Inhibited Hepatitis C Virus RNA Replication Research Articles

Roseoside Is a Bioactive Compound in Kirengeshoma koreana Nakai Extract with Potent In Vitro Antiviral Activity Against Hepatitis C Virus.

Hepatitis C virus (HCV) is a pathogen that causes cirrhosis and hepatocellular carcinoma through chronic hepatitis C. This study focused on the anti-HCV activity of a 70% ethanol extract of Kirengeshoma koreana Nakai (KKE) and its bioactive chemical constituent(s). The KKE and its n-butanol (n-BuOH) fraction induced a significant reduction in HCV RNA levels without inducing cytotoxicity. A high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis revealed the presence of roseoside in the n-butanol fraction of the KKE, which inhibited HCV RNA replication in a concentration- and time-dependent manner without exerting cytotoxicity. Consistent with in silico molecular docking analysis data, roseoside targets and inhibits HCV NS5A/B replicase. Collectively, our findings demonstrate that roseoside is a chemical constituent in KKE that interferes with HCV replication by targeting NS5A/B replicase.

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.

Phytochemical analysis of Berberis lyceum methanolic extract and its antiviral activity through the restoration of MAPK signaling pathway modulated by HCV NS5A

Objective: To evaluate the antiviral activity and phytochemicals of selected plant extracts and their effect on the mitogen-activated protein kinase (MAPK) signaling pathway modulated by hepatitis C virus (HCV) nonstructural protein 5A (NS5A). Methods: A total of ten plant extracts were initially screened for their toxicities against HepG2 cells. The non-toxic plants were tested for their inhibitory effect on the expression of HCV NS5A at both mRNA and protein levels using real-time PCR and Western blotting assays, respectively. The differential expression of the genes associated with MAPK pathway in the presence of NS5A gene and plant extract was measured through real-time PCR. Subsequently, the identification of secondary metabolites was carried out by phytochemical and HPLC analysis. Results: The phytochemical profiling of Berberis lyceum revealed the presence of alkaloids, phenols, saponins, tannins, flavonoids, carbohydrates, terpenoids, steroids, and glycosides. Similarly, quercetin, myricetin, gallic acid, caffeic acid, and ferulic acid were identified through HPLC analysis. The methanolic extract of Berberis lyceum strongly inhibited HCV RNA replication with an IC50 of 11.44 µg/mL. RT-PCR and Western blotting assays showed that the extract reduced the expression of HCV NS5A in a dose- dependent manner. Berberis lyceum extract also attenuated NS5A- induced dysregulation of the MAPK signaling pathway. Conclusions: Our findings suggest that Berberis lyceum extract strongly inhibits HCV propagation by reducing HCV NS5A- induced perturbation of MAPK signaling.

RACK1 mediates rewiring of intracellular networks induced by hepatitis C virus infection.

Hepatitis C virus (HCV) is a positive-strand RNA virus replicating in a membranous replication organelle composed primarily of double-membrane vesicles (DMVs) having morphological resemblance to autophagosomes. To define the mechanism of DMV formation and the possible link to autophagy, we conducted a yeast two-hybrid screening revealing 32 cellular proteins potentially interacting with HCV proteins. Among these was the Receptor for Activated Protein C Kinase 1 (RACK1), a scaffolding protein involved in many cellular processes, including autophagy. Depletion of RACK1 strongly inhibits HCV RNA replication without affecting HCV internal ribosome entry site (IRES) activity. RACK1 is required for the rewiring of subcellular membranous structures and for the induction of autophagy. RACK1 binds to HCV nonstructural protein 5A (NS5A), which induces DMV formation. NS5A interacts with ATG14L in a RACK1 dependent manner, and with the ATG14L-Beclin1-Vps34-Vps15 complex that is required for autophagosome formation. Both RACK1 and ATG14L are required for HCV DMV formation and viral RNA replication. These results indicate that NS5A participates in the formation of the HCV replication organelle through interactions with RACK1 and ATG14L.

Notch signaling suppresses CD14+ monocytes cells activity in patients with chronic hepatitis C

Hepatitis C virus (HCV) infection always leads to chronic hepatitis via dysregulation of host immunity. Notch signaling also modulates the response of monocytes/macrophages. Thus, we aimed to investigate the regulatory role of Notch signaling to CD14+ monocytes. Forty patients with chronic hepatitis C and twenty normal controls (NC) were enrolled. CD14+ monocytes and CD4+ T cells were purified from peripheral bloods. Notch receptors' mRNA expression in CD14+ monocytes was semi-quantified by real-time PCR. Cytokine production by CD14+ monocytes in response to γ-secretase inhibitor (GSI) was investigated by ELISA. GSI-induced CD14+ monocytes activity to HCV clearance in Huh7.5 cells and to CD4+ T cell differentiation was also assessed in direct and indirect contact co-culture system. Notch1 mRNA relative level was approximately 10-fold elevated in CD14+ monocytes from chronic hepatitis C patients when compared with NC. GSI stimulation resulted in enhanced cytokines production by CD14+ monocytes from chronic hepatitis C patients. GSI-stimulated CD14+ monocytes from chronic hepatitis C patients induced suppression of HCV RNA replication in both direct and indirect contact co-culture system of CD14+ monocytes and HCVcc-infected Huh7.5 cells, and this process was accompanied by elevation of interferon-γ production but not increased target cell death. Moreover, GSI stimulation also enhanced CD14+ monocytes-induced Th1 and Th17 cells activation, and this process required direct cell-to-cell contact. Effective antiviral therapy down-regulated Notch1 mRNA expression and promoted cytokine production by CD14+ monocytes from chronic hepatitis C. Current data revealed an important immunoregulatory property of Notch signaling to CD14+ monocytes in chronic HCV infection.

Inhibition Mechanisms of Hepatitis C Virus Infection by Caffeic Acid and Tannic Acid.

Previously, we reported that coffee extract and its constituents, caffeic acid (CA) and p-coumaric acid, inhibit infection by the hepatitis C virus (HCV). In the present report, we identified another coffee-related compound, tannic acid (TA), which also inhibits HCV infection. We systematically evaluated which steps of the viral lifecycle were affected by CA and TA. TA substantially inhibits HCV RNA replication and egression, while CA does not. The infectivity of the HCV pretreated with CA or TA was almost lost. Cellular attachment of HCV particles and their interaction with apolipoprotein E, which is essential for HCV infectivity, were significantly reduced by CA. These results indicate that CA inhibits HCV entry via its direct effect on viral particles and TA inhibits HCV RNA replication and particle egression as well as entry into host cells. Taken together, our findings may provide insights into CA and TA as potential anti-HCV strategies.

Hepatitis C Virus-Specific T Cell Receptor mRNA-Engineered Human T Cells: Impact of Antigen Specificity on Functional Properties.

Therapy with genetically modified autologous T cells has shown great promise in cancer therapy. For an efficient control of hepatitis C virus (HCV) infection, cytotoxic T cells (CTL) are pivotal, but persistence of activated T cells may lead to liver toxicity. Here, anti-HCV T cell receptors (TCRs) recognizing the HCV nonstructural (NS) NS3 or NS5 viral peptide target were examined by mRNA transfection of human peripheral blood lymphocytes (PBLs) derived from healthy donors as well as chronically infected HCV patients. Immunological analysis shows that while the CTLs expressing the NS5-specific TCR reduced HCV RNA replication by a noncytotoxic mechanism, the NS3-specific TCR-redirected CTLs were polyfunctional and inhibited HCV RNA replication through antigen-specific cytotoxicity. Transcriptome signatures from these two types of CTL responses revealed uniquely expressed gene clusters upon encountering hepatoma target cells presenting endogenously expressed HCV proteins. The NS3 TCR induced a rapid expression of apoptotic signaling pathways and formation of embryonic gene clusters, whereas the NS5A TCR activation induced extended proliferative and metabolic pathways as the HCV target cells survived. Our results provide detailed insights into basic HCV T cell immunology and have clinical relevance for redirecting T cells to target virally infected hepatoma cells.IMPORTANCE Due to the protective ability of HCV-specific T cells and the hepatotoxic potential that they possess, there is a great need for the understanding of the functional aspects of HCV-specific T cells. To circumvent the low level of precursor frequency in patients, we engineered primary CD8+ T cells by mRNA TCR vectors to confer HCV specificity to new T cells. HCV TCRs that differ in antigen specificity and polyfunctionality were examined. mRNA TCR engineering of peripheral blood lymphocytes from healthy donors or chronically infected HCV patients resulted in strikingly high levels of HCV TCR expression and HCV-specific responses. While a cytotoxicity response from a polyfunctional T cell activation caused hepatotoxicity and the rapid induction of apoptotic signaling pathways, the noncytotoxic T cell activation showed extended proliferative, metabolic pathways and persistence of HCV target cells. Our results provide detailed insights into basic HCV T cell immunology and have clinical relevance for immune protection of HCV-associated diseases.

In Silico Design and Experimental Validation of siRNAs Targeting Conserved Regions of Multiple Hepatitis C Virus Genotypes.

RNA interference (RNAi) is a post-transcriptional gene silencing mechanism that mediates the sequence-specific degradation of targeted RNA and thus provides a tremendous opportunity for development of oligonucleotide-based drugs. Here, we report on the design and validation of small interfering RNAs (siRNAs) targeting highly conserved regions of the hepatitis C virus (HCV) genome. To aim for therapeutic applications by optimizing the RNAi efficacy and reducing potential side effects, we considered different factors such as target RNA variations, thermodynamics and accessibility of the siRNA and target RNA, and off-target effects. This aim was achieved using an in silico design and selection protocol complemented by an automated MysiRNA-Designer pipeline. The protocol included the design and filtration of siRNAs targeting highly conserved and accessible regions within the HCV internal ribosome entry site, and adjacent core sequences of the viral genome with high-ranking efficacy scores. Off-target analysis excluded siRNAs with potential binding to human mRNAs. Under this strict selection process, two siRNAs (HCV353 and HCV258) were selected based on their predicted high specificity and potency. These siRNAs were tested for antiviral efficacy in HCV genotype 1 and 2 replicon cell lines. Both in silico-designed siRNAs efficiently inhibited HCV RNA replication, even at low concentrations and for short exposure times (24h); they also exceeded the antiviral potencies of reference siRNAs targeting HCV. Furthermore, HCV353 and HCV258 siRNAs also inhibited replication of patient-derived HCV genotype 4 isolates in infected Huh-7 cells. Prolonged treatment of HCV replicon cells with HCV353 did not result in the appearance of escape mutant viruses. Taken together, these results reveal the accuracy and strength of our integrated siRNA design and selection protocols. These protocols could be used to design highly potent and specific RNAi-based therapeutic oligonucleotide interventions.

Betulinic acid exerts anti‐hepatitis C virus activity via the suppression of NF‐κB‐ and MAPK‐ERK1/2‐mediated COX‐2 expression

This study was designed to evaluate the effect of betulinic acid (BA), extracted from Avicennia marina, on the replication of hepatitis C virus (HCV) and to investigate the mechanism of this BA-mediated anti-HCV activity. HCV replicon and infectious systems were used to evaluate the anti-HCV activity of BA. Exogenous COX-2 or knock-down of COX-2 expression was used to investigate the role of COX-2 in the anti-HCV activity of BA. The effects of BA on the phosphorylation of NF-κB and on kinases in the MAPK signalling pathway were determined. The anti-HCV activity of BA in combination with other HCV inhibitors was also determined to assess its use as an anti-HCV supplement. BA inhibited HCV replication in both Ava5 replicon cells and in a cell culture-derived infectious HCV particle system. Treatment with a combination of BA and IFN-α, the protease inhibitor telaprevir or the NS5B polymerase inhibitor sofosbuvir resulted in the synergistic suppression of HCV RNA replication. Exogenous overexpression of COX-2 gradually attenuated the inhibitory effect of BA on HCV replication, suggesting that BA reduces HCV replication by suppressing the expression of COX-2. In particular, BA down-regulated HCV-induced COX-2 expression by reducing the phosphorylation of NF-κB and ERK1/2 of the MAPK signalling pathway. BA inhibits HCV replication by suppressing the NF-κB- and ERK1/2-mediated COX-2 pathway and may serve as a promising compound for drug development or as a potential supplement for use in the treatment of HCV-infected patients.

Synthesis of Novel 4′α-Trifluoromethyl-2′β-C-Methyl-Carbodine Analogs as Anti-Hepatitis C Virus Agents

Novel 4 ′α-trifluoromethyl-2 ′β-methyl carbocyclic nucleoside analogs have been prepared and evaluated for inhibition of hepatitis C virus (HCV) RNA replication in cell cultures. Construction of cyclopentene intermediate 10a was achieved via sequential Johnson–Claisen orthoester rearrangement and ring-closing metathesis starting from the α-trifluoromethyl-α,β-unsaturated ester 5. Stereoselective dihydroxylation and desilylation yielded the target carbodine analogs. The synthesized nucleoside analogs mentioned above (18 and 19) were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line (LucNeo#2). However, the synthesized nucleosides showed neither significant antiviral activity nor toxicity up to 50 μM.

Pharmacokinetics of a Cholesterol-conjugated Aptamer Against the Hepatitis C Virus (HCV) NS5B Protein.

Hepatitis C virus (HCV) is the major cause of progressive liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Previously, we reported that a 29 nucleotide-long 2'-F pyrimidine modified RNA aptamer against the HCV nonstructural protein 5B efficiently inhibited HCV replication and suppressed HCV infectious virus particle formation in a cell culture system. In this study, we modified this aptamer through conjugation of cholesterol for in vivo availability. This cholesterol-conjugated aptamer (chol-aptamer) efficiently entered the cell and inhibited HCV RNA replication, without any alteration in gene expression profiling including innate immune response-related genes. Moreover, systemic administration of the chol-aptamer was well tolerated without any abnormalities in mice. To evaluate the pharmacokinetics of the chol-aptamer in vivo, dose proportionality, bioavailability, and pharmacokinetic parameters were evaluated by noncompartmental analyses in normal BALB/c mice. Population analysis was performed using nonlinear mixed effects modeling. Moreover, the pharmacokinetics of two different routes (intravenous, IV, versus intraperitoneal, IP) were compared. Cholesterol conjugation showed dose proportionality, extended the time that the aptamer was in the plasma, and enhanced aptamer exposure to the body. Noticeably, the IV route was more suitable than the IP route due to the chol-aptamer remaining in the plasma for a longer period of time.

Inhibition of Hepatitis C Virus Infection by DNA Aptamer against NS2 Protein

NS2 protein is essential for hepatitis C virus (HCV) replication. NS2 protein was expressed and purified. Aptamers against NS2 protein were raised and antiviral effects of the aptamers were examined. The molecular mechanism through which the aptamers exert their anti-HCV activity was investigated. The data showed that aptamer NS2-3 inhibited HCV RNA replication in replicon cell line and infectious HCV cell culture system. NS2-3 and another aptamer NS2-2 were demonstrated to inhibit infectious virus production without cytotoxicity in vitro. They did not affect hepatitis B virus replication. Interferon beta (IFN-β) and interferon-stimulated genes (ISGs) were not induced by the aptamers in HCV-infected hepatocytes. Furthermore, our study showed that N-terminal region of NS2 protein is involved in the inhibition of HCV infection by NS2-2. I861T within NS2 is the major resistance mutation identified. Aptamer NS2-2 disrupts the interaction of NS2 with NS5A protein. The data suggest that NS2-2 aptamer against NS2 protein exerts its antiviral effects through binding to the N-terminal of NS2 and disrupting the interaction of NS2 with NS5A protein. NS2-specific aptamer is the first NS2 inhibitor and can be used to understand the mechanisms of virus replication and assembly. It may be served as attractive candidates for inclusion in the future HCV direct-acting antiviral combination therapies.

4′-Substituted pyrimidine nucleosides lacking 5′-hydroxyl function as potential anti-HCV agents

Hepatitis C virus (HCV) infection is one of the major health problems worldwide. If left untreated, it leads to liver cirrhosis, liver cancer and death. Herein, we report synthesis and anti-HCV activity of a new class of pyrimidine nucleosides possessing a 4′-carboxymethyl (9–16, 21 and 23) or 4′-carboxamide function (17–19 and 24). Among these, 10–12 (EC50=33.1–42.4μM), 14 and 21 (EC50=43.4–59.5μM) exhibited potent activity in HCV-1a replicon cells without any toxicity to parent Huh-7 cells (CC50=>829–1055μM). The anti-HCV activities demonstrated by this unusual class of compounds were superior to that of ribavirin (EC50=81.9μM). Further, the most active analog, 12, was found to interact synergistically with ribavirin to inhibit HCV RNA replication.

Peroxisome proliferator-activated receptor delta antagonists inhibit hepatitis C virus RNA replication

It has been reported that ligand-mediated transcription factor peroxisome proliferator-activated receptor alpha (hPPARα) is involved in hepatitis C virus (HCV) RNA replication, whereas hPPARγ is not, and the effect of hPPARδ is unknown. Here, we show that hPPARδ-selective antagonists effectively inhibit HCV RNA replication. We describe the design, synthesis and pharmacological evaluation of a series of biphenyl-4-carboxylic acid-type hPPARδ antagonists, including previously reported compounds, as candidate anti-HCV agents. A representative compound (4c) dose-dependently inhibited HCV RNA replication (EC50 0.22μM), while exhibiting relatively weak cytotoxicity to the host cells (CC50 2.5μM). It also showed an additive and dose-dependent effect on the inhibition of HCV RNA replication by pegylated interferon alpha (Peg-IFNα) alone and by both Peg-IFNα and ribavirin (currently the clinical treatment of choice for HCV infection). Thus, combination of a hPPARδ antagonist with current therapy may improve the efficacy of treatment for HCV infection.

The antimalarial ferroquine is an inhibitor of hepatitis C virus

Hepatitis C virus (HCV) is a major cause of chronic liver disease. Despite recent success in improving anti‐HCV therapy, additional progress is still needed to develop cheaper and interferon (IFN)‐free treatments. Here, we report that ferroquine (FQ), an antimalarial ferrocenic analog of chloroquine, is a novel inhibitor of HCV. FQ potently inhibited HCV infection of hepatoma cell lines by affecting an early step of the viral life cycle. The antiviral activity of FQ on HCV entry was confirmed with pseudoparticles expressing HCV envelope glycoproteins E1 and E2 from six different genotypes. In addition to its effect on HCV entry, FQ also inhibited HCV RNA replication, albeit at a higher concentration. We also showed that FQ has no effect on viral assembly and virion secretion. Using a binding assay at 4°C, we showed that FQ does not prevent attachment of the virus to the cell surface. Furthermore, virus internalization was not affected by FQ, whereas the fusion process was impaired in the presence of FQ as shown in a cell‐cell fusion assay. Finally, virus with resistance to FQ was selected by sequential passage in the presence of the drug, and resistance was shown to be conferred by a single mutation in E1 glycoprotein (S327A). By inhibiting cell‐free virus transmission using a neutralizing antibody, we also showed that FQ inhibits HCV cell‐to‐cell spread between neighboring cells. Combinations of FQ with IFN, or an inhibitor of HCV NS3/4A protease, also resulted in additive to synergistic activity. Conclusion: FQ is a novel, interesting anti‐HCV molecule that could be used in combination with other direct‐acting antivirals. (Hepatology 2013)

Inhibition of hepatitis C virus NS5B polymerase by S-trityl-l-cysteine derivatives

Structure-based studies led to the identification of a constrained derivative of S-trityl-l-cysteine (STLC) scaffold as a candidate inhibitor of hepatitis C virus (HCV) NS5B polymerase. A panel of STLC derivatives were synthesized and investigated for their activity against HCV NS5B. Three STLC derivatives, 9, F-3070, and F-3065, were identified as modest HCV NS5B inhibitors with IC50 values between 22.3 and 39.7 μM. F-3070 and F-3065 displayed potent inhibition of intracellular NS5B activity in the BHK-NS5B-FRLuc reporter and also inhibited HCV RNA replication in the Huh7/Rep-Feo1b reporter system. Binding mode investigations suggested that the STLC scaffold can be used to develop new NS5B inhibitors by further chemical modification at one of the trityl phenyl group.

Anti‐hepatitis C virus activity of 3‐hydroxy caruilignan C from Swietenia macrophylla stems

Chronic hepatitis C virus (HCV) infection ultimately leads to chronic hepatitis, hepatic cirrhosis and hepatocellular carcinoma (HCC). As the standard treatment is not completely efficacious, a safer and more effective agent against HCV infection needs to be developed. In this report, we demonstrated that 3-hydroxy caruilignan C (3-HCL-C) isolated from Swietenia macrophylla stems exhibited high anti-HCV activity at both protein and RNA levels at nontoxic concentrations, with an EC(50) value of 10.5 ± 1.2 μm. Combinations of 3-HCL-C and interferon-α (IFN-α), an HCV NS5B polymerase inhibitor (2'-C-methylcytidine; NM-107) or an HCV NS3/4A protease inhibitor (Telaprevir; VX-950) increased the suppression of HCV RNA replication. The results suggested that 3-HCL-C may be a potential anti-viral agent. We then demonstrated that 3-HCL-C interfered with HCV replication by inducing IFN-stimulated response element transcription and IFN-dependent anti-viral gene expression.

Expression of an IRF-3 fusion protein and mouse estrogen receptor, inhibits hepatitis C viral replication in RIG-I-deficient Huh 7.5 cells

Interferon Regulatory Factor-3 (IRF-3) plays a central role in the induction of interferon (IFN) production and succeeding interferon-stimulated genes (ISG) expression en route for restraining hepatitis C virus (HCV) infection. Here, we established a stable Huh7.5-IRF3ER cell line expressing a fusion protein of IRF-3 and mouse estrogen receptor (ER) to examine IFN production and anti-HCV effects of IRF-3 in retinoic acid inducible-gene-I (RIG-I) deficient Huh 7.5 cells. Homodimerization of the IRF-3ER fusion protein was detected by Western blotting after treatment with the estrogen receptor agonist 4-hydrotamoxifen (4-HT) in Huh7.5-IRF3ER cells. Expression of IFN-α, IFN-β, and their inhibitory effects on HCV replication were demonstrated by real-time polymerase chain reaction (PCR). Peak expression of IFN-α and IFN-β was achieved 24-hours post 4-HT treatment, coinciding with the appearance of phosphorylated signal transducer and activator of transcription (STAT) proteins. Additionally, HCV viral replication declined in time-dependent fashion. In previous studies, a novel IFN-mediated pathway regulating expression of 1-8U and heterogeneous nuclear ribonucleoprotein M (hnRNP M) inhibited HCV internal ribosomal entry site (IRES)-dependent translation. When expression of ISGs such as 1-8U and hnRNP M were measured in 4-HT-treated Huh7.5-IRF3ER cells, both genes were positively regulated by activation of the IRF-3ER fusion protein. In conclusion, the anti-HCV effects of IRF-3ER homodimerization inhibited HCV RNA replication as well as HCV IRES-dependent translation in Huh7.5-IRF3ER cells. The results of this study indicate that IRF-3ER homodimerization is a key step to restore IFN expression in Huh7.5-IRF3ER cells and in achieving its anti-HCV effects.

Suppression of hepatitis C virus by the flavonoid quercetin is mediated by inhibition of NS3 protease activity

Phytochemicals exert antiviral activity and may play a potential therapeutic role in hepatitis C virus (HCV) infection. In this work, we aimed to isolate NS3 inhibitors from traditional Indian medicinal plants that were found, in our earlier study, to inhibit HCV NS3 protease activity and to evaluate their potential to inhibit HCV replication. A potent inhibitory effect of NS3 catalytic activity was obtained with Embelia ribes plant extracts. Quercetin, a ubiquitous plant flavonoid, was identified as the active substance in the fractioned extract. It was found to inhibit NS3 activity in a specific dose-dependent manner in an in vitro catalysis assay. Quercetin inhibited HCV RNA replication as analysed in the subgenomic HCV RNA replicon system. It also inhibited HCV infectious virus production in the HCV infectious cell culture system (HCVcc), as analysed by the focus-forming unit reduction assay and HCV RNA real-time PCR. The inhibitory effect of quercetin was also obtained when using a model system in which NS3 engineered substrates were introduced in NS3-expressing cells, providing evidence that inhibition in vivo could be directed to the NS3 and do not involve other HCV proteins. Our work demonstrates that quercetin has a direct inhibitory effect on the HCV NS3 protease. These results point to the potential of quercetin as a natural nontoxic anti-HCV agent reducing viral production by inhibiting both NS3 and heat shock proteins essential for HCV replication.

The effects of NS5A inhibitors on NS5A phosphorylation, polyprotein processing and localization

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multi-functional protein that is expressed in basally phosphorylated (p56) and in hyperphosphorylated (p58) forms. NS5A phosphorylation has been implicated in regulating multiple aspects of HCV replication. We recently reported the identification of a class of compounds that potently inhibit HCV RNA replication by targeting NS5A. Although the precise mechanism of inhibition of these compounds is not well understood, one activity that has been described is their ability to block expression of the hyperphosphorylated form of NS5A. Here, we report that an NS5A inhibitor impaired hyperphosphorylation without affecting basal phosphorylation at the C-terminal region of NS5A. This inhibitor activity did not require NS5A domains II and III and was distinct from that of a cellular kinase inhibitor that also blocked NS5A hyperphosphorylation, results that are consistent with an inhibitor-binding site within the N-terminal region of NS5A. In addition, we observed that an NS5A inhibitor promoted the accumulation of an HCV polyprotein intermediate, suggesting that inhibitor binding to NS5A may occur prior to the completion of polyprotein processing. Finally, we observed that NS5A p56 and p58 separated into different membrane fractions during discontinuous sucrose gradient centrifugation, consistent with these NS5A phosphoforms performing distinct replication functions. The p58 localization pattern was disrupted by an NS5A inhibitor. Collectively, our results suggest that NS5A inhibitors probably impact several aspects of HCV expression and regulation. These findings may help to explain the exceptional potency of this class of HCV replication complex inhibitors.