(−)-Epigallocatechin-3-gallate (EGCG) is the most abundant catechin in green tea In this study, we found that hepatitis C virus (HCV) infection was significantly suppressed by EGCG in an HCV cell culture (HCVcc) system using a JFH1-GFP chimeric virus, with a 50 % effective concentration (EC50) of 179 μM The inhibitory activity of EGCG was confirmed by monitoring HCV RNA and protein expression levels in Huh751 cells infected with the JFH1 virus Moreover, we demonstrated that the inhibitory mechanisms of EGCG were attributable to the suppression of both the HCV entry and RNA replication steps, although EGCG had little effect on translation directed by the viral internal ribosome entry site (IRES) Furthermore, HCV could be rapidly eliminated from cell cultures after two and five passages in the presence of 50 and 25 μM EGCG, respectively These results indicate that EGCG is a potential candidate as a preventive and antiviral drug for HCV infection

(−)-Epigallocatechin-3-gallate inhibits the replication cycle of hepatitis C virus

DNA methyltransferases 1 and 3B are required for hepatitis C virus infection in cell culture

Publisher Summary This chapter provides an overview of progress made towards identifying and developing the inhibitors of hepatitis C virus (HCV). It focuses on specifically targeted inhibitors of HCV, including HCV NS2 protease inhibitors, HCV NS3/4A protease inhibitors, HCV NS3 helicase inhibitors, HCV NS4B replication factor inhibitors, HCV NS5A replication factor inhibitors, and HCV NS5B polymerase inhibitors. The NS2 protein is essential for the production of infectious virus. The crystal structure of the protease catalytic domain has revealed it to be a cysteine protease that functions as a dimer. The two composite active sites interact cooperatively, with the catalytic histidine and glutamate residues from one domain working in concert with the catalytic cysteine from the second domain. Although the majority of compounds in clinical studies are still in the early stages of development, the first steps towards developing a combination of specific antiviral agents to treat HCV unresponsive to standard of care(SOC), either as add-on or replacement therapy, have been taken. With compounds acting at multiple sites in the virus replication cycle in development, there is reason to be optimistic about the potential to identify effective combinations that offer higher rates of therapeutic cure of HCV infection resistant to the current SOC.

Chapter 20 Progress towards the Discovery and Development of Specifically Targeted Inhibitors of Hepatitis C Virus

OBJECTIVES Hepatitis C virus (HCV) infection is a serious health problem that causes chronic infection in up to 85% of cases. HCV nonstructural (NS) cysteine protease, NS2/3, is required for viral replication in vivo. Cystatin C is a naturally occurring cysteine protease inhibitor in human cells. We aimed to investigate the relation between serum levels of cystatin C and HCV viremia in treatment-naive children with chronic hepatitis C. METHODS Serum cystatin C levels were measured in 27 children with chronic hepatitis C and determined their relation with liver functions, histopathological parameters, and hepatitis C viral load. Serum cystatin C was compared with that of 25 age- and sex-matched healthy controls. RESULTS Cystatin C was significantly higher in patients than in controls (1.4 ± 0.47 vs 0.99 ± 0.49; P = 0.006), and in those with low viremia than in those with moderate viremia (1.55 ± 0.41 vs 0.99 ± 0.43; P = 0.013). Cystatin C was not correlated with histopathological findings in liver biopsy (P > 0.05 for all). In addition, there was no significant difference of cystatin C levels in patients with normal versus those with elevated transaminases (P > 0.05). Of importance, cystatin C correlated negatively with viral load (P < 0.0001). CONCLUSIONS Cystatin C levels correlated negatively with HCV viremia. This finding may reflect an inhibitory effect of cystatin C on HCV replication through inhibiting its NS2/3 and tempting for further studies for cystatin C as a possible adjuvant therapy for HCV infection.

Serum cystatin C correlates negatively with viral load in treatment-naïve children with chronic hepatitis C.

Molecular covariation of highly polymorphic viruses is thought to have crucial effects on viral replication and fitness. This study employs association rule data mining of hepatitis C virus (HCV) sequences to search for specific evolutionary covariation and then tests functional relevance on HCV replication. Data mining is performed between nucleotides in the untranslated regions 5' and 3'UTR, and the amino acid residues in the non-structural proteins NS2, NS3 and NS5B. Results indicate covariance of the 243(rd) nucleotide of the 5'UTR with the 14(th), 41(st), 76(th), 110(th), 211(th) and 212(th) residues of NS2 and with the 71(st), 175(th) and 621(st) residues of NS3. Real-time experiments using an HCV subgenomic system to quantify viral replication confirm replication regulation for each covariant pair between 5'UTR₂₄₃ and NS2-41, -76, -110, -211, and NS3-71, -175. The HCV subgenomic system with/without the NS2 region shows that regulatory effects vanish without NS2, so replicative modulation mediated by HCV 5'UTR₂₄₃ depends on NS2. Strong binding of the NS2 variants to HCV RNA correlates with reduced HCV replication whereas weak binding correlates with restoration of HCV replication efficiency, as determined by RNA-protein immunoprecipitation assay band intensity. The dominant haplotype 5'UTR₂₄₃-NS2-41-76-110-211-NS3-71-175 differs according to the HCV genotype: G-Ile-Ile-Ile-Gly-Ile-Met for genotype 1b and A-Leu-Val-Leu-Ser-Val-Leu for genotypes 1a, 2a and 2b. In conclusion, 5'UTR₂₄₃ co-varies with specific NS2/3 protein amino acid residues, which may have significant structural and functional consequences for HCV replication. This unreported mechanism involving HCV replication possibly can be exploited in the development of advanced anti-HCV medication.

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Novel nucleotide and amino acid covariation between the 5'UTR and the NS2/NS3 proteins of hepatitis C virus: bioinformatic and functional analyses.

Chronic hepatitis C virus (HCV) infection often leads to liver cancer. The HCV NS2 protein is a hydrophobic transmembrane protein that associates with several cellular proteins in mammalian cells. In this report, we investigated the function of NS2 protein on HCV replication and translation by using a transient cell-based expression system. Cells co-transfected with pcDNA3.1 (-)-NS2 and the dual-luciferase reporter construct containing the HCV IRES were used to detect the effect of NS2 protein on HCV translation. Cells co-transfected with pcDNA3.1(-)-NS2, pcDNA-NS5B and a reporter plasmid were used to detect the effect of NS2 protein on HCV replication. The results showed that HCV NS2 protein up-regulated HCV IRES-dependent translation in a specific and dose-dependent manner in Huh7 cells but not in HeLa and HepG2 cells, and NS2 protein inhibited NS5B RdRp activity in a dose-independent manner in all three cell lines. These findings may suggest a novel mechanism by which HCV modulates its NS5B replication and IRES-dependent translation and facilitates virus persistence.

Xulin Chen

Papers

Hepatitis C virus (HCV) NS2 protein up-regulates HCV IRES-dependent translation and down-regulates NS5B RdRp activity