Antiviral effect of peptoids on hepatitis B virus infection in cell culture.

Although antimicrobial peptides have been shown to inactivate viruses through disruption of their viral envelopes, clinical use of such peptides has been hampered by a number of factors, especially their enzymatically unstable structures. To overcome the shortcomings of antimicrobial peptides, peptoids (sequence-specific N-substituted glycine oligomers) mimicking antimicrobial peptides have been developed. We aimed to demonstrate the antiviral effects of antimicrobial peptoids against hepatitis B virus (HBV) in cell culture. The anti-HBV activity of antimicrobial peptoids was screened and evaluated in an infection system involving the HBV reporter virus and HepG2.2.15-derived HBV. By screening with the HBV reporter virus infection system, three (TM1, TM4, and TM19) of 12 peptoids were identified as reducing the infectivity of HBV, though they did not alter the production levels of HBs antigen in cell culture. These peptoids were not cytotoxic at the evaluated concentrations. Among these peptoids, TM19 was confirmed to reduce HBV infection most potently in a HepG2.2.15-derived HBV infection system that closely demonstrates authentic HBV infection. In cell culture, the most effective administration of TM19 was virus treatment at the infection step, but the reduction in HBV infectivity by pre-treatment or post-treatment of cells with TM19 was minimal. The disrupting effect of TM19 targeting infectious viral particles was clarified in iodixanol density gradient analysis. In conclusion, the peptoid TM19 was identified as a potent inhibitor of HBV. This peptoid prevents HBV infection by disrupting viral particles and is a candidate for a new class of anti-HBV reagents.

Production and Purification of Cell Culture-generated Hepatitis B Virus by Transient Transfection and Density Gradient.

An efficient cell culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral agents. Currently, for HBV infection assays in cell culture, HBV genome-integrated cell line-derived viruses are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against anti-viral agents. To detect the infection of cell culture-generated HBV (HBVcc) by the transient transfection of the HBV molecular clone, a large amount of purified viruses is needed, because such viruses exhibit limited infection efficiencies in cell culture. Here, we describe how to generate and purify HBVcc by the transient transfection of HBV molecular clones. This system provides a powerful tool for studying the infection and propagation of HBV and for developing anti-viral agents against HBV.

Performance evaluation of in vitro diagnostic kits for hepatitis B virus infection using the regional reference panel of Japan.

BACKGROUND: Hepatitis B virus (HBV) infection is a global public health concern. Precise and sensitive detection of viral markers, including HBV DNA and HBs antigen (Ag), is essential to determine HBV infection. METHODS: The sensitivities and specificities of 5 HBV DNA and 14 HBsAg kits were evaluated using World Health Organization International Standards (WHO IS) and the Regional Reference Panel (RRP) consisting of 64 HBsAg-negative and 80 HBsAg-positive specimens. RESULTS: All 5 HBV DNA kits detected HBV DNA in the WHO IS at a concentration of 10 IU/mL. The sensitivity and specificity to the RRP were 98.8-100% and 96.9-100%, respectively. HBV DNA titers were well correlated among the 5 kits regardless of HBV genotype. However, discordance of the HBV DNA titer was found in 5 specimens measured by CAP/CTM HBV v2.0. Among 12 automated HBsAg kits, the minimum detectable concentrations in the WHO IS varied from 0.01 to 0.1 IU/mL. Two lateral flow assays were positive for WHO IS concentrations greater than or equal to 1.0 and 0.1 IU/mL, respectively. When analyzed by the RRP, 12 automated kits exhibited a sensitivity of 98.8-100%, and 2 lateral flow assays showed sensitivities of 93.8% and 100%. The specificities of HBsAg kits were 100%. In the quantification of HBsAg, some kits showed a poor correlation of measurements with each other and showed up to a 1.7-fold difference in the regression coefficient of HBsAg titers. There were variations in the correlations of measurements among HBsAg kits when analyzed by genotype. CONCLUSIONS: Five HBV DNA kits showed sufficient sensitivity and specificity to determine HBV infection. HBV DNA titers were compatible with each other irrespective of HBV genotypes. HBsAg kits had enough sensitivity and specificity to screen for HBV infection. One of the lateral flow assays had a nearly equivalent sensitivity to that of the automated HBsAg kit. HBsAg titers quantified by the evaluated kits were not compatible across the kits. Genotype-dependent amino acid variations might affect the quantification of HBsAg titers.

Neutralization of hepatitis B virus with vaccine-escape mutations by hepatitis B vaccine with large-HBs antigen.

Although the current hepatitis B (HB) vaccine comprising small-HBs antigen (Ag) is potent and safe, attenuated prophylaxis against hepatitis B virus (HBV) with vaccine-escape mutations (VEMs) has been reported. We investigate an HB vaccine consisting of large-HBsAg that overcomes the shortcomings of the current HB vaccine. Yeast-derived large-HBsAg is immunized into rhesus macaques, and the neutralizing activities of the induced antibodies are compared with those of the current HB vaccine. Although the antibodies induced by the current HB vaccine cannot prevent HBV infection with VEMs, the large-HBsAg vaccine-induced antibodies neutralize those infections. The HBV genotypes that exhibited attenuated neutralization via these vaccines are different. Here, we show that the HB vaccine consisting of large-HBsAg is useful to compensate for the shortcomings of the current HB vaccine. The combined use of these HB vaccines may induce antibodies that can neutralize HBV strains with VEMs or multiple HBV genotypes.

Development and Use of a Kinetical and Real-Time Monitoring System to Analyze the Replication of Hepatitis C Virus.

In microbiological research, it is important to understand the time course of each step in a pathogen's lifecycle and changes in the host cell environment induced by infection. This study is the first to develop a real-time monitoring system that kinetically detects luminescence reporter activity over time without sampling cells or culture supernatants for analyzing the virus replication. Subgenomic replicon experiments with hepatitis C virus (HCV) showed that transient translation and genome replication can be detected separately, with the first peak of translation observed at 3-4 h and replication beginning around 20 h after viral RNA introduction into cells. From the bioluminescence data set measured every 30 min (48 measurements per day), the initial rates of translation and replication were calculated, and their capacity levels were expressed as the sums of the measured signals in each process, which correspond to the areas on the kinetics graphs. The comparison of various HuH-7-derived cell lines showed that the bioluminescence profile differs among cell lines, suggesting that both translation and replication capacities potentially influence differences in HCV susceptibility. The effects of RNA mutations within the 5' UTR of the replicon on viral translation and replication were further analyzed in the system developed, confirming that mutations to the miR-122 binding sites primarily reduce replication activity rather than translation. The newly developed real-time monitoring system should be applied to the studies of various viruses and contribute to the analysis of transitions and progression of each process of their life cycle.

The kinesin KIF4 mediates HBV/HDV entry through the regulation of surface NTCP localization and can be targeted by RXR agonists in vitro.

Intracellular transport via microtubule-based dynein and kinesin family motors plays a key role in viral reproduction and transmission. We show here that Kinesin Family Member 4 (KIF4) plays an important role in HBV/HDV infection. We intended to explore host factors impacting the HBV life cycle that can be therapeutically addressed using siRNA library transfection and HBV/NLuc (HBV/NL) reporter virus infection in HepG2-hNTCP cells. KIF4 silencing resulted in a 3-fold reduction in luciferase activity following HBV/NL infection. KIF4 knockdown suppressed both HBV and HDV infection. Transient KIF4 depletion reduced surface and raised intracellular NTCP (HBV/HDV entry receptor) levels, according to both cellular fractionation and immunofluorescence analysis (IF). Overexpression of wild-type KIF4 but not ATPase-null KIF4 mutant regained the surface localization of NTCP and significantly restored HBV permissiveness in these cells. IF revealed KIF4 and NTCP colocalization across microtubule filaments, and a co-immunoprecipitation study revealed that KIF4 interacts with NTCP. KIF4 expression is regulated by FOXM1. Interestingly, we discovered that RXR agonists (Bexarotene, and Alitretinoin) down-regulated KIF4 expression via FOXM1-mediated suppression, resulting in a substantial decrease in HBV-Pre-S1 protein attachment to HepG2-hNTCP cell surface and subsequent HBV infection in both HepG2-hNTCP and primary human hepatocyte (PXB) (Bexarotene, IC50 1.89 ± 0.98 µM) cultures. Overall, our findings show that human KIF4 is a critical regulator of NTCP surface transport and localization, which is required for NTCP to function as a receptor for HBV/HDV entry. Furthermore, small molecules that suppress or alleviate KIF4 expression would be potential antiviral candidates targeting HBV and HDV entry.

Amino Acid Polymorphism in Hepatitis B Virus Associated With Functional Cure.

BACKGROUND & AIMS: To provide an adequate treatment strategy for chronic hepatitis B, it is essential to know which patients are expected to have a good prognosis and which patients do not require therapeutic intervention. Previously, we identified the substitution of isoleucine to leucine at amino acid 97 (I97L) in the hepatitis B core region as a key predictor among patients with stable hepatitis. In this study, we attempted to identify the point at which I97L affects the hepatitis B virus (HBV) life cycle and to elucidate the underlying mechanisms governing the stabilization of hepatitis. METHODS: To confirm the clinical features of I97L, we used a cohort of hepatitis B e antigen-negative patients with chronic hepatitis B infected with HBV-I97 wild-type (wt) or HBV-I97L. The effects of I97L on viral characteristics were evaluated by in vitro HBV production and infection systems with the HBV reporter virus and cell culture-generated HBV. RESULTS: The ratios of reduction in hepatitis B surface antigen and HBV DNA were higher in patients with HBV-I97L than in those with HBV-I97wt. HBV-I97L exhibited lower infectivity than HBV-I97wt in both infection systems with reporter HBV and cell culture-generated HBV. HBV-I97L virions exhibiting low infectivity primarily contained a single-stranded HBV genome. The lower efficiency of cccDNA synthesis was demonstrated after infection of HBV-I97L or transfection of the molecular clone of HBV-I97L. CONCLUSIONS: The I97L substitution reduces the level of cccDNA through the generation of immature virions with single-stranded genomes. This I97L-associated low efficiency of cccDNA synthesis may be involved in the stabilization of hepatitis.

Inhibition of hepatitis C virus by vitamin D.

Until the development of direct-acting antivirals (DAAs), interferon (IFN)-based therapy had been the primary treatment strategy for patients with chronic hepatitis C, even though this therapy has a therapeutic limitations and considerable side effects. Therefore, many efforts have been made to improve the efficacy of treatment. Several clinical studies have clearly shown that supplementation with vitamin D of IFN-based therapy improves treatment efficacy. To clarify the molecular mechanisms of the effect of vitamin D on IFN-based therapy, several researchers have performed basic research with cell culture models of hepatitis C virus (HCV). Consequently, two vitamin D3 metabolites, 25-hydroxyvitamin D3 (25-(OH)D3) and 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3), have been suggested to have anti-HCV effects. 25-(OH)D3 inhibits HCV production by suppressing infectious virus assembly through reducing apolipoprotein expression, while 1α,25-(OH)2D3 inhibits HCV production by modulating IFN signaling and/or inducing various host factors associated with the inhibition of viral genome replication. In addition, an antimicrobial peptide, LL-37, which is known to be partly regulated by vitamin D, was also reported to exhibit an anti-HCV effect by disrupting infectious viral particles directly. In conclusion, vitamin D3 supplementation improves the response rate of IFN-based therapy via the direct and/or indirect anti-HCV effects of vitamin D3 metabolites.

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

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.

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

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 MafF 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-1ß (IL-1ß) or tumor necrosis factor alpha (TNF-α) treatment in both HepG2 and PXB cells, in an NF-κB-dependent manner. Consistently, MafF 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. IMPORTANCE 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-1ß 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.

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

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.

Performance Evaluation of In Vitro Screening and Diagnostic Kits for Hepatitis C Virus Infection.

AIM: A reliable kit with high sensitivity and specificity is indispensable for diagnosing hepatitis C virus (HCV) infection. Detection kits for anti-HCV antibodies (anti-HCV) are used for screening, and quantification kits for HCV RNA and HCV antigen (Ag) are used for the definite diagnosis of HCV infection or the evaluation of the pathological condition of and therapeutic effects in patients with chronic hepatitis C. Several kits are currently available for these purposes and are provided for clinical use in Japan. In this study, we aimed to evaluate the performance of these kits. METHODS: We used International Standards for HCV RNA and HCV Ag and a regional reference panel to evaluate the performance of thirteen anti-HCV, five HCV RNA, and two HCV Ag kits. RESULTS: All specimens in the regional reference panel were diagnosed correctly by all anti-HCV kits, although the distributions of the quantified values varied, and the ratios of titer classification were not identical across kits. All HCV RNA kits quantified the International Standard with minimum deviation and diagnosed the specimens of the reference panel correctly. The quantified values of the International Standard by two HCV Ag kits were inconsistent. HCV Ag titers of some specimens were underestimated owing to the amino acid polymorphisms in comparison with HCV RNA titers. CONCLUSIONS: The evaluation with International Standards and the regional reference panel was useful for assessing the quality of screening and diagnostic kits for HCV infection, and such quality control is essential for the clinical usage of these kits.

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

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.

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

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.

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

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.

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.

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.

Establishment of Replication-Competent HCV Strain with Minimum Modifications.

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.

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

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 D3 (25-(OH)D3), 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)D3 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.

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

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 EC50 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.

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

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) -1ß as an inducer of expression from the Ski2 promoter. IL-1ß 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-1ß. 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-1ß 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.