Masayuki Sudoh

Intracellular-diced dsRNA has enhanced efficacy for silencing HCV RNA and overcomes variation in the viral genotype

RNA interference (RNAi) can be used to inhibit viral replication in mammalian cells and therefore could be a powerful new antiviral therapy. Small interfering RNA (siRNA) may be effective for RNAi, but there are some technical problems that must be solved in each case, for example, predicting the effective siRNA target site and targeting heterogeneous sequences in a virus population. We show here that diced siRNA generated from long double-stranded RNA (dsRNA) is highly effective for inducing RNAi in HuH-7 cells harboring hepatitis C virus (HCV) replicons and can overcome variations in the HCV genotype. However, in mammalian cells, long dsRNA induced an interferon response and caused cell death. Here we describe an improvement of this method, U6 promoter-driven expression of long hairpin-RNA with multiple point mutations in the sense strand. This can efficiently silence HCV RNA replication and HCV protein expression without triggering the interferon response or cell death normally caused by dsRNA. In conclusion, intracellular-diced dsRNA efficiently induces RNAi, and, despite the high rate of mutation in HCV, it should be a feasible therapeutic strategy for silencing HCV RNA.

Proanthocyanidin from Blueberry Leaves Suppresses Expression of Subgenomic Hepatitis C Virus RNA

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. While searching for new natural anti-HCV agents in agricultural products, we found a potent inhibitor of HCV RNA expression in extracts of blueberry leaves when examined in an HCV subgenomic replicon cell culture system. This activity was observed in a methanol extract fraction of blueberry leaves and was purified by repeated fractionations in reversed-phase high-performance liquid chromatography. The final purified fraction showed a 63-fold increase in specific activity compared with the initial methanol extracts and was composed only of carbon, hydrogen, and oxygen. Liquid chromatography/mass-ion trap-time of flight analysis and butanol-HCl hydrolysis analysis of the purified fraction revealed that the blueberry leaf-derived inhibitor was proanthocyanidin. Furthermore, structural analysis using acid thiolysis indicated that the mean degree of polymerization of the purified proanthocyanidin was 7.7, consisting predominantly of epicatechin. Proanthocyanidin with a polymerization degree of 8 to 9 showed the greatest potency at inhibiting the expression of subgenomic HCV RNA. Purified proanthocyanidin showed dose-dependent inhibition of expression of the neomycin-resistant gene and the NS-3 protein gene in the HCV subgenome in replicon cells. While characterizing the mechanism by which proanthocyanidin inhibited HCV subgenome expression, we found that heterogeneous nuclear ribonucleoprotein A2/B1 showed affinity to blueberry leaf-derived proanthocyanidin and was indispensable for HCV subgenome expression in replicon cells. These data suggest that proanthocyanidin isolated from blueberry leaves may have potential usefulness as an anti-HCV compound by inhibiting viral replication.

Cloning of the chitin synthase 3 gene from Candida albicans and its expression during yeast-hyphal transition

The chitin synthase 3 gene (CACHS3) has been cloned from Candida albicans. The yeast CAL1 gene encoding the chitin synthase 3 of Saccharomyces cerevisiae was used as a probe for the isolation of the gene from C. albicans. The CAL1 homolog was identified in Southern blots of C. albicans genomic DNA and cloned from a C. albicans genomic DNA library. The nucleotide sequences of two partial clones were determined and combined giving a total length of 4610 bp. A continuous open reading frame of 3525 by encoding a predicted protein of 1175 amino acids and molecular mass of 131 850 daltons was identified. A comparison of the deduced amino acid sequences of CAL1 and the Candida chitin synthase 3 protein showed 59.3% identitiy. Southern blot analysis indicates that the CACHS3 gene is present in a single copy in the genome and maps to chromosome I. Northern blot analysis shows that expression of chitin synthase 3 gene is dramatically increased during the transition from the yeast to the hyphal form of C. albicans. This change in transcription level strongly suggests that CACHS3 may play a role in Candida morphogenesis.

The yeast Chs4 protein stimulates the trypsin-sensitive activity of chitin synthase 3 through an apparent protein-protein interaction.

Inducible overexpression of the CHS4 gene under the control of the GAL1 promoter increased Chs3p (chitin synthase 3) activity in Saccharomyces cerevisiae several fold. Approximately half of the Chs3p activity in the membranes of cells overexpressing Chs4p was extracted using CHAPS and cholesteryl hemisuccinate. The detergent-extractable Chs3p activity appeared to be non-zymogenic because incubation with trypsin decreased enzyme activity in both the presence and absence of the substrate, UDP-N-acetylglucosamine. Western blotting confirmed that Chs3p was extracted from membranes by CHAPS and cholesteryl hemisuccinate and revealed that Chs4p was also solubilized using these detergents. Yeast two-hybrid analysis with truncated Chs4p demonstrated that the region of Chs4p between amino acids 269 and 563 is indispensable not only for eliciting the non-zymogenic activity of Chs3p but also for binding of Chs4p to Chs3p. Neither the EF-hand motif nor a possible prenylation site in Chs4p was required for these activities. Thus, it was demonstrated that stimulation of non-zymogenic Chs3p activity by Chs4p requires the amino acid region from 269 to 563 of Chs4p, and it seems that Chs4p activates Chs3p through protein-protein interaction.

An improved mouse model that rapidly develops fibrosis in non-alcoholic steatohepatitis

Non‐alcoholic steatohepatitis (NASH) is a progressive fibrotic disease, the pathogenesis of which has not been fully elucidated. One of the most common models used in NASH research is a nutritional model where NASH is induced by feeding a diet deficient in both methionine and choline. However, the dietary methionine‐/choline‐deficient model in mice can cause severe weight loss and liver atrophy, which are not characteristics of NASH seen in human patients. Exclusive, long‐term feeding with a high‐fat diet (HFD) produced fatty liver and obesity in mice, but the HFD for several months did not affect fibrosis. We aimed to establish a mouse model of NASH with fibrosis by optimizing the methionine content in the HFD. Male mice were fed a choline‐deficient, L‐amino acid‐defined, high‐fat diet (CDAHFD) consisting of 60 kcal% fat and 0.1% methionine by weight. After 1–14 weeks of being fed CDAHFD, the mice were killed. C57BL/6J mice maintained or gained weight when fed CDAHFD, while A/J mice showed a steady decline in body weight (of up to 20% of initial weight). In both strains of mice, plasma levels of alanine aminotransferase increased from week 1, when hepatic steatosis was also observed. By week 6, C57BL/6J mice had developed enlarged fatty liver with fibrosis as assessed by Massons trichrome staining and by hydroxyproline assay. Therefore, this improved CDAHFD model may be a mouse model of rapidly progressive liver fibrosis and be potentially useful for better understanding human NASH disease and in the development of efficient therapies for this condition.

Self-Enhancement of Hepatitis C Virus Replication by Promotion of Specific Sphingolipid Biosynthesis

Lipids are key components in the viral life cycle that affect host-pathogen interactions. In this study, we investigated the effect of HCV infection on sphingolipid metabolism, especially on endogenous SM levels, and the relationship between HCV replication and endogenous SM molecular species. We demonstrated that HCV induces the expression of the genes (SGMS1 and 2) encoding human SM synthases 1 and 2. We observed associated increases of both total and individual sphingolipid molecular species, as assessed in human hepatocytes and in the detergent-resistant membrane (DRM) fraction in which HCV replicates. SGMS1 expression had a correlation with HCV replication. Inhibition of sphingolipid biosynthesis with a hepatotropic serine palmitoyltransferase (SPT) inhibitor, NA808, suppressed HCV-RNA production while also interfering with sphingolipid metabolism. Further, we identified the SM molecular species that comprise the DRM fraction and demonstrated that these endogenous SM species interacted with HCV nonstructural 5B polymerase to enhance viral replication. Our results reveal that HCV alters sphingolipid metabolism to promote viral replication, providing new insights into the formation of the HCV replication complex and the involvement of host lipids in the HCV life cycle.

Identification of a Novel Inhibitor Specific to the Fungal Chitin Synthase INHIBITION OF CHITIN SYNTHASE 1 ARRESTS THE CELL GROWTH, BUT INHIBITION OF CHITIN SYNTHASE 1 AND 2 IS LETHAL IN THE PATHOGENIC FUNGUS CANDIDA ALBICANS

As in Saccharomyces cerevisiae, the pathogenic fungus Candida albicans harbors three chitin synthases called CaChs1p, CaChs2p, and CaChs3p, which are structurally and functionally analogous to the S. cerevisiae ScChs2p, ScChs1p, and ScChs3p, respectively. In S. cerevisiae,ScCHS1, ScCHS2, and ScCHS3 are all non-essential genes; only the simultaneous disruption ofScCHS2 and ScCHS3 is lethal. The fact that a null mutation of the CaCHS1 is impossible, however, implies that CaCHS1 is required for the viability of C. albicans. To gain more insight into the physiological importance of CaCHS1, we identified and characterized a novel inhibitor that was highly specific to CaChs1p. RO-09-3143 inhibited CaChs1p with a K i value of 0.55 nm in a manner that was non-competitive to the substrate UDP-N-acetylglucosamine. RO-09-3143 also hampered the growth of the C. albicans cells with an MIC50value of 0.27 μm. In the presence of RO-09-3143, theC. albicans cells failed to form septa and displayed an aberrant morphology, confirming the involvement of the C. albicans Chs1p in septum formation. Although the effect of RO-09-3143 on the wild-type C. albicans was fungistatic, it caused cell death in the cachs2Δ null mutants but not in the cachs3Δ null mutants. Thus, it appears that inC. albicans, inhibition of CaChs1p causes cell growth arrest, but simultaneous inhibition of CaChs1p and CaChs2p is lethal.

Raloxifene hydrochloride is an adjuvant antiviral treatment of postmenopausal women with chronic hepatitis C: A randomized trial

BACKGROUND & AIMS Early menopause in women with chronic hepatitis C virus (HCV) infection is associated with a low likelihood of a sustained virological response (SVR) in conjunction with their antiviral treatment. This is potentially related to their reduced estrogen secretion. The study was done to determine whether selective estrogen receptor modulator administration might improve the efficacy of the current standard of care (SOC) treatment, pegylated interferon (PegIFN) α2a plus ribavirin (RBV), for postmenopausal women. METHODS One hundred and twenty-three postmenopausal women with genotype 1b chronic hepatitis C were randomly assigned to one of two treatment groups: raloxifene hydrochloride (RLX) (60 mg/day) plus SOC (PegIFNα2a 180 μg/week and RBV 600-1,000 mg/day) (n=62) or SOC only (n=61). Genotyping was performed of the polymorphism in the interleukin-28B (IL28B) gene region (rs8099917) of DNA collected from each patient. RESULTS One RLX-treated patient discontinued RLX because of a systemic rash following 2 weeks of treatment. Twenty-four weeks after treatment, the SVR rate was significantly higher for RLX plus SOC patients (61.3%) than for SOC only patients (34.4%) (p=0.0051). Further, the SVR rate was significantly higher for RLX plus SOC patients with IL28B TT (72.5%) than for SOC only patients with IL28B TT (39.2%) (p=0.0014), but no such relationship was observed in patients carrying the minor IL28B allele. CONCLUSIONS RLX improved the efficacy of SOC in the treatment of postmenopausal women with chronic hepatitis C. RLX shows promise as an adjuvant to the standard antiviral treatment of such patients.

Augmentation of DHCR24 expression by hepatitis C virus infection facilitates viral replication in hepatocytes

BACKGROUND & AIMS We characterized the role of 24-dehydrocholesterol reductase (DHCR24) in hepatitis C virus infection (HCV). DHCR24 is a cholesterol biosynthetic enzyme and cholesterol is a major component of lipid rafts, which is reported to play an important role in HCV replication. Therefore, we examined the potential of DHCR24 as a target for novel HCV therapeutic agents. METHODS We examined DHCR24 expression in human hepatocytes in both the livers of HCV-infected patients and those of chimeric mice with human hepatocytes. We targeted DHCR24 with siRNA and U18666A which is an inhibitor of both DHCR24 and cholesterol synthesis. We measured the level of HCV replication in these HCV replicon cell lines and HCV infected cells. U18666A was administrated into chimeric mice with humanized liver, and anti-viral effects were assessed. RESULTS Expression of DHCR24 was induced by HCV infection in human hepatocytes in vitro, and in human hepatocytes of chimeric mouse liver. Silencing of DHCR24 by siRNA decreased HCV replication in replicon cell lines and HCV JFH-1 strain-infected cells. Treatment with U18666A suppressed HCV replication in the replicon cell lines. Moreover, to evaluate the anti-viral effect of U18666A in vivo, we administrated U18666A with or without pegylated interferon to chimeric mice and observed an inhibitory effect of U18666A on HCV infection and a synergistic effect with interferon. CONCLUSIONS DHCR24 is an essential host factor which augmented its expression by HCV infection, and plays a significant role in HCV replication. DHCR24 may serve as a novel anti-HCV drug target.

In vivo therapeutic potential of Dicer-hunting siRNAs targeting infectious hepatitis C virus.

The development of RNA interference (RNAi)-based therapy faces two major obstacles: selecting small interfering RNA (siRNA) sequences with strong activity, and identifying a carrier that allows efficient delivery to target organs. Additionally, conservative region at nucleotide level must be targeted for RNAi in applying to virus because hepatitis C virus (HCV) could escape from therapeutic pressure with genome mutations. In vitro preparation of Dicer-generated siRNAs targeting a conserved, highly ordered HCV 5′ untranslated region are capable of inducing strong RNAi activity. By dissecting the 5′-end of an RNAi-mediated cleavage site in the HCV genome, we identified potent siRNA sequences, which we designate as Dicer-hunting siRNAs (dh-siRNAs). Furthermore, formulation of the dh-siRNAs in an optimized multifunctional envelope-type nano device inhibited ongoing infectious HCV replication in human hepatocytes in vivo. Our efforts using both identification of optimal siRNA sequences and delivery to human hepatocytes suggest therapeutic potential of siRNA for a virus.