showed that silencing of led to a 6-fold increase in luciferase activity after HBV/NL infection. Overexpression of MafF reduced HBV core promoter transcriptional activity, which was relieved upon mutation of the putative MafF binding region. Loss of MafF expression through CRISPR/Cas9 editing (in HepG2-hNTCP-C4 cells) or siRNA silencing (in primary hepatocytes [PXB cells]) induced HBV core RNA and HBV pregenomic RNA (pgRNA) levels, respectively, after HBV infection. MafF physically binds to the HBV core promoter and competitively inhibits HNF-4α binding to an overlapping sequence in the HBV enhancer II sequence (EnhII), as seen by chromatin immunoprecipitation (ChIP) analysis. MafF expression was induced by interleukin-1β (IL-1β) or tumor necrosis factor alpha (TNF-α) treatment in both HepG2 and PXB cells, in an NF-κB-dependent manner. Consistently, expression levels were significantly enhanced and positively correlated with the levels of these cytokines in patients with chronic HBV infection, especially in the immune clearance phase. HBV is a leading cause of chronic liver diseases, infecting about 250 million people worldwide. HBV has developed strategies to escape interferon-dependent innate immune responses. Therefore, the identification of other anti-HBV mechanisms is important for understanding HBV pathogenesis and developing anti-HBV strategies. MafF was shown to suppress transcription from the HBV core promoter, leading to significant suppression of the HBV life cycle. Furthermore, MafF expression was induced in chronic HBV patients and in primary human hepatocytes (PXB cells). This induction correlated with the levels of inflammatory cytokines (IL-1β and TNF-α). These data suggest that the induction of MafF contributes to the host's antiviral defense by suppressing transcription from selected viral promoters. Our data shed light on a novel role for MafF as an anti-HBV host restriction factor.
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http://dx.doi.org/10.1128/JVI.00767-21DOI Listing
July 2021

N-Terminal PreS1 Sequence Regulates Efficient Infection of Cell-Culture-Generated Hepatitis B Virus.

Hepatology 2021 02 31;73(2):520-532. Epub 2020 Oct 31.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

Background And Aims: An efficient cell-culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral reagents. Currently, for the HBV infection assay in cell culture, viruses derived from HBV genome-integrated cell lines of HepG2.2.15 or HepAD-38 are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against antiviral reagents. HBV obtained by the transient transfection of the ordinary HBV molecular clone has limited infection efficiencies in cell culture.

Approach And Results: We found that an 11-amino-acid deletion (d11) in the preS1 region enhances the infectivity of cell-culture-generated HBV (HBVcc) to sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. Infection of HBVcc derived from a d11-introduced genotype C strain (GTC-d11) was ~10-fold more efficient than infection of wild-type GTC (GTC-wt), and the number of infected cells was comparable between GTC-d11- and HepG2.2.15-derived viruses when inoculated with the same genome equivalents. A time-dependent increase in pregenomic RNA and efficient synthesis of covalently closed circular DNA were detected after infection with the GTC-d11 virus. The involvement of d11 in the HBV large surface protein in the enhanced infectivity was confirmed by an HBV reporter virus and hepatitis D virus infection system. The binding step of the GTC-d11 virus onto the cell surface was responsible for this efficient infection.

Conclusions: This system provides a powerful tool for studying the infection and propagation of HBV in cell culture and also for developing the antiviral strategy against HBV infection.
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http://dx.doi.org/10.1002/hep.31308DOI Listing
February 2021

Anti-viral effects of interferon-λ3 on hepatitis B virus infection in cell culture.

Hepatol Res 2020 Mar 5;50(3):283-291. Epub 2020 Jan 5.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

Aim: Interferon (IFN)-λ3 is known to have antiviral effects against various pathogens. Recently, it has been reported that the production of IFN-λ3 in colon cells after the administration of nucleotide analogs is expected to reduce hepatitis B surface antigen in chronic hepatitis B patients. Here, we aimed to prove the antiviral effects of IFN-λ3 on hepatitis B virus (HBV) by using an in vitro HBV production and infection system.

Methods: We used HepG2.2.15-derived HBV as an inoculum and the replication-competent molecular clone of HBV as a replication model.

Results: By administering IFN-λ3 to HepG2 cells transfected with the HBV molecular clone, the production of hepatitis B surface antigen and hepatitis B core-related antigen was reduced dose-dependently. IFN-λ3 treatment also reduced the number of HBV-positive cells and the synthesis of covalently closed circular DNA after infection of HepG2.2.15-derived HBV to sodium taurocholate cotransporting polypeptide-transduced HepG2 cells. The inhibitory effect on HBV infection by IFN-λ3 was confirmed by using a recombinant a HBV reporter virus system. To elucidate the underlying mechanisms of the anti-HBV effect of IFN-λ3, we assessed the transcription of HBV RNA and the production of core-associated HBV DNA in HBV molecular clone-transfected HepG2 cells, and found that both parameters were reduced by IFN-λ3.

Conclusions: We observed that the administration of IFN-λ3 inhibits HBV infection and the production of HBV proteins at the HBV RNA transcription level. This finding provides novel insight into the treatment of chronic hepatitis B patients with the administration or induction of IFN-λ3.
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http://dx.doi.org/10.1111/hepr.13449DOI Listing
March 2020

Hepatitis B Virus Genotype-Dependent Vulnerability of Infected Cells to Immune Reaction in the Early Phase of Infection.

Front Microbiol 2019 18;10:2427. Epub 2019 Oct 18.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

Infection with hepatitis B virus (HBV) genotype (GT)-A has been reported to predispose patients to chronic infection. To explore the immune responses in infection with different HBV genotypes and clarify the genotype-dependent pathogenicity, a system mimicking the immune reaction during the early phase of HBV infection is indispensable. To this end, we established a coculture system with the replication-competent HBV molecular clone-transfected HepG2 cells and immortalized human natural killer (NK) cells, NK-92MI. Using this system, we evaluated HBV genotype dependency in NK functions and cell death of HBV positive HepG2 cells induced by NK cells or administration of tumor necrosis factor (TNF) by use of flow cytometry. After coculture with NK cells, we found that GT-A-positive HepG2 cells exhibited lower susceptibility to NK cell-induced cell death than GT-B- or GT-C-positive HepG2 cells. The NK responses of degranulation and cytokine production were not different among transfected HBV genotypes in cocultured cells. The expression levels of death receptors in HBV-transfected HepG2 cells were not different. In GT-A-positive cells, a similar low susceptibility was detected by the external administration of TNF, although relatively higher susceptibility was observed in GT-B- and GT-C-positive cells than in GT-A-positive cells. The activation of caspase signaling was revealed to be responsible for this genotype-dependent susceptibility. In conclusion, our results indicate that the HBV genotype does not influence the NK cell function itself but rather cell vulnerability through the TNF signal pathway. This observation may explain the high chronicity rate of HBV GT-A strains even in adult infections.
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http://dx.doi.org/10.3389/fmicb.2019.02427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6813626PMC
October 2019

Evaluation of in vitro screening and diagnostic kits for hepatitis B virus infection.

J Clin Virol 2019 08 29;117:37-42. Epub 2019 May 29.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan. Electronic address:

Background: For the diagnosis of hepatitis B virus (HBV) infection, the detection and quantification of hepatitis B surface antigen (HBsAg) and HBV DNA are used. Several kits are available for this purpose, and there is a growing need for the evaluation of these kits because their performance may be affected by HBV genotype- or strain-specific polymorphisms.

Objectives And Study Design: In this study, we used International Standards and the established regional reference panel to evaluate the performance of two HBV DNA quantitative kits, five HBsAg qualitative kits, seven HBsAg quantitative kits and three rapid immune-chromatographic tests for HBsAg.

Results: The quantification values of two HBV DNA quantitative kits exhibited excellent correlation. In the evaluation of HBsAg qualitative and quantitative kits, the titers of several specimens in the HBV-positive panel were below the detection limits of a few kits, and the specimens were determined as HBV-negative. Notably, the quantitative kit results exhibited low correlation values. However, when these data were analyzed for each genotype, the correlations improved. These results suggest that the HBsAg quantification data are influenced by HBV genotypes. The novel rapid immune-chromatographic test exhibited the comparable level of sensitivity to the HBsAg quantitative kits.

Conclusions: We evaluated the performance of kits for the detection of HBV infection. The HBV DNA quantification data correlated with an excellent agreement, whereas the HBsAg quantification data were affected by HBV genotype. Such evaluations will be useful for estimating the quality of currently available and new HBV assay kits, and for the quality control of these kits.
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http://dx.doi.org/10.1016/j.jcv.2019.05.011DOI Listing
August 2019

Combinations of two drugs among NS3/4A inhibitors, NS5B inhibitors and non-selective antiviral agents are effective for hepatitis C virus with NS5A-P32 deletion in humanized-liver mice.

J Gastroenterol 2019 May 25;54(5):449-458. Epub 2019 Jan 25.

Gastroenterology and Hepatology, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka, 565-0871, Japan.

Background: The emergence of a deletion mutant at hepatitis C virus (HCV) NS5A-P32 (P32del) has recently been reported in a subset of chronic hepatitis C patients who experience virologic failure after direct-acting antiviral drug (DAA) treatment. This mutation confers extremely high resistance to NS5A inhibitors. No effective treatment has been established for cases with this mutation.

Methods: We used a JFH1-based recombinant virus with NS5A from a genotype 1b strain to introduce a P32del mutation. We inoculated human hepatocyte chimeric mice with sera from a patient with ledipasvir/sofosbuvir therapy failure carrying a genotype 1b HCV with NS5A L31M and P32del or from a DAA-naïve patient carrying wild-type virus.

Results: JFH1-based chimeric viruses with P32del showed sufficient levels of replication for in vitro assay despite the suppression of viral growth and infectious virus production. Variants with P32del exhibited severe resistance to all tested NS5A inhibitors, including daclatasvir, ledipasvir, elbasvir and velpatasvir, but were as susceptible to NS3/4A inhibitors, NS5B inhibitors, interferon alfa-2b, and ribavirin as wild-type viruses in the in vitro assay. The P32del mutant virus caused persistent infection in all inoculated chimeric mice with high viral titer and frequency. The virus was resistant to the ledipasvir/GS-558093 (a nucleotide analog inhibitor of NS5B polymerase) regimen but susceptible to either simeprevir plus GS-558093 or peg-interferon alfa-2b, compared to the wild-type virus.

Conclusion: Therapies combining at least two drugs among NS3/4A inhibitors, NS5B inhibitors and non-selective antiviral agents may be effective for HCV-infected patients with NS5A-P32del.
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http://dx.doi.org/10.1007/s00535-018-01541-xDOI Listing
May 2019

Establishment of Replication-Competent HCV Strain with Minimum Modifications.

Methods Mol Biol 2019 ;1911:73-83

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

The HCV cell culture system, consisting of the JFH-1 strain and HuH-7 cells, has been broadly used to assess the complete HCV life cycle in cultured cells. However, being able to use multiple HCV strains in such a system is vital for future studies of this virus. We recently established a novel HCV cell culture system using another HCV genotype 2a strain, J6CF, which replicates in chimpanzees but not in cultured cells. We identified effective cell culture-adaptive mutations and established a replication-competent J6CF strain with minimum modifications in cultured cells. The strategy of how we established the replication-competent HCV strain and how we identified the effective cell culture-adaptive mutations is described here and could prove useful for establishing other replication-competent HCV strains.
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http://dx.doi.org/10.1007/978-1-4939-8976-8_4DOI Listing
June 2019

Vitamin D derivatives inhibit hepatitis C virus production through the suppression of apolipoprotein.

Antiviral Res 2018 12 16;160:55-63. Epub 2018 Oct 16.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan. Electronic address:

Supplementation with vitamin D (VD) has been reported to improve the efficacy of interferon-based therapy for chronic hepatitis C. We found that 25-hydroxyvitamin D (25-(OH)D), one of the metabolites of VD, has antiviral effects by inhibiting the infectious virus production of the hepatitis C virus (HCV). In this study, to clarify the underlying mechanisms of the anti-HCV effects, we searched VD derivatives that have anti-HCV effects and identified the common target molecule in the HCV life cycle by using an HCV cell culture system. After infection of Huh-7.5.1 cells with cell culture-generated HCV, VD derivatives were added to culture media, and the propagation of HCV was assessed by measuring the HCV core antigen levels in culture media and cell lysates. To determine the step in the HCV life cycle affected by these compounds, the single-cycle virus production assay was used with a CD81-negative cell line. Of the 14 structural derivatives of VD, an anti-HCV effect was detected in 9 compounds. Cell viability was not affected by these effective compounds. The 2 representative VD derivatives inhibited the infectious virus production in the single-cycle virus production assay. Treatment with these compounds and 25-(OH)D suppressed the expression of apolipoprotein A1 and C3, which are known to be involved in infectious virus production of HCV, and the knockdown of these apolipoproteins reduced infectious virus production. In conclusion, we identified several compounds with anti-HCV activity by screening VD derivatives. These compounds reduce the infectious virus production of HCV by suppressing the expression of apolipoproteins in host cells.
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http://dx.doi.org/10.1016/j.antiviral.2018.10.014DOI Listing
December 2018

Evaluation of antiviral effects of novel NS5A inhibitors in hepatitis C virus cell culture system with full-genome infectious clones.

Antiviral Res 2018 10 15;158:161-170. Epub 2018 Aug 15.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan. Electronic address:

Nonstructural protein 5A (NS5A) inhibitors of hepatitis C virus (HCV) are known to have potent anti-viral effects; however, these inhibitors have limited activities on strains with resistant-associated substitutions or non-genotype 1 strains. To overcome these shortcomings, novel NS5A inhibitors have been developed and approved for clinical application. The aim of this study was to evaluate the anti-viral effect of novel NS5A inhibitors (derivatives of odalasvir) on HCV genotype 2 strains in a cell culture system. Chimeric JFH-1 viruses replaced with NS5A of genotypes 1 and 2 were utilized to assess the genotype-specific potencies of NS5A inhibitors. We also examined full-genome infectious clones of JFH-1, J6cc, and J8cc to confirm the effects of NS5A inhibitors on genotype 2 strains. All chimeric viruses were capable of replication at similar levels in cell culture. We examined the anti-viral effects of derivatives of the novel NS5A inhibitor and compared with the first-generation NS5A inhibitor, daclatasvir (DCV). These compounds inhibited replication of chimeric JFH-1 viruses with NS5A of genotypes 1 and 2 at low concentrations in comparison with DCV. The EC values of J6cc and J8cc to these compounds were more than 100-fold lower than that of DCV. By long-term culture in the presence of these compounds, we obtained highly resistant variants and identified the responsible substitutions. In conclusion, novel NS5A inhibitors displayed improved potency against HCV genotype 2 strains compared with DCV. However, the activity of these compounds was impaired by emerging resistance-associated substitutions.
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http://dx.doi.org/10.1016/j.antiviral.2018.08.008DOI Listing
October 2018

IL-1β/ATF3-mediated induction of Ski2 expression enhances hepatitis B virus x mRNA degradation.

Biochem Biophys Res Commun 2018 09 25;503(3):1854-1860. Epub 2018 Jul 25.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, 162-8640, Japan. Electronic address:

Hepatitis B virus (HBV) -x protein is a transcriptional regulator required for the HBV life cycle. HBx also induces complications in the host such as hepatocellular carcinoma. We previously showed that HBx mRNA is degraded by the Ski2/RNA exosome complex. In the present study, we report the regulation of this system through the control of Ski2 expression. We identified interleukin (IL) -1β as an inducer of expression from the Ski2 promoter. IL-1β induced the expression of ATF3 transcription factor, which in turn binds to cyclic AMP-responsive element sequence in the Ski2 promoter and is responsible for Ski2 promoter induction by IL-1β. We previously reported that Ski2 expression increases HBx mRNA degradation; consistent with those data, we showed here that HBx mRNA is degraded in response to IL-1β treatment. Interestingly, HBx also significantly induced Ski2 expression. To our knowledge, this is the first report to show activation of the Ski2/RNA exosome complex by both the host and HBV. Understanding the regulation of the Ski2/RNA exosome system is expected to facilitate prevention of HBx-mediated complications through targeting the posttranscriptional degradation of HBx mRNA; and will also help shedding a light on the role of RNA decay systems in inflammation.
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http://dx.doi.org/10.1016/j.bbrc.2018.07.126DOI Listing
September 2018

Evaluation of in vitro screening and diagnostic kits for hepatitis C virus infection.

J Clin Virol 2018 08 11;105:97-102. Epub 2018 Jun 11.

Department of Safety Research on Blood and Biological Products, National Institute of Infectious Diseases, Tokyo, Japan. Electronic address:

Background: To detect infection by hepatitis C virus (HCV), a reliable kit with high sensitivity and specificity is indispensable. Detection kits for anti-HCV antibodies (anti-HCV) are used for screening, and quantification kits for HCV RNA and core antigen are used for definite diagnosis of HCV infection.

Objectives: We evaluated the performance of these kits using International Standards and a regional reference panel with HCV negative and positive specimens.

Study Design: In vitro diagnostic kits (10 anti-HCV, two HCV RNA, and three HCV core antigen) were included.

Results: Nearly all specimens in the regional reference panel were correctly identified by all anti-HCV detection kits (one false-positive was observed in one kit). Both HCV RNA quantification kits also correctly identified and quantified HCV RNA titers, without genotype-specific differences. Among the HCV core antigen kits, International Standard values were inconsistent. The sensitivities of these kits were insufficient to detect HCV in positive specimens in the regional reference panel.

Conclusions: In vitro diagnostic kits assessing anti-HCV and HCV RNA have sufficient sensitivities and specificities to screen and detect HCV infection. However, HCV core antigen quantification kits have some limitations in their sensitivities and consistencies for diagnosis of HCV infection. Quality control with International Standards and a regional reference panel is important to maintain the performances of diagnostic kits for HCV infection and to verify the clinical reliability of these kits.
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http://dx.doi.org/10.1016/j.jcv.2018.06.008DOI Listing
August 2018

Interferon sensitivity-determining region of hepatitis C virus influences virus production and interferon signaling.

Oncotarget 2018 Jan 21;9(5):5627-5640. Epub 2017 Dec 21.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

The number of amino acid substitutions in the interferon (IFN) sensitivity-determining region (ISDR) of hepatitis C virus (HCV) NS5A is a strong predictor for the outcome of IFN-based treatment. To assess the involvement of ISDR in the HCV life cycle and to clarify the molecular mechanisms influencing IFN susceptibility, we used recombinant JFH-1 viruses with NS5A of the genotype 1b Con1 strain (JFH1/5ACon1) and with NS5A ISDR containing 7 amino acid substitutions (JFH1/5ACon1/i-7mut), and compared the virus propagation and the induction of interferon-stimulated genes (ISGs). By transfecting RNAs of these strains into HuH-7-derived cells, we found that the efficiency of infectious virus production of JFH1/5ACon1/i-7mut was attenuated compared with JFH1/5ACon1. After transfecting full-length HCV RNA into HepaRG cells, the mRNA expression of ISGs was sufficiently induced by IFN treatment in JFH1/5ACon1/i-7mut-transfected but not in JFH1/5ACon1-transfected cells. These data suggested that the NS5A-mediated inhibition of ISG induction was deteriorated by amino acid substitutions in the ISDR. In conclusion, using recombinant JFH-1 viruses, we demonstrated that HCV NS5A is associated with infectious virus production and the inhibition of IFN signaling, and amino acid substitutions in the NS5A ISDR deteriorate these functions. These observations explain the strain-specific evasion of IFN signaling by HCV.
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http://dx.doi.org/10.18632/oncotarget.23562DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5814163PMC
January 2018

Resistance mutations of hepatitis B virus in entecavir-refractory patients.

Hepatol Commun 2017 04 9;1(2):110-121. Epub 2017 Mar 9.

Department of Virology II National Institute of Infectious Diseases Tokyo Japan.

The emergence of resistance mutations in the reverse transcriptase gene of hepatitis B virus (HBV) is associated with treatment failure. Entecavir (ETV) is one of the most potent anti-HBV reagents; it has a very low resistance rate and is used as the first-line treatment for chronic hepatitis B. In this study, we isolated HBVs in 4 ETV-refractory patients (2 with viral breakthrough, 1 with partial virological response, and 1 with flare-up) and assessed ETV resistance using replication-competent 1.38-fold HBV genome-length molecular clones. The full genome sequences of infected HBVs in ETV-refractory patients were determined. The HBV molecular clones were generated with the patient-derived sequences. After transfection of these molecular clones into HepG2 cells, viral replications and ETV susceptibilities were evaluated by measuring the amount of intracellular core-particle-associated HBV DNA using Southern blotting and real-time polymerase chain reaction. Among these cases, ETV-resistant variants were detected in 2 patients with viral breakthrough and responsible amino acid mutations in reverse transcriptase were successfully identified in these variants. No ETV-resistant mutation was detected in the other cases. The identified ETV-resistant mutations did not confer resistance to tenofovir disoproxil fumarate. : The HBV replication model with patient-derived sequences is useful for assessing replication efficiency, susceptibility to anti-HBV reagents, and responsible resistance mutations and can aid in choosing the appropriate treatment strategy for treatment-failure cases of chronic hepatitis B. ( 2017;1:110-121).
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http://dx.doi.org/10.1002/hep4.1022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5721430PMC
April 2017

Amino Acid Mutations in the NS4A Region of Hepatitis C Virus Contribute to Viral Replication and Infectious Virus Production.

J Virol 2017 02 31;91(4). Epub 2017 Jan 31.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan

Hepatitis C virus (HCV) strain JFH-1, which belongs to genotype 2a, replicates autonomously in cultured cells, whereas another genotype 2a strain, J6CF, does not. Previously, we found that replacement of the NS3 helicase and NS5B-to-3'X regions of J6CF with those of JFH-1 confers J6CF replication competence. In this study, we aimed to identify the minimum modifications within these genomic regions needed to establish replication-competent J6CF. We previously identified 4 mutations in the NS5B-to-3'X region that could be used instead of replacement of this region to confer J6CF replication competence. Here, we induced cell culture-adaptive mutations in J6CF by the long-term culture of J6CF/JFH-1 chimeras composed of JFH-1 NS5B-to-3'X or individual parts of this but not the NS3 helicase region. After 2 months of culture, efficient HCV replication and infectious virus production in chimeric RNA-transfected cells were observed, and several amino acid mutations in NS4A were identified in replicating HCV genomes. The introduction of NS4A mutations into the J6CF/JFH-1 chimeras enhanced viral replication and infectious virus production. Immunofluorescence microscopy demonstrated that some of these mutations altered the subcellular localization of the coexpressed NS3 protein and affected the interaction between NS3 and NS4A. Finally, introduction of the most effective NS4A mutation, A1680E, into J6CF contributed to its replication competence in cultured cells when introduced in conjunction with four previously identified adaptive mutations in the NS5B-to-3'X region. In conclusion, we identified an adaptive mutation in NS4A that confers J6CF replication competence when introduced in conjunction with 4 mutations in NS5B-to-3'X and established a replication-competent J6CF strain with minimum essential modifications in cultured cells.

Importance: The HCV cell culture system using the JFH-1 strain and HuH-7 cells can be used to assess the complete HCV life cycle in cultured cells. This cell culture system has been used to develop direct-acting antivirals against HCV, and the ability to use various HCV strains within this system is important for future studies. In this study, we aimed to establish a novel HCV cell culture system using another HCV genotype 2a strain, J6CF, which replicates in chimpanzees but not in cultured cells. We identified an effective cell culture-adaptive mutation in NS4A and established a replication-competent J6CF strain in cultured cells with minimum essential modifications. The described strategy can be used in establishing a novel HCV cell culture system, and the replication-competent J6CF clone composed of the minimum essential modifications needed for cell culture adaptation will be valuable as another representative of genotype 2a strains.
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http://dx.doi.org/10.1128/JVI.02124-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5286874PMC
February 2017

Completion of the Entire Hepatitis C Virus Life Cycle in Vero Cells Derived from Monkey Kidney.

mBio 2016 06 14;7(3). Epub 2016 Jun 14.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan

Unlabelled: A hepatitis C virus (HCV) cell culture system incorporating the JFH-1 strain and the human hepatoma cell line HuH-7 enabled the production of infectious HCV particles. Several host factors were identified as essential for HCV replication. Supplementation of these factors in nonhepatic human cell lines enabled HCV replication and particle production. Vero cells established from monkey kidney are commonly used for the production of vaccines against a variety of viruses. In this study, we aimed to establish a novel Vero cell line to reconstruct the HCV life cycle. Unmodified Vero cells did not allow HCV infection or replication. The expression of microRNA 122 (miR-122), an essential factor for HCV replication, is notably low in Vero cells. Therefore, we supplemented Vero cells with miR-122 and found that HCV replication was enhanced. However, Vero cells that expressed miR-122 still did not allow HCV infection. We supplemented HCV receptor molecules and found that scavenger receptor class B type I (SRBI) was essential for HCV infection in Vero cells. The supplementation of apolipoprotein E (ApoE), a host factor important for virus production, enabled the production of infectious virus in Vero cells. Finally, we created a Vero cell line that expressed the essential factors miR-122, SRBI, and ApoE; the entire HCV life cycle, including infection, replication, and infectious virus production, was completed in these cells. In conclusion, we demonstrated that miR-122, SRBI, and ApoE were necessary and sufficient for the completion of the entire HCV life cycle in nonhuman, nonhepatic Vero cells.

Importance: HCV is a major cause of chronic liver diseases worldwide, and an effective prophylactic HCV vaccine is needed. For safety reasons, the current HCV cell culture system using HuH-7 cells, which was established from a hepatocellular carcinoma, is not suitable for the production of a vaccine against HCV. A robust HCV production system using non-cancer-derived cells is indispensable for this purpose. In this study, we wanted to establish a novel HCV cell culture system using Vero cells, which are widely used in the production of vaccines against different viruses. We identified the minimum essential host factors for the completion of the entire HCV life cycle in Vero cells to develop a novel HCV cell culture system. A cell culture system that uses Vero cells will be useful not only for HCV vaccine production but also for the further elucidation of the mechanisms of various HCV-host interactions.
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http://dx.doi.org/10.1128/mBio.00273-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4916372PMC
June 2016

Antimicrobial peptide LL-37 attenuates infection of hepatitis C virus.

Hepatol Res 2016 Aug 4;46(9):924-32. Epub 2016 Feb 4.

Department of Virology II, National Institute of Infectious Diseases, Tokyo.

Aim: Although recent studies indicate that supplementation with vitamin D (VD) potentiates a sustained viral response by interferon-based therapy to chronic hepatitis C, detailed mechanisms are not fully defined. The production of cathelicidin, an antimicrobial peptide, has been demonstrated to be part of the VD-dependent antimicrobial pathway in innate immunity. Cathelicidin is known to directly kill or inhibit the growth of microbial pathogens including mycobacteria and viruses.

Methods: We used a hepatitis C virus (HCV) cell culture system to clarify the anti-HCV effects of the human cathelicidin, LL-37. HuH-7 cells were administrated with LL-37 and infected with cell culture-generated HCV (HCVcc). HCV propagation was estimated by measuring the level of HCV core antigen (Ag).

Results: Treatment with LL-37 resulted in decreased intra- and extracellular levels of HCV core Ag, suggesting inhibition of HCV propagation. To assess the effects of LL-37 on HCV replication, JFH-1 subgenomic replicon RNA-transfected cells were treated with LL-37. However, inhibition of HCV replication was not detected by this assay. To clarify the effects on HCV infection, we treated HCVcc with LL-37 and removed the antimicrobial peptide prior to use of the virus in infection. This exposure of HCVcc to LL-37 diminished the infectivity titers in a dose-dependent fashion. Iodixanol density gradient analysis revealed that the peak fraction of infectivity titer was eliminated by LL-37 treatment.

Conclusion: The VD-associated antimicrobial peptide LL-37 attenuated the infectivity of HCV. This anti-HCV effect of LL-37 may explain the contribution of VD to the improved efficacy of interferon-based therapy.
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http://dx.doi.org/10.1111/hepr.12627DOI Listing
August 2016

Amino Acid Polymorphisms in Hepatitis C Virus Core Affect Infectious Virus Production and Major Histocompatibility Complex Class I Molecule Expression.

Sci Rep 2015 Sep 14;5:13994. Epub 2015 Sep 14.

Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan.

Amino acid (aa) polymorphisms in the hepatitis C virus (HCV) genotype 1b core protein have been reported to be a potent predictor for poor response to interferon (IFN)-based therapy and a risk factor for hepatocarcinogenesis. We investigated the effects of these polymorphisms with genotype 1b/2a chimeric viruses that contained polymorphisms of Arg/Gln at aa 70 and Leu/Met at aa 91. We found that infectious virus production was reduced in cells transfected with chimeric virus RNA that had Gln at aa 70 (aa70Q) compared with RNA with Arg at aa 70 (aa70R). Using flow cytometry analysis, we confirmed that HCV core protein accumulated in aa70Q clone transfected cells, and it caused a reduction in cell-surface expression of major histocompatibility complex (MHC) class I molecules induced by IFN treatment through enhanced protein kinase R phosphorylation. We could not detect any effects due to the polymorphism at aa 91. In conclusion, the polymorphism at aa 70 was associated with efficiency of infectious virus production, and this deteriorated virus production in strains with aa70Q resulted in the intracellular accumulation of HCV proteins and attenuation of MHC class I molecule expression. These observations may explain the strain-associated resistance to IFN-based therapy and hepatocarcinogenesis of HCV.
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http://dx.doi.org/10.1038/srep13994DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4568458PMC
September 2015

Hepatitis C virus entry is impaired by claudin-1 downregulation in diacylglycerol acyltransferase-1-deficient cells.

J Virol 2014 Aug 4;88(16):9233-44. Epub 2014 Jun 4.

Laboratory of Immunology and Infectious Diseases, Graduate School of Medical Science and Engineering, KAIST, Daejeon, Republic of Korea

Unlabelled: Diacylglycerol acyltransferase-1 (DGAT1) is involved in the assembly of hepatitis C virus (HCV) by facilitating the trafficking of the HCV core protein to the lipid droplet. Here, we abrogated DGAT1 expression in Huh-7.5 cells by using either the transcription activator-like effector nuclease (TALEN) or lentivirus vector short hairpin RNA (shRNA) and achieved complete long-term silencing of DGAT1. HCV entry was severely impaired in DGAT1-silenced Huh-7.5 cell lines, which showed markedly diminished claudin-1 (CLDN1) expression. In DGAT1-silenced cell lines, the forced expression of CLDN1 restored HCV entry, implying that the downregulation of CLDN1 is a critical factor underlying defective HCV entry. The expression of the gene coding for hepatocyte nuclear factor 4α (HNF4α) and other hepatocyte-specific genes was also reduced in DGAT1-silenced cell lines. After DGAT1 gene rescue, CLDN1 expression was preserved, and HCV entry was restored. Strikingly, after DGAT1 silencing, CLDN1 expression and HCV entry were also restored by low-dose palmitic acid treatment, indicating that the downregulation of CLDN1 was associated with altered fatty acid homeostasis in the absence of DGAT1. Our findings provide novel insight into the role of DGAT1 in the life cycle of HCV.

Importance: In this study, we report the novel effect of complete silencing of DGAT1 on the entry of HCV. DGAT1 was recently reported as a host factor of HCV, involved in the assembly of HCV by facilitating the trafficking of the HCV core protein to lipid droplets. We achieved complete and long-term silencing of DGAT1 by either TALEN or repeated transduction of lentivirus shRNA. We found that HCV entry was severely impaired in DGAT1-silenced cell lines. The impairment of HCV entry was caused by CLDN1 downregulation, and the expression of HNF4α and other hepatocyte-specific genes was also downregulated in DGAT1-silenced cell lines. Our results suggest new roles of DGAT1 in human liver-derived cells: maintaining intracellular lipid homeostasis and affecting HCV entry by modulating CLDN1 expression.
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http://dx.doi.org/10.1128/JVI.01428-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4136266PMC
August 2014

Single strain isolation method for cell culture-adapted hepatitis C virus by end-point dilution and infection.

PLoS One 2014 21;9(5):e98168. Epub 2014 May 21.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

The hepatitis C virus (HCV) culture system has enabled us to clarify the HCV life cycle and essential host factors for propagation. However, the virus production level of wild-type JFH-1 (JFH-1/wt) is limited, and this leads to difficulties in performing experiments that require higher viral concentrations. As the cell culture-adapted JFH-1 has been reported to have robust virus production, some mutations in the viral genome may play a role in the efficiency of virus production. In this study, we obtained cell culture-adapted virus by passage of full-length JFH-1 RNA-transfected Huh-7.5.1 cells. The obtained virus produced 3 log-fold more progeny viruses as compared with JFH-1/wt. Several mutations were identified as being responsible for robust virus production, but, on reverse-genetics analysis, the production levels of JFH-1 with these mutations did not reach the level of cell culture-adapted virus. By using the single strain isolation method by end-point dilution and infection, we isolated two strains with additional mutations, and found that these strains have the ability to produce more progeny viruses. On reverse-genetics analysis, the strains with these additional mutations were able to produce robust progeny viruses at comparable levels as cell culture-adapted JFH-1 virus. The strategy used in this study will be useful for identifying strains with unique characteristics, such as robust virus production, from a diverse population, and for determining the responsible mutations for these characteristics.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0098168PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4029950PMC
January 2015

Involvement of hepatitis C virus NS5A hyperphosphorylation mediated by casein kinase I-α in infectious virus production.

J Virol 2014 Jul 23;88(13):7541-55. Epub 2014 Apr 23.

Department of Infectious Diseases, Hamamatsu University School of Medicine, Handayama, Higashi-ku, Hamamatsu, Japan

Unlabelled: Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) possesses multiple functions in the viral life cycle. NS5A is a phosphoprotein that exists in hyperphosphorylated and basally phosphorylated forms. Although the phosphorylation status of NS5A is considered to have a significant impact on its function, the mechanistic details regulating NS5A phosphorylation, as well as its exact roles in the HCV life cycle, are still poorly understood. In this study, we screened 404 human protein kinases via in vitro binding and phosphorylation assays, followed by RNA interference-mediated gene silencing in an HCV cell culture system. Casein kinase I-α (CKI-α) was identified as an NS5A-associated kinase involved in NS5A hyperphosphorylation and infectious virus production. Subcellular fractionation and immunofluorescence confocal microscopy analyses showed that CKI-α-mediated hyperphosphorylation of NS5A contributes to the recruitment of NS5A to low-density membrane structures around lipid droplets (LDs) and facilitates its interaction with core protein and the viral assembly. Phospho-proteomic analysis of NS5A with or without CKI-α depletion identified peptide fragments that corresponded to the region located within the low-complexity sequence I, which is important for CKI-α-mediated NS5A hyperphosphorylation. This region contains eight serine residues that are highly conserved among HCV isolates, and subsequent mutagenesis analysis demonstrated that serine residues at amino acids 225 and 232 in NS5A (genotype 2a) may be involved in NS5A hyperphosphorylation and hyperphosphorylation-dependent regulation of virion production. These findings provide insight concerning the functional role of NS5A phosphorylation as a regulatory switch that modulates its multiple functions in the HCV life cycle.

Importance: Mechanisms regulating NS5A phosphorylation and its exact function in the HCV life cycle have not been clearly defined. By using a high-throughput screening system targeting host protein kinases, we identified CKI-α as an NS5A-associated kinase involved in NS5A hyperphosphorylation and the production of infectious virus. Our results suggest that the impact of CKI-α in the HCV life cycle is more profound on virion assembly than viral replication via mediation of NS5A hyperphosphorylation. CKI-α-dependent hyperphosphorylation of NS5A plays a role in recruiting NS5A to low-density membrane structures around LDs and facilitating its interaction with the core for new virus particle formation. By using proteomic approach, we identified the region within the low-complexity sequence I of NS5A that is involved in NS5A hyperphosphorylation and hyperphosphorylation-dependent regulation of infectious virus production. These findings will provide novel mechanistic insights into the roles of NS5A-associated kinases and NS5A phosphorylation in the HCV life cycle.
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http://dx.doi.org/10.1128/JVI.03170-13DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054430PMC
July 2014

A subclone of HuH-7 with enhanced intracellular hepatitis C virus production and evasion of virus related-cell cycle arrest.

PLoS One 2012 20;7(12):e52697. Epub 2012 Dec 20.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

Hepatitis C virus (HCV) cell culture system with JFH-1 strain and HuH-7 cells enabled us to produce infectious HCV particles in vitro, and such system is useful to explore the anti-HCV compounds and to develop the vaccine against HCV. In the present study, we describe the derivation of a cell line that permits improved production of HCV particles. Specifically, we characterized several subclones that were isolated from the original HuH-7 cell line by limiting dilution. These HuH-7 subclones displayed a notable range of HCV production levels following transfection by full-genome JFH-1 RNA. Among these subclones, HuH-7T1 produced HCV more efficiently than other subclones and Huh-7.5.1 that is known to be highly permissive for HCV replication. Upon transfection with full-genome RNA, HCV production was increased ten-fold in HuH-7T1 compared to Huh-7.5.1. This increase in viral production correlated with increased efficiency of intracellular infectious virus production. Furthermore, HCV replication did not induce cell cycle arrest in HuH-7T1, whereas it did in Huh-7.5.1. Consequently, the use of HuH-7T1 as host cells could provide increased population of HCV-positive cells and elevated viral titer. In conclusion, we isolated a HuH-7 subclone, HuH-7T1, that supports efficient HCV production. High efficiency of intracellular infectious virus production and evasion of cell cycle arrest were important for this phenotype. We expect that the use of this cell line will facilitate analysis of the underlying mechanisms for HCV particle assembly and the cell cycle arrest caused by HCV.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0052697PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3527576PMC
June 2013

Novel cell culture-adapted genotype 2a hepatitis C virus infectious clone.

J Virol 2012 Oct 11;86(19):10805-20. Epub 2012 Jul 11.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

Although the recently developed infectious hepatitis C virus system that uses the JFH-1 clone enables the study of whole HCV viral life cycles, limited particular HCV strains have been available with the system. In this study, we isolated another genotype 2a HCV cDNA, the JFH-2 strain, from a patient with fulminant hepatitis. JFH-2 subgenomic replicons were constructed. HuH-7 cells transfected with in vitro transcribed replicon RNAs were cultured with G418, and selected colonies were isolated and expanded. From sequencing analysis of the replicon genome, several mutations were found. Some of the mutations enhanced JFH-2 replication; the 2217AS mutation in the NS5A interferon sensitivity-determining region exhibited the strongest adaptive effect. Interestingly, a full-length chimeric or wild-type JFH-2 genome with the adaptive mutation could replicate in Huh-7.5.1 cells and produce infectious virus after extensive passages of the virus genome-replicating cells. Virus infection efficiency was sufficient for autonomous virus propagation in cultured cells. Additional mutations were identified in the infectious virus genome. Interestingly, full-length viral RNA synthesized from the cDNA clone with these adaptive mutations was infectious for cultured cells. This approach may be applicable for the establishment of new infectious HCV clones.
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http://dx.doi.org/10.1128/JVI.07235-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3457305PMC
October 2012

Japanese reference panel of blood specimens for evaluation of hepatitis C virus RNA and core antigen quantitative assays.

J Clin Microbiol 2012 Jun 11;50(6):1943-9. Epub 2012 Apr 11.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

An accurate and reliable quantitative assay for hepatitis C virus (HCV) is essential for measuring viral propagation and the efficacy of antiviral therapy. There is a growing need for domestic reference panels for evaluation of clinical assay kits because the performance of these kits may vary with region-specific genotypes or polymorphisms. In this study, we established a reference panel by selecting 80 donated blood specimens in Japan that tested positive for HCV. Using this panel, we quantified HCV viral loads using two HCV RNA kits and five core antigen (Ag) kits currently available in Japan. The data from the two HCV RNA assay kits showed excellent correlation. All RNA titers were distributed evenly across a range from 3 to 7 log IU/ml. Although the data from the five core Ag kits also correlated with RNA titers, the sensitivities of individual kits were not sufficient to quantify viral load in all samples. As calculated by the correlation with RNA titers, the theoretical lower limits of detection by these core Ag assays were higher than those for the detection of RNA. Moreover, in several samples in our panel, core Ag levels were underestimated compared to RNA titers. Sequence analysis in the HCV core region suggested that polymorphisms at amino acids 47 to 49 of the core Ag were responsible for this underestimation. The panel established in this study will be useful for estimating the quality of currently available and upcoming HCV assay kits; such quality control is essential for clinical usage of these kits.
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http://dx.doi.org/10.1128/JCM.00487-12DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3372108PMC
June 2012

25-Hydroxyvitamin D3 suppresses hepatitis C virus production.

Hepatology 2012 Oct;56(4):1231-9

Division of Gastroenterology, Department of Medicine, Showa University School of Medicine, Tokyo, Japan.

Unlabelled: Because the current interferon (IFN)-based treatment for hepatitis C virus (HCV) infection has a therapeutic limitation and side effects, a more efficient therapeutic strategy is desired. Recent studies show that supplementation of vitamin D significantly improves sustained viral response via IFN-based therapy. However, mechanisms and an active molecular form of vitamin D for its anti-HCV effects have not been fully clarified. To address these questions, we infected HuH-7 cells with cell culture-generated HCV in the presence or absence of vitamin D(3) or its metabolites. To our surprise, 25-hydroxyvitamin D(3) [25(OH)D(3) ], but not vitamin D(3) or 1,25-dihydroxyvitamin D(3) , reduced the extra- and intracellular levels of HCV core antigen in a concentration-dependent manner. Single-cycle virus production assay with a CD81-negative cell line reveals that the inhibitory effect of 25(OH)D(3) is at the level of infectious virus assembly but not entry or replication. Long-term 25(OH)D(3) treatment generates a HCV mutant with acquired resistance to 25(OH)D(3) , and this mutation resulting in a N1279Y substitution in the nonstructural region 3 helicase domain is responsible for the resistance.

Conclusion: 25(OH)D(3) is a novel anti-HCV agent that targets an infectious viral particle assembly step. This finding provides insight into the improved efficacy of anti-HCV treatment via the combination of vitamin D(3) and IFN. Our results also suggest that 25(OH)D(3) , not vitamin D(3) , is a better therapeutic option in patients with hepatic dysfunction and reduced enzymatic activity for generation of 25(OH)D(3) .
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http://dx.doi.org/10.1002/hep.25763DOI Listing
October 2012

Production of infectious chimeric hepatitis C virus genotype 2b harboring minimal regions of JFH-1.

J Virol 2012 Feb 7;86(4):2143-52. Epub 2011 Dec 7.

Department of Virology II, National Institute of Infectious Diseases, Shinjuku-ku, Tokyo, Japan.

To establish a cell culture system for chimeric hepatitis C virus (HCV) genotype 2b, we prepared a chimeric construct harboring the 5' untranslated region (UTR) to the E2 region of the MA strain (genotype 2b) and the region of p7 to the 3' UTR of the JFH-1 strain (genotype 2a). This chimeric RNA (MA/JFH-1.1) replicated and produced infectious virus in Huh7.5.1 cells. Replacement of the 5' UTR of this chimera with that from JFH-1 (MA/JFH-1.2) enhanced virus production, but infectivity remained low. In a long-term follow-up study, we identified a cell culture-adaptive mutation in the core region (R167G) and found that it enhanced virus assembly. We previously reported that the NS3 helicase (N3H) and the region of NS5B to 3' X (N5BX) of JFH-1 enabled replication of the J6CF strain (genotype 2a), which could not replicate in cells. To reduce JFH-1 content in MA/JFH-1.2, we produced a chimeric viral genome for MA harboring the N3H and N5BX regions of JFH-1, combined with a JFH-1 5' UTR replacement and the R167G mutation (MA/N3H+N5BX-JFH1/R167G). This chimeric RNA replicated efficiently, but virus production was low. After the introduction of four additional cell culture-adaptive mutations, MA/N3H+N5BX-JFH1/5am produced infectious virus efficiently. Using this chimeric virus harboring minimal regions of JFH-1, we analyzed interferon sensitivity and found that this chimeric virus was more sensitive to interferon than JFH-1 and another chimeric virus containing more regions from JFH-1 (MA/JFH-1.2/R167G). In conclusion, we established an HCV genotype 2b cell culture system using a chimeric genome harboring minimal regions of JFH-1. This cell culture system may be useful for characterizing genotype 2b viruses and developing antiviral strategies.
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http://dx.doi.org/10.1128/JVI.05386-11DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3302416PMC
February 2012

Development of recombinant hepatitis C virus with NS5A from strains of genotypes 1 and 2.

Biochem Biophys Res Commun 2011 Jul 6;410(3):404-9. Epub 2011 Jun 6.

Department of Virology II, National Institute of Infectious Diseases, Tokyo 162-8640, Japan.

Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) plays multiple and diverse roles in the viral lifecycle, and is currently recognized as a novel target for anti-viral therapy. To establish an HCV cell culture system with NS5A of various strains, recombinant viruses were generated by replacing NS5A of strain JFH-1 with those of strains of genotypes 1 (H77; 1a and Con1; 1b) and 2 (J6CF; 2a and MA; 2b). All these recombinant viruses were capable of replication and infectious virus production. The replacement of JFH-1 NS5A with those of genotype 1 strains resulted in similar or slightly reduced virus production, whereas replacement with those of genotype 2 strains enhanced virus production as compared with JFH-1 wild-type. A single cycle virus production assay with a CD81-negative cell line revealed that the efficient virus production elicited by replacement with genotype 2 strains depended on enhanced viral assembly, and that substitutions in the C-terminus of NS5A were responsible for this phenotype. Pulse-chase assays revealed that these substitutions in the C-terminus of NS5A were possibly associated with accelerated cleavage kinetics at the NS5A-NS5B site. Using this cell culture system with NS5A-substituted recombinant viruses, the anti-viral effects of an NS5A inhibitor were then examined. A 300- to 1000-fold difference in susceptibility to the inhibitor was found between strains of genotypes 1 and 2. This system will facilitate not only a better understanding of strain-specific roles of NS5A in the HCV lifecycle, but also enable the evaluation of genotype and strain dependency of NS5A inhibitors.
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http://dx.doi.org/10.1016/j.bbrc.2011.05.144DOI Listing
July 2011

In vivo adaptation of hepatitis C virus in chimpanzees for efficient virus production and evasion of apoptosis.

Hepatology 2011 Aug 26;54(2):425-33. Epub 2011 Jun 26.

Department of Virology II, National Institute of Infectious Diseases, Tokyo, Japan.

Unlabelled: Hepatitis C virus (HCV) employs various strategies to establish persistent infection that can cause chronic liver disease. Our previous study showed that both the original patient serum from which the HCV JFH-1 strain was isolated and the cell culture-generated JFH-1 virus (JFH-1cc) established infection in chimpanzees, and that infected JFH-1 strains accumulated mutations after passage through chimpanzees. The aim of this study was to compare the in vitro characteristics of JFH-1 strains emerged in each chimpanzee at early and late stages of infection, as it could provide an insight into the phenomenon of viral persistence. We generated full-genome JFH-1 constructs with the mutations detected in patient serum-infected (JFH-1/S1 and S2) and JFH-1cc-infected (JFH-1/C) chimpanzees, and assessed their effect on replication, infectious virus production, and regulation of apoptosis in cell culture. The extracellular HCV core antigen secreted from JFH-1/S1-, S2-, and C-transfected HuH-7 cells was 2.5, 8.9, and 2.1 times higher than that from JFH-1 wild-type (JFH-1/wt) transfected cells, respectively. Single cycle virus production assay with a CD81-negative cell line revealed that the strain JFH-1/S2, isolated from the patient serum-infected chimpanzee at a later time point of infection, showed lower replication and higher capacity to assemble infectious virus particles. This strain also showed productive infection in human hepatocyte-transplanted mice. Furthermore, the cells harboring this strain displayed lower susceptibility to the apoptosis induced by tumor necrosis factor α or Fas ligand compared with the cells replicating JFH-1/wt.

Conclusion: The ability of lower replication, higher virus production, and less susceptibility to cytokine-induced apoptosis may be important for prolonged infection in vivo. Such control of viral functions by specific mutations may be a key strategy for establishing persistent infection.
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http://dx.doi.org/10.1002/hep.24399DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3145027PMC
August 2011
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