Novel Neplanocin A Derivatives as Selective Inhibitors of Hepatitis B Virus with a Unique Mechanism of Action.

Novel neplanocin A derivatives have been identified as potent and selective inhibitors of hepatitis B virus (HBV) replication in vitro. These include (1S,2R,5R)-5-(5-bromo-4-methyl-7H-pyrrolo[2,3-d]-pyrimidin-7-yl)-3-(hydroxymethyl)cyclopent-3-ene-1,2-diol (AR-II-04-26) and (1S,2R,5R)-5-(4-amino-3-iodo-1H-pyrazolo[3,4-d]pyrimidin-1-yl)-3-(hydroxylmethyl)cyclopent-3-ene-1,2-diol (MK-III-02-03). The 50% effective concentrations of AR-II-04-26 and MK-III-02-03 were 0.77 ± 0.23 and 0.83 ± 0.36 µM in HepG2.2.15.7 cells, respectively. These compounds reduced intracellular HBV RNA levels in HepG2.2.15.7 cells and infected primary human hepatocytes. Accordingly, they could reduce HBs and HBe antigen production in the culture supernatants, which was not observed with clinically approved anti-HBV nucleosides and nucleotides (reverse transcriptase inhibitors). The neplanocin A derivatives also inhibited HBV RNA derived from cccDNA. In addition, unlike neplanocin A itself, the compounds did not inhibit S-adenosyl-l-homocysteine hydrolase activity. Thus, it appears that the mechanism of action of AR-II-04-26 and MK-III-02-03 differs from that of the clinically approved anti-HBV agents. Although their exact mechanism (target molecule) remains to be elucidated, the novel neplanocin A derivatives are considered promising candidate drugs for inhibition of HBV replication.

Induction of HOX Genes by Hepatitis C Virus Infection via Impairment of Histone H2A Monoubiquitination.

Hepatitis C virus (HCV) infection causes liver pathologies, including hepatocellular carcinoma (HCC). Homeobox (HOX) gene products regulate embryonic development and are associated with tumorigenesis, although the regulation of HOX genes by HCV infection has not been clarified in detail. We examined the effect of HCV infection on HOX gene expression. In this study, HCV infection induced more than half of the HOX genes and reduced the level of histone H2A monoubiquitination on lysine 119 (K119) (H2Aub), which represses HOX gene promoter activity. HCV infection also promoted proteasome-dependent degradation of RNF2, which is an E3 ligase mediating H2A monoubiquitination as a component of polycomb repressive complex 1. Since full-genomic replicon cells but not subgenomic replicon cells exhibited reduced RNF2 and H2Aub levels and induction of HOX genes, we focused on the core protein. Expression of the core protein reduced the amounts of RNF2 and H2Aub and induced HOX genes. Treatment with LY-411575, which can reduce HCV core protein expression via signal peptide peptidase (SPP) inhibition without affecting other viral proteins, dose-dependently restored the amounts of RNF2 and H2Aub in HCV-infected cells and impaired the induction of HOX genes and production of viral particles but not viral replication. The chromatin immunoprecipitation assay results also indicated infection- and proteasome-dependent reductions in H2Aub located in HOX gene promoters. These results suggest that HCV infection or core protein induces HOX genes by impairing histone H2A monoubiquitination via a reduction in the RNF2 level.IMPORTANCE Recently sustained virologic response can be achieved by direct-acting antiviral (DAA) therapy in most hepatitis C patients. Unfortunately, DAA therapy does not completely eliminate a risk of hepatocellular carcinoma (HCC). Several epigenetic factors, including histone modifications, are well known to contribute to hepatitis C virus (HCV)-associated HCC. However, the regulation of histone modifications by HCV infection has not been clarified in detail. In this study, our data suggest that HCV infection or HCV core protein expression impairs monoubiquitination of histone H2A K119 in the homeobox (HOX) gene promoter via destabilization of RNF2 and then induces HOX genes. Several lines of evidence suggest that the expression of several HOX genes is dysregulated in certain types of tumors. These findings reveal a novel mechanism of HCV-related histone modification and may provide information about new targets for diagnosis and prevention of HCC occurrence.

Deep sequencing analysis of serum hepatitis B virus-RNA during nucleot(s)ide analogue therapy.

AIM: Recently, serum hepatitis B virus (HBV)-RNA has been reported to be detectable even when HBV particle production is inhibited by nucleot(s)ide analogues (NAs). However, the dynamics of the HBV-RNA sequence compared with those of HBV-DNA during the emergence of antiviral resistance are yet to be elucidated. METHODS: First, we quantified serum HBV-RNA in 181 infected patients, and its relationships with clinical characteristics as well as HBV markers were investigated. Next, we undertook simultaneous deep sequencing of HBV-RNA/HBV-DNA and their dynamics among four patients receiving NA therapy who were experiencing viral breakthrough. RESULTS: Serum HBV-RNA was detected in 25% (31/123) of cases among patients with HBV without NAs, and the detection rate was significantly high in hepatitis B e antigen-positive cases with high viral activity. In patients with chronic hepatitis, hepatitis B core-related antigen was significantly correlated with serum HBV-RNA irrespective of NA use. In the analysis of the four patients experiencing viral breakthrough, no NA resistance mutation was detected in the serum HBV-RNA immediately before the breakthrough. However, NA-resistant sequences appeared at the rates of 0%, 3%, 14%, and 100%, and the NA-resistant HBV-RNA sequence rate was correlated with the peak HBV-DNA titer multiplied by the HBV-DNA detection duration during the breakthrough (R2 = 0.978) observed before redisappearance of HBV-DNA following the addition of new NA. CONCLUSION: Serum HBV-RNA could reflect the transcriptional activity of covalently closed circular DNA and hepatitis B core-related antigen. The dynamics of HBV-RNA could help understanding of the turnover process of HBV covalently closed circular DNA in the liver.

Cancer-related genetic changes in multistep hepatocarcinogenesis and their correlation with imaging and histological findings.

AIM: The landscape of cancer-related genetic aberrations in hepatocellular carcinoma (HCC) has gradually become clear through recent next-generation sequencing studies. However, it remains unclear how genetic aberrations correlate with imaging and histological findings. METHODS: Using 117 formalin-fixed paraffin-embedded specimens of primary liver tumors, we undertook targeted next-generation sequencing of 50 cancer-related genes and digital polymerase chain reaction of hTERT. After classifying tumors into several imaging groups by hierarchal clustering with the information from gadoxetic acid enhanced magnetic resonance imaging, contrast-enhanced computed tomography, contrast-enhanced ultrasound, and diffusion-weighted imaging magnetic resonance imaging, the correlation between genetic aberrations and imaging and histology were investigated. RESULTS: Most frequent mutations were hTERT (61.5%), followed by TP53 (42.7%), RB1 (24.8%), and CTNNB1 (18.8%). Liver tumors were classified into six imaging groups/grades, and the prevalence of hTERT mutations tended to increase with the advancement of imaging/histological grades (P = 0.026 and 0.13, respectively), whereas no such tendency was evident for TP53 mutation (P = 0.78 and 1.00, respectively). Focusing on the mutations in each tumor, although the variant frequency (VF) of hTERT did not change (P = 0.36 and 0.14, respectively) in association with imaging/histological grades, TP53 VF increased significantly (P = 0.004 and <0.001, respectively). In multivariate analysis, stage III or IV (hazard ratio, 3.64; P = 0.003), TP53 VF ≥ 50% (hazard ratio, 3.79; P = 0.020) was extracted as an independent risk for recurrence in primary HCC patients. CONCLUSIONS: Increased prevalence of hTERT mutation and increased TP53 mutation VF are characteristic features of HCC progression, diagnosed with imaging/histological studies.

Roles of the 5' Untranslated Region of Nonprimate Hepacivirus in Translation Initiation and Viral Replication.

The 5' untranslated region (UTR) of hepatitis C virus (HCV), which is composed of four domains (I, II, III, and IV) and a pseudoknot, is essential for translation and viral replication. Equine nonprimate hepacivirus (EHcV) harbors a 5' UTR consisting of a large 5'-terminal domain (I); three additional domains (I', II, and III), which are homologous to domains I, II, and III, respectively, of HCV; and a pseudoknot, in the order listed. In this study, we investigated the roles of the EHcV 5' UTR in translation and viral replication. The internal ribosome entry site (IRES) activity of the EHcV 5' UTR was lower than that of the HCV 5' UTR in several cell lines due to structural differences in domain III. Domains I and III of EHcV were functional in the HCV 5' UTR in terms of IRES activity and the replication of the subgenomic replicon (SGR), although domain II was not exchangeable between EHcV and HCV for SGR replication. Furthermore, the region spanning domains I and I' of EHcV (the 5'-proximal EHcV-specific region) improved RNA stability and provided the HCV SGR with microRNA 122 (miR-122)-independent replication capability, while EHcV domain I alone improved SGR replication and RNA stability irrespective of miR-122. These data suggest that the region spanning EHcV domains I and I' improves RNA stability and viral replication regardless of miR-122 expression. The 5'-proximal EHcV-specific region may represent an inherent mechanism to facilitate viral replication in nonhepatic tissues.IMPORTANCE EHcV is the closest viral homolog to HCV among other hepaciviruses. HCV exhibits a narrow host range and liver-specific tropism, while epidemiological reports suggest that EHcV infects the liver and respiratory organs in horses, donkeys, and dogs. However, the mechanism explaining the differences in host or organ tropism between HCV and EHcV is unknown. In this study, our data suggest that the 5' untranslated region (UTR) of EHcV is composed of an internal ribosome entry site (IRES) element that is functionally exchangeable with HCV IRES elements. Furthermore, the 5'-proximal EHcV-specific region enhances viral replication and RNA stability in a miR-122-independent manner. Our data suggest that the region upstream of domain II in the EHcV 5' UTR contributes to the differences in tissue tropism observed between these hepaciviruses.

Hepatitis B virus (HBV)-infected patients with low hepatitis B surface antigen and high hepatitis B core-related antigen titers have a high risk of HBV-related hepatocellular carcinoma.

AIM: Although the viral markers hepatitis B surface antigen (HBsAg) and hepatitis B core-related antigen (HbcrAg) could reflect intrahepatic hepatitis B virus (HBV) replication activity and constitute important biomarkers for hepatocellular carcinoma (HCC), the value of using these two markers in combination for assessing HCC risk has not been clarified in detail. METHODS: Four hundred and forty-nine consecutive patients with chronic HBV infection were included in the study and the association of HBsAg and HBcrAg with HCC risk was investigated cross-sectionally, as well as longitudinally. RESULTS: When the high value cut-offs of HBsAg and HBcrAg were defined as 3.0 log IU/mL and 3.0 log U/mL, respectively, patients with a history of HCC were found frequently in the low HBsAg group (P = 0.002) and high HBcrAg group (P < 0.001). When HBsAg and HBcrAg were combined, an HCC history was most frequent in the subset with low HBsAg and high HBcrAg, among the HBeAg-negative patients (odds ratio [OR], 7.83; P < 0.001), irrespective of nucleos(t) ide analogue (NA) therapy (NA: OR, 4.76; P < 0.001; non-NA: OR, 9.60; P < 0.001). In a longitudinal analysis of the subsequent development of HCC, carried out on the 338 patients without an HCC history at enrollment, HCC developed significantly more frequently in the low HBsAg/high HBcrAg group (P = 0.005). CONCLUSIONS: Patients with low HBsAg/high HBcrAg values are at high risk of developing HBV-related HCC, according to this cross-sectional and longitudinal analysis, indicating that the combination of HBsAg and HBcrAg values is an excellent biomarker for assessing HCC risk.

Hepatitis B virus prevents excessive viral production via reduction of cell death-inducing DFF45-like effectors.

The relationship between hepatitis B virus (HBV) infection and lipid accumulation remains largely unknown. In this study, we investigated the effect of HBV propagation on lipid droplet growth in HBV-infected cells and HBV-producing cell lines, HepG2.2.15 and HBV-inducible Hep38.7-Tet. The amount of intracellular triglycerides was significantly reduced in HBV-infected and HBV-producing cells compared with HBV-lacking control cells. Electron and immunofluorescent microscopic analyses showed that the average size of a single lipid droplet (LD) was significantly less in the HBV-infected and HBV-producing cells than in the HBV-lacking control cells. Cell death-inducing DFF45-like effectors (CIDEs) B and C (CIDEB and CIDEC), which are involved in LD expansion for the improvement of lipid storage, were expressed at a significantly lower level in HBV-infected or HBV-producing cells than in HBV-lacking control cells, while CIDEA was not detected in those cells regardless of HBV production. The activity of the CIDEB and CIDEC gene promoters was impaired in HBV-infected or HBV-producing cells compared to HBV-lacking control cells, while CIDEs potentiated HBV core promoter activity. The amount of HNF4α, that can promote the transcription of CIDEB was significantly lower in HBV-producing cells than in HBV-lacking control cells. Knockout of CIDEB or CIDEC significantly reduced the amount of supernatant HBV DNA, intracellular viral RNA and nucleocapsid-associated viral DNA, while the expression of CIDEB or CIDEC recovered HBV production in CIDEB- or CIDEC-knockout cells. These results suggest that HBV regulates its own viral replication via CIDEB and CIDEC.

Hepatocyte Factor JMJD5 Regulates Hepatitis B Virus Replication through Interaction with HBx.

UNLABELLED: Hepatitis B virus (HBV) is a causative agent for chronic liver diseases such as hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). HBx protein encoded by the HBV genome plays crucial roles not only in pathogenesis but also in replication of HBV. Although HBx has been shown to bind to a number of host proteins, the molecular mechanisms by which HBx regulates HBV replication are largely unknown. In this study, we identified jumonji C-domain-containing 5 (JMJD5) as a novel binding partner of HBx interacting in the cytoplasm. DNA microarray analysis revealed that JMJD5-knockout (JMJD5KO) Huh7 cells exhibited a significant reduction in the expression of transcriptional factors involved in hepatocyte differentiation, such as HNF4A, CEBPA, and FOXA3. We found that hydroxylase activity of JMJD5 participates in the regulation of these transcriptional factors. Moreover, JMJD5KO Huh7 cells exhibited a severe reduction in HBV replication, and complementation of HBx expression failed to rescue replication of a mutant HBV deficient in HBx, suggesting that JMJD5 participates in HBV replication through an interaction with HBx. We also found that replacing Gly(135) with Glu in JMJD5 abrogates binding with HBx and replication of HBV. Moreover, the hydroxylase activity of JMJD5 was crucial for HBV replication. Collectively, these results suggest that direct interaction of JMJD5 with HBx facilitates HBV replication through the hydroxylase activity of JMJD5. IMPORTANCE: HBx protein encoded by hepatitis B virus (HBV) plays important roles in pathogenesis and replication of HBV. We identified jumonji C-domain-containing 5 (JMJD5) as a novel binding partner to HBx. JMJD5 was shown to regulate several transcriptional factors to maintain hepatocyte function. Although HBx had been shown to support HBV replication, deficiency of JMJD5 abolished contribution of HBx in HBV replication, suggesting that HBx-mediated HBV replication is largely dependent on JMJD5. We showed that hydroxylase activity of JMJD5 in the C terminus region is crucial for expression of HNF4A and replication of HBV. Furthermore, a mutant JMJD5 with Gly(135) replaced by Glu failed to interact with HBx and to rescue the replication of HBV in JMJD5-knockout cells. Taken together, our data suggest that interaction of JMJD5 with HBx facilitates HBV replication through the hydroxylase activity of JMJD5.

Four Aromatic Sulfates with an Inhibitory Effect against HCV NS3 Helicase from the Crinoid Alloeocomatella polycladia.

Bioassay-guided separation of a lipophilic extract of the crinoid Alloeocomatella polycladia, inhibiting the activity of HCV NS3 helicase, yielded two groups of molecules: cholesterol sulfate and four new aromatic sulfates 1-4. The structures of the aromatics were elucidated by spectroscopic analysis in addition to theoretical studies. The aromatic sulfates 1-4 showed moderate inhibition against NS3 helicase with IC50 values of 71, 95, 7, and 5 µM, respectively.

Deep sequencing and phylogenetic analysis of variants resistant to interferon-based protease inhibitor therapy in chronic hepatitis induced by genotype 1b hepatitis C virus.

UNLABELLED: Because of recent advances in deep sequencing technology, detailed analysis of hepatitis C virus (HCV) quasispecies and their dynamic changes in response to direct antiviral agents (DAAs) became possible, although the role of quasispecies is not fully understood. In this study, to clarify the evolution of viral quasispecies and the origin of drug-resistant mutations induced by interferon (IFN)-based protease inhibitor therapy, the nonstructural-3 (NS3) region of genotype 1b HCV in 34 chronic hepatitis patients treated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) was subjected to a deep sequencing study coupled with phylogenetic analysis. Twenty-six patients (76.5%) achieved a sustained viral response (SVR), while 8 patients did not (non-SVR; 23.5%). When the complexity of the quasispecies was expressed as the mutation frequency or Shannon entropy value, a significant decrease in the IFNL3 (rs8099917) TT group and a marginal decrease in the SVR group were found soon (12 h) after the introduction of treatment, whereas there was no decrease in the non-SVR group and no significant decrease in mutation frequency in the IFNL3 TG/GG group. In the analysis of viral quasispecies composition in non-SVR patients, major populations greatly changed, accompanied by the appearance of resistance, and the compositions were unlikely to return to the pretreatment composition even after the end of therapy. Clinically TVR-resistant variants were observed in 5 non-SVR patients (5/8, 62.5%), all of which were suspected to have acquired resistance by mutations through phylogenetic analysis. In conclusion, results of the study have important implications for treatment response and outcome in interferon-based protease inhibitor therapy. IMPORTANCE: In the host, hepatitis C virus (HCV) consists of a variety of populations (quasispecies), and it is supposed that dynamic changes in quasispecies are closely related to pathogenesis, although this is poorly understood. In this study, recently developed deep sequencing technology was introduced, and changes in quasispecies associated with telaprevir (TVR)/pegylated interferon (PEG-IFN)/ribavirin (RBV) triple therapy and their clinical significance were investigated extensively by phylogenetic tree analysis. Through this study, the associations among treatment response, changes in viral quasispecies complexity in the early stage of treatment, changes in the quasispecies composition, and origin of TVR-resistant variant HCV were elucidated.

Inhibitory effect of presenilin inhibitor LY411575 on maturation of hepatitis C virus core protein, production of the viral particle and expression of host proteins involved in pathogenicity.

Hepatitis C virus (HCV) core protein is responsible for the formation of infectious viral particles and induction of pathogenicity. The C-terminal transmembrane region of the immature core protein is cleaved by signal peptide peptidase (SPP) for maturation of the core protein. SPP belongs to the family of presenilin-like aspartic proteases. Some presenilin inhibitors are expected to suppress HCV infection and production; however, this anti-HCV effect has not been investigated in detail. In this study, presenilin inhibitors were screened to identify anti-HCV compounds. Of the 13 presenilin inhibitors tested, LY411575 was the most potent inhibitor of SPP-dependent cleavage of HCV core protein. Production of intracellular core protein and supernatant infectious viral particles from HCV-infected cells was significantly impaired by LY411575 in a dose-dependent manner (half maximum inhibitory concentration = 0.27 µM, cytotoxic concentration of the extracts to cause death to 50% of viable cells > 10 µM). No effect of LY411575 on intracellular HCV RNA in the subgenomic replicon cells was detected. LY411575 synergistically promoted daclatasvir-dependent inhibition of viral production, but not that of viral replication. Furthermore, LY411575 inhibited HCV-related production of reactive oxygen species and expression of NADPH oxidases and vascular endothelial growth factor. Taken together, our data suggest that LY411575 suppresses HCV propagation through SPP inhibition and impairs host gene expressions related to HCV pathogenicity.

Hallmarks of hepatitis C virus in equine hepacivirus.

UNLABELLED: Equine hepacivirus (EHcV) has been identified as a closely related homologue of hepatitis C virus (HCV) in the United States, the United Kingdom, and Germany, but not in Asian countries. In this study, we genetically and serologically screened 31 serum samples obtained from Japanese-born domestic horses for EHcV infection and subsequently identified 11 PCR-positive and 7 seropositive serum samples. We determined the full sequence of the EHcV genome, including the 3' untranslated region (UTR), which had previously not been completely revealed. The polyprotein of a Japanese EHcV strain showed approximately 95% homology to those of the reported strains. HCV-like cis-acting RNA elements, including the stem-loop structures of the 3' UTR and kissing-loop interaction were deduced from regions around both UTRs of the EHcV genome. A comparison of the EHcV and HCV core proteins revealed that Ile(190) and Phe(191) of the EHcV core protein could be important for cleavage of the core protein by signal peptide peptidase (SPP) and were replaced with Ala and Leu, respectively, which inhibited intramembrane cleavage of the EHcV core protein. The loss-of-function mutant of SPP abrogated intramembrane cleavage of the EHcV core protein and bound EHcV core protein, suggesting that the EHcV core protein may be cleaved by SPP to become a mature form. The wild-type EHcV core protein, but not the SPP-resistant mutant, was localized on lipid droplets and partially on the lipid raft-like membrane in a manner similar to that of the HCV core protein. These results suggest that EHcV may conserve the genetic and biological properties of HCV. IMPORTANCE: EHcV, which shows the highest amino acid or nucleotide homology to HCV among hepaciviruses, was previously reported to infect horses from Western, but not Asian, countries. We herein report EHcV infection in Japanese-born horses. In this study, HCV-like RNA secondary structures around both UTRs were predicted by determining the whole-genome sequence of EHcV. Our results also suggest that the EHcV core protein is cleaved by SPP to become a mature form and then is localized on lipid droplets and partially on lipid raft-like membranes in a manner similar to that of the HCV core protein. Hence, EHcV was identified as a closely related homologue of HCV based on its genetic structure as well as its biological properties. A clearer understanding of the epidemiology, genetic structure, and infection mechanism of EHcV will assist in elucidating the evolution of hepaciviruses as well as the development of surrogate models for the study of HCV.

Identification of Antiviral Agents Targeting Hepatitis B Virus Promoter from Extracts of Indonesian Marine Organisms by a Novel Cell-Based Screening Assay.

The current treatments of chronic hepatitis B (CHB) face a limited choice of vaccine, antibody and antiviral agents. The development of additional antiviral agents is still needed for improvement of CHB therapy. In this study, we established a screening system in order to identify compounds inhibiting the core promoter activity of hepatitis B virus (HBV). We prepared 80 extracts of marine organisms from the coral reefs of Indonesia and screened them by using this system. Eventually, two extracts showed high inhibitory activity (>95%) and low cytotoxicity (66% to 77%). Solvent fractionation, column chromatography and NMR analysis revealed that 3,5-dibromo-2-(2,4-dibromophenoxy)-phenol (compound 1) and 3,4,5-tribromo-2-(2,4-dibromophenoxy)-phenol (compound 2), which are classified as polybrominated diphenyl ethers (PBDEs), were identified as anti-HBV agents in the extracts. Compounds 1 and 2 inhibited HBV core promoter activity as well as HBV production from HepG2.2.15.7 cells in a dose-dependent manner. The EC50 values of compounds 1 and 2 were 0.23 and 0.80 µM, respectively, while selectivity indexes of compound 1 and 2 were 18.2 and 12.8, respectively. These results suggest that our cell-based HBV core promoter assay system is useful to determine anti-HBV compounds, and that two PBDE compounds are expected to be candidates of lead compounds for the development of anti-HBV drugs.

Identification of Hydroxyanthraquinones as Novel Inhibitors of Hepatitis C Virus NS3 Helicase.

Hepatitis C virus (HCV) is an important etiological agent of severe liver diseases, including cirrhosis and hepatocellular carcinoma. The HCV genome encodes nonstructural protein 3 (NS3) helicase, which is a potential anti-HCV drug target because its enzymatic activity is essential for viral replication. Some anthracyclines are known to be NS3 helicase inhibitors and have a hydroxyanthraquinone moiety in their structures; mitoxantrone, a hydroxyanthraquinone analogue, is also known to inhibit NS3 helicase. Therefore, we hypothesized that the hydroxyanthraquinone moiety alone could also inhibit NS3 helicase. Here, we performed a structure-activity relationship study on a series of hydroxyanthraquinones by using a fluorescence-based helicase assay. Hydroxyanthraquinones inhibited NS3 helicase with IC50 values in the micromolar range. The inhibitory activity varied depending on the number and position of the phenolic hydroxyl groups, and among different hydroxyanthraquinones examined, 1,4,5,8-tetrahydroxyanthraquinone strongly inhibited NS3 helicase with an IC50 value of 6 µM. Furthermore, hypericin and sennidin A, which both have two hydroxyanthraquinone-like moieties, were found to exert even stronger inhibition with IC50 values of 3 and 0.8 µM, respectively. These results indicate that the hydroxyanthraquinone moiety can inhibit NS3 helicase and suggest that several key chemical structures are important for the inhibition.

Deep-sequencing analysis of the association between the quasispecies nature of the hepatitis C virus core region and disease progression.

Variation of core amino acid (aa) 70 of hepatitis C virus (HCV) has been shown recently to be closely correlated with liver disease progression, suggesting that the core region might be present as a quasispecies during persistent infection and that this quasispecies nature might have an influence on the progression of disease. In our investigation, the subjects were 79 patients infected with HCV genotype 1b (25 with chronic hepatitis [CH], 29 with liver cirrhosis [LC], and 25 with hepatocellular carcinoma [HCC]). Deep sequencing of the HCV core region was carried out on their sera by using a Roche 454 GS Junior pyrosequencer. Based on a plasmid containing a cloned HCV sequence (pCV-J4L6S), the background error rate associated with pyrosequencing, including the PCR procedure, was calculated as 0.092 ± 0.005/base. Deep sequencing of the core region in the clinical samples showed a mixture of "mutant-type" Q/H and "wild-type" R at the core aa 70 position in most cases (71/79 [89.9%]), and the ratio of mutant residues to R in the mixture increased as liver disease advanced to LC and HCC. Meanwhile, phylogenetic analysis of the almost-complete core region revealed that the HCV isolates differed genetically depending on the mutation status at core aa 70. We conclude that the core aa 70 mixture ratio, determined by deep sequencing, reflected the status of liver disease, demonstrating a significant association between core aa 70 and disease progression in CH patients infected with HCV genotype 1b.

Cholesterol sulfate as a potential inhibitor of hepatitis C virus NS3 helicase.

Hepatitis C virus nonstructural protein 3 (NS3) helicase is a promising target for developing new therapeutics. In this study, we identified cholesterol sulfate (CS) as a novel NS3 helicase inhibitor (IC50 = 1.7 ± 0.2 µM with a Hill coefficient of 3.9) by screening the extracts from marine organisms. The lack of the sulfate group, sterol structure or alkyl side chain of CS diminished the inhibition, suggesting that an anion binding and hydrophobic region in NS3 may be a target site of CS. It was further found that CS partly inhibits NS3-RNA binding activity, but exerted no or less inhibition against ATPase and serine protease activities. Moreover, we demonstrated that CS probably does not bind to RNA. Our findings suggest that CS may inhibit NS3 helicase not by abolishing the other NS3 activities but by inducing conformational changes via interaction with possible allosteric sites of NS3.

Identification and biochemical characterization of halisulfate 3 and suvanine as novel inhibitors of hepatitis C virus NS3 helicase from a marine sponge.

Hepatitis C virus (HCV) is an important etiological agent that is responsible for the development of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV nonstructural protein 3 (NS3) helicase is a possible target for novel drug development due to its essential role in viral replication. In this study, we identified halisulfate 3 (hal3) and suvanine as novel NS3 helicase inhibitors, with IC50 values of 4 and 3 µM, respectively, from a marine sponge by screening extracts of marine organisms. Both hal3 and suvanine inhibited the ATPase, RNA binding, and serine protease activities of NS3 helicase with IC50 values of 8, 8, and 14 µM, and 7, 3, and 34 µM, respectively. However, the dengue virus (DENV) NS3 helicase, which shares a catalytic core (consisting mainly of ATPase and RNA binding sites) with HCV NS3 helicase, was not inhibited by hal3 and suvanine, even at concentrations of 100 µM. Therefore, we conclude that hal3 and suvanine specifically inhibit HCV NS3 helicase via an interaction with an allosteric site in NS3 rather than binding to the catalytic core. This led to the inhibition of all NS3 activities, presumably by inducing conformational changes.

PBDE: structure-activity studies for the inhibition of hepatitis C virus NS3 helicase.

The helicase portion of the hepatitis C virus nonstructural protein 3 (NS3) is considered one of the most validated targets for developing direct acting antiviral agents. We isolated polybrominated diphenyl ether (PBDE) 1 from a marine sponge as an NS3 helicase inhibitor. In this study, we evaluated the inhibitory effects of PBDE (1) on the essential activities of NS3 protein such as RNA helicase, ATPase, and RNA binding activities. The structure-activity relationship analysis of PBDE (1) against the HCV ATPase revealed that the biphenyl ring, bromine, and phenolic hydroxyl group on the benzene backbone might be a basic scaffold for the inhibitory potency.

Serum RANTES level influences the response to pegylated interferon and ribavirin therapy in chronic hepatitis C.

AIM: Prediction of treatment responses to pegylated interferon (PEG IFN) plus ribavirin (RBV) therapy is uncertain for genotype 1b chronic hepatitis C. METHODS: In this study, 96 patients were investigated for the correlation between 36 pretreatment serum chemokine/cytokine levels and PEG IFN/RBV treatment efficacy by a sandwich enzyme-linked immunoassay (ELISA) and a bead array. RESULTS: First, chemokines/cytokines were measured semiquantitatively by sandwich ELISA in 31 randomly-selected patients and the serum regulated on activation normal T-cell expressed and secreted (RANTES) level was found to be significantly higher in the sustained virological response (SVR) group than the non-SVR group (P = 0.048). Precise RANTES measurement in all 96 patients using a bead array confirmed this correlation (P = 0.002). However, the genetic RANTES haplotype was not significantly related to the serum level. The serum RANTES level was extracted by multivariate analysis (odds ratio = 4.09, 95% confidence interval = 1.02-16.5, P = 0.048) as an independent variable contributing to SVR. CONCLUSION: The serum RANTES level is an important determinant influencing the virological response to PEG IFN/RBV therapy in chronic hepatitis C.

Inhibition of hepatitis C virus NS3 helicase by manoalide.

The hepatitis C virus (HCV) causes one of the most prevalent chronic infectious diseases in the world, hepatitis C, which ultimately develops into liver cancer through cirrhosis. The NS3 protein of HCV possesses nucleoside triphosphatase (NTPase) and RNA helicase activities. As both activities are essential for viral replication, NS3 is proposed as an ideal target for antiviral drug development. In this study, we identified manoalide (1) from marine sponge extracts as an RNA helicase inhibitor using a high-throughput screening photoinduced electron transfer (PET) system that we previously developed. Compound 1 inhibits the RNA helicase and ATPase activities of NS3 in a dose-dependent manner, with IC(50) values of 15 and 70 µM, respectively. Biochemical kinetic analysis demonstrated that 1 does not affect the apparent K(m) value (0.31 mM) of NS3 ATPase activity, suggesting that 1 acts as a noncompetitive inhibitor. The binding of NS3 to single-stranded RNA was inhibited by 1. Manoalide (1) also has the ability to inhibit the ATPase activity of human DHX36/RHAU, a putative RNA helicase. Taken together, we conclude that 1 inhibits the ATPase, RNA binding, and helicase activities of NS3 by targeting the helicase core domain conserved in both HCV NS3 and DHX36/RHAU.