Antiviral action of interferon-alpha against hepatitis C virus replicon and its modulation by interferon-gamma and interleukin-8

BACKGROUND AND AIM: Interferon-alpha (IFN-alpha) based therapy is the main treatment used to control hepatitis C virus (HCV) infection. The aim of this study was to understand the mechanisms of IFN-alpha inhibition of HCV replication and the resistance of HCV to IFN-alpha therapy, and improve the efficiency of HCV treatment. METHODS: The inhibitory effects of IFN-alpha on a HCV replicon system were examined and the potential regulatory effects of interferon-gamma (IFN-gamma) and interleukin-8 (IL-8) on the antiviral actions of IFN-alpha were also investigated in this report. RESULTS: The results showed that IFN-alpha can effectively inhibit the replication of HCV replicon. Pretreatment of HCV replicon cells with IFN-gamma could significantly potentiate the inhibitory effects of IFN-alpha on the HCV replicon. Direct addition of IL-8 to the culture medium of HCV replicon cells could partially rescue the HCV replicon from the inhibition of IFN-alpha, which may be the result of IL-8 down-regulation of interferon-stimulated genes. CONCLUSION: Our study demonstrated that IFN-gamma has synergistic antiviral effects with IFN-alpha; whereas IL-8 can attenuate the anti-HCV actions of IFN-alpha and is associated with HCV resistance to interferon-alpha therapy.     

Antiviral action of ribavirin in chronic hepatitis C

BACKGROUND & AIMS: In the patients with chronic hepatitis C, the addition of ribavirin to interferon (IFN)-alpha significantly increases the virologic responses. Our aim was to assess the antiviral action of ribavirin on hepatitis C virus (HCV) as a function of ribavirin pharmacokinetics and to evaluate the influence of this antiviral effect on IFN-alpha efficacy. METHODS: Forty-five patients with chronic hepatitis C (genotype 1b) received various schedules of IFN-alpha and/or ribavirin administration. Frequent blood sampling was performed for HCV RNA kinetics and ribavirin pharmacokinetics assessment. RESULTS: Ribavirin monotherapy induced a significant, moderate, early, and transient viral load decrease in approximately half of the patients. The occurrence of this effect was associated with longer ribavirin clearance half-lives and higher serum ribavirin concentrations. Ribavirin antiviral effect partly reduced the rebound preceding the second IFN-alpha injection in patients receiving standard IFN-alpha 3 times per week plus ribavirin. The magnitude of the rebound was inversely related to ribavirin concentrations. These patients subsequently experienced a slow, but significant, second slope of viral decrease and cleared HCV RNA. The addition of ribavirin to daily IFN-alpha monotherapy did not have any impact on the second phase of viral decline. CONCLUSIONS: Ribavirin exerts a significant, moderate, and transient antiviral effect in a significant proportion of patients with chronic hepatitis C. The antiviral effect of ribavirin correlates with ribavirin pharmacokinetics and is partly responsible for the improved efficacy of the combination of standard IFN-alpha and ribavirin compared with IFN-alpha monotherapy by increasing the incidence of the initial response.     

Mycophenolic acid inhibits hepatitis C virus replication and acts in synergy with cyclosporin A and interferon-alpha

BACKGROUND & AIMS: Chronic hepatitis C virus (HCV) infection is the leading indication for liver transplantation. Clinical evidence suggests that particular immunosuppressive agents can have an influence on HCV recurrence. Cyclosporine A (CsA) specifically inhibits HCV replication through blocking the viral RNA polymerase enzyme NS5B. In this study, we investigated the effect of mycophenolic acid (MPA) and other immunosuppressants on HCV replication. METHODS: MPA and other compounds were tested in vitro using an HCV-replication model containing a luciferase reporter gene. RESULTS: At clinically relevant concentrations (1.0-6.0 microg/mL), MPA inhibited HCV replication to approximately 75%. CsA and interferon (IFN)-alpha also showed inhibition in a dose-dependent manner. In these short-term (18 hours) experiments, MPA did not inhibit cell proliferation or induce cell death, which could have accounted for the antiviral effect. In contrast to the antiviral activity of MPA against West Nile virus, the effect of MPA on HCV replication was guanosine independent. When combined, MPA and CsA showed significant synergistic inhibition of replication, reaching maximum inhibition of approximately 90% at the highest doses. Synergistic effects were observed with suboptimal concentrations of IFN-alpha with MPA or CsA. The kinetics of HCV inhibition by MPA, CsA, and IFN-alpha were clearly distinct, with earliest effects seen with IFN-alpha. No specific inhibitory effects were observed with tacrolimus or rapamycin. CONCLUSIONS: The immunosuppressive drug MPA is as potent as CsA as an inhibitor of HCV replication. MPA was shown to have a distinct anti-HCV mechanism of action, independent of cell proliferation and guanosine depletion.     

Suppression of hepatitis C virus replication by cyclosporin a is mediated by blockade of cyclophilins

BACKGROUND & AIMS: Cyclosporin A specifically suppresses hepatitis C virus (HCV) replication in vitro at clinically achievable concentrations. In this study, we investigated the mechanisms of action of cyclosporin A against HCV replication. METHODS: The in vitro effects of cyclosporin A on HCV replication were analyzed using an HCV replicon system that expresses chimeric luciferase reporter protein. RESULTS: The significant effects of cyclosporin A on expression of an HCV replicon and the absence of such effects of FK506, which shares mechanisms of action with cyclosporin A, suggested the involvement of intracellular ligands of cyclosporin A, the cyclophilins. Transient and stable knockdown of the expression of cytoplasmic cyclophilins A, B, and C by short hairpin RNA-expressing vectors suppressed HCV replication significantly. A cyclosporin analogue, cyclosporin D, which lacks immunosuppressive activity but exhibits cyclophilin binding, induced a similar suppression of HCV replication. Furthermore, cyclosporin A treatment of Huh7 cells induced an unfolded protein response exemplified by expression of cellular BiP/GRP78. Treatment of cells with thapsigargin and mercaptoethanol, which induce the unfolded protein responses, suppressed HCV replication, suggesting that the cyclosporin-induced unfolded protein responses might contribute to the suppression of HCV protein processing and replication. CONCLUSIONS: The anti-HCV activity of cyclosporin A is mediated through a specific blockade of cyclophilins, and these molecules may constitute novel targets for anti-HCV therapeutics.     

Synergistic inhibition of intracellular hepatitis C virus replication by combination of ribavirin and interferon- alpha

Treatment of hepatitis C virus (HCV) infection with interferon (IFN)- alpha and ribavirin combination therapy results in superior clinical antiviral responses than does monotherapy with IFN. To explore the virological basis of the effects of combination therapy, we analyzed the effects of IFN- alpha and ribavirin, singly and in combination, on intracellular HCV replication by use of an HCV replicon system. A new replicon that expressed a selectable chimeric reporter protein comprising firefly luciferase and neomycin phosphotransferase was constructed. The replicon was highly sensitive to IFN-alpha (50% inhibitory concentration [IC(50)], 0.5 U/mL). Therapy with ribavirin showed weak suppression of HCV replication at a lower concentration (IC(50), 126 mu mol/L). The nucleotide sequence diversity of the replicon was increased significantly by therapy with ribavirin, suggesting that error-prone HCV replication was induced by the drug. Importantly, use of a clinically achievable concentration of ribavirin (approximately 10 mu mol/L) in combination with IFN showed strong synergistic inhibitory effects on HCV replication. Our results suggest that the direct effects of ribavirin on the genetic stability of the HCV subgenome and its synergistic action combined, with IFN-alpha, may explain the improved clinical responses to combination therapy.     

A cell-based, high-throughput screen for small molecule regulators of hepatitis C virus replication

BACKGROUND & AIMS: Only half of patients with chronic hepatitis C virus (HCV) infection experience sustained virologic response to pegylated-interferon and ribavirin, which cause numerous side effects. Thus, the identification of more effective and better tolerated agents is a high priority. We applied chemical biology to screen small molecules that regulate HCV. METHODS: We first optimized the Huh7/Rep-Feo replicon cell line for the 384-well microplate format and used this line to screen a large library of well-characterized, known biologically active compounds using automated technology. After identifying several molecules capable of either stimulating or inhibiting HCV replication in this primary screen, we then validated our hit compounds using a full-length HCV replicon cell line in secondary screens. RESULTS: We identified and validated a number of antiviral and proviral agents, including HMG-CoA reductase inhibitors (antiviral) and corticosteroids (proviral). The finding of increased replication associated with corticosteroids suggests that these agents directly promote viral replication independent of their suppressive effects on the immune response. The finding of antiviral activity associated with the HMG-CoA reductase inhibitors implies an important role for lipid metabolism in the viral life cycle. CONCLUSIONS: We have developed a simple, reproducible, and reliable cell-based high-throughput screening assay system using an HCV replicon model to identify small molecules that regulate HCV replication. This method can be used to identify not only putative antiviral agents, but also cellular regulators of viral replication.     

Gene expression associated with interferon alfa antiviral activity in an HCV replicon cell line

Interferon alfa (IFN-alpha)-based treatment is the only therapeutic option for chronic hepatitis C viral infection. However, the molecular mechanisms of IFN-alpha antiviral activity are not completely understood. The recent development of an HCV replicon cell culture system provides a feasible experimental model to investigate the molecular details of IFN-induced direct antiviral activity in hepatocytes. In this report, we show that IFN-alpha can effectively inhibit HCV subgenomic RNA replication and suppress viral nonstructural protein synthesis. Using cDNA microarray analysis, we also show that the replicon cells have different gene expression profile compared with the parental hepatoma cells (Huh7). IFN-alpha can induce a number of responsive genes in the replicon cells. One of the genes, 6-16 (G1P3), can enhance IFN-alpha antiviral efficacy. In addition, we demonstrate that IFN-alpha can significantly activate STAT3 in hepatoma cells, suggesting that this pathway plays a role in IFN-alpha signaling. In conclusion, our results indicate that IFN-alpha antiviral activity is associated with activation of STAT3-signaling pathway and intracellular gene activation. Our results also suggest that IFN-alpha-induced target genes may play an important role in IFN-alpha anti-HCV activity.     

Interferons alpha, beta, gamma each inhibit hepatitis C virus replication at the level of internal ribosome entry site-mediated translation.

Interferon (IFN)-alpha is the standard therapy for the treatment of chronic hepatitis C, but the mechanisms underlying its antiviral action are not well understood. In this report, we demonstrated that IFN-alpha, -beta and -gamma inhibit replication of the hepatitis C virus (HCV) in a cell culture model at concentrations between 10 and 100 IU/ml. We demonstrated that the antiviral actions each of each these IFNs are targeted to the highly conserved 5' untranslated region of the HCV genome, and that they directly inhibit translation from a chimeric clone between full-length HCV genome and green fluorescent protein (GFP). This effect is not limited to HCV internal ribosome entry site (IRES), since these IFNs also inhibit translation of the encephalomyocardititis virus (EMCV) chimeric mRNA in which GFP is expressed by IRES-dependent mechanisms (pCITE-GFP). These IFNs had minimal effects on the expression of mRNAs from clones in which translation is not IRES dependent. We conclude that IFN-alpha, -beta and -gamma inhibit replication of sub-genomic HCV RNA in a cell culture model by directly inhibiting two internal translation initiation sites of HCV- and EMCV-IRES sequences present in the dicistronic HCV sub-genomic RNA. Results of this in vitro study suggest that selective inhibition of IRES-mediated translation of viral polyprotein is a general mechanism by which IFNs inhibits HCV replication.     

Mycophenolic acid augments interferon-stimulated gene expression and inhibits hepatitis C Virus infection in vitro and in vivo

Mycophenolic acid (MPA) is a highly effective immunosuppressant that has broad antiviral activity against different viruses and can act in synergy with interferon-α (IFN-α) on hepatitis C virus (HCV) replication. MPA is a potent inosine monophosphate dehydrogenase (IMPDH) inhibitor but the antiviral mechanisms are less understood. The aim of this study was to investigate the inhibition of HCV infection by MPA and the molecular basis for its synergy with IFN-α. The role of IMPDH and interferon-stimulated genes (ISGs) was investigated in two HCV models using gain- or loss-of-function approaches. The in vivo effect of MPA treatment was studied in NOD/SCID mice engrafted with HCV replicon cells. Potent antiviral effects of MPA at clinically relevant concentrations were observed with both the subgenomic and JFH1-derived infectious HCV models. MPA treatment in mice resulted in a specific and robust inhibition of HCV replication. Ectopic expression of an MPA-resistant IMPDH2 mutant in HCV host cells completely reversed the antiproliferative effect of MPA but only partially affected the antiviral potency. However, similar to ribavirin, MPA induced expression of multiple antiviral ISGs, including interferon regulatory factor 1 (IRF1). Cotreatment of MPA with IFN-α resulted in additive effects on ISG expression and enhanced IFN-induced luciferase reporter activity. Knockdown of IRF1, but not IFITM3, significantly attenuated the inhibition of HCV replication by MPA. CONCLUSION: MPA exerts a potent anti-HCV effect in vitro and in mice and acts in synergy with IFN-α. MPA's antiviral activity partially depends on IMPDH but also involves stimulation of ISGs, providing a molecular basis for its synergy with IFN-α.     

An antioxidant resveratrol significantly enhanced replication of hepatitis C virus

AIM:To elucidate the effect of antioxidants,resveratrol (RVT)and astaxanthin(AXN),on hepatitis C virus(HCV) replication. METHODS:We investigated the effect of recent popular antioxidant supplements on replication of the HCV replicon system OR6.RVT is a strong antioxidant and a kind of polyphenol that inhibits replication of various viruses.AXN is also a strong antioxidant.The replication of HCV RNA was assessed by the luciferase reporter assay.An additive effect of antioxidants on antiviral effects of inter...     

含EGFP报告基因单顺反子HCV亚基因组复制子载体的构建和表达

目的构建含增强型绿色荧光蛋白(EGFP)报告基因的HCV复制子表达载体,并实现其在细胞中的复制表达。方法用分子生物学基因克隆技术对HCV 2a型复制子的基因进行改造,用EGFP基因替代HCV基因组中的包膜基因(E1和E2)体外构建重组单顺反子HCV亚基因组复制子真核表达质粒pcDNA-JFH1-EGFP,经限制性内切酶酶切分析和测序鉴定;脂质体介导转染人肝癌细胞系Huh-7细胞,用荧光显微镜观察EGFP表达,采用半定量RT-PCR方法检测重组复制子的HCV RNA负链,采用Western blot检测HCV NS3蛋白的复制表达,并观察IFN-α对重组质粒表达的HCV RNA复制的抑制作用。结果构建的4个重组质粒酶切分析与预期相符,HCV亚基因复制子表达载体中未发生EGFP和HCV编码区读码框架改变,转染重组载体Huh-7细胞检测到HCV负链及EGFP和HCV NS3蛋白表达。转染后48h,1 000IU/ml和2 000IU/ml IFN-α处理的细胞HCV RNA表达水平分别为未处理组的20.0%和7.6%。结论含EGFP报告基因的单顺反子HCV亚基因组复制子表达载体pcDNA-JFH1-EGFP构建成功,在Huh-7细胞中能有效复制表达,为进一步研究HCV提供了实验平台。     

Two flavonoids extracts from Glycyrrhizae radix inhibit in vitro hepatitis C virus replication

Aim:  Traditional herbal medicines have been used for several thousand years in China and other Asian countries. In this study we screened herbal drugs and their purified compounds, using the Feo replicon system, to determine their effects on in vitro HCV replication.
Methods:  We screened herbal drugs and their purified extracts for the activities to suppress hepatitis C virus (HCV) replication using an HCV replicon system that expressed chimeric firefly luciferase reporter and neomycin phosphotransferase (Feo) genes. We tested extracts and 13 purified compounds from the following herbs: Glycyrrhizae radix ; Rehmanniae radix ; Paeoniae radix ; Artemisiae capillari spica ; and Rhei rhizoma .
Results:  The HCV replication was significantly and dose-dependently suppressed by two purified compounds, isoliquiritigenin and glycycoumarin, which were from Glycyrrhizae radix . Dose-effect analyses showed that 50% effective concentrations were 6.2 ± 1.0 µg/mL and 15.5 ± 0.8 µg/mL for isoliquiritigenin and glycycoumarin, respectively. The MTS assay did not show any effect on cell growth and viability at these effective concentrations, indicating that the effects of the two compounds were specific to HCV replication. These two compounds did not affect the HCV IRES-dependent translation nor did they show synergistic action with interferon-alpha.
Conclusion:  Two purified herbal extracts, isoliquiritigenin and glycycoumarin, specifically suppressed in vitro HCV replication. Further elucidation of their mechanisms of action and evaluation of in vivo effects and safety might constitute a new anti-HCV therapeutics.     

Hepatitis C virus core protein is a potent inhibitor of RNA silencing-based antiviral response

BACKGROUND & AIMS: Persistent infection with hepatitis C virus (HCV) leads to chronic hepatitis and hepatocellular carcinoma (HCC). RNA interference (RNAi) may act as a host antiviral response against viral RNA. METHODS: The effects of RNAi on both the replicative intermediates and the internal ribosome entry site (IRES) of HCV were studied by using HCV-related short interfering RNA (siRNA) detection assay. The mechanism that permits HCV to escape RNAi was studied by using RNAi assay materials. RESULTS: These studies demonstrate that the Dicer, an RNase enzyme that generates short siRNA, can target and digest both the IRES and the replicative intermediate of HCV into siRNA of approximately 22 nucleotides. Further studies also show that Dicer can inhibit the replication of the HCV subgenomic replicon. However, the HCV core protein inhibits this RNAi and rescues the replication of the HCV subgenomic replicon through a direct interaction with Dicer. CONCLUSIONS: RNAi is a limiting factor for HCV infection, and the core protein suppresses the RNA silencing-based antiviral response. This ability of the core protein to counteract the host defense may lead to a persistent viral infection and may contribute to the pathogenesis of HCV.     

Hepatitis C virus expression suppresses interferon signaling by degrading STAT1

BACKGROUND & AIMS: The molecular mechanisms by which hepatitis C virus (HCV) antagonizes the antiviral actions of interferon (IFN) have not been fully characterized. Specifically, how HCV proteins impact on IFN signaling components has yet to be elucidated. We used an HCV cell-based expression model to examine the interaction between HCV protein expression and host type I IFN signaling components in the Jak-STAT kinase pathway. METHODS: Full-length HCV and HCV subgenomic constructs corresponding to structural and each of the nonstructural proteins were transiently transfected into Huh-T7 cells. HCV expression was monitored by an HCV core antigen enzyme-linked immunosorbent assay. STAT1, P-STAT1, and HCV protein expression was investigated with immunoprecipitation and Western blots. RESULTS: Overexpression and small interfering RNA studies showed that STAT1 was indispensable for control of HCV expression. STAT1 and P-STAT1 expression were markedly reduced in HCV-transfected cells. Full-length HCV, HCV core/E1/E2, and NS3-4A were each associated with decreased STAT1 expression, which was attributable to proteasome-dependent degradation of STAT1. HCV core, but not HCV E1, E2, NS3, NS4, or NS5, bound to STAT1. STAT2 expression was not affected by HCV. CONCLUSIONS: HCV expression selectively degrades STAT1 and reduces P-STAT1 accumulation in the nucleus in a proteasome-dependent manner. HCV core protein binds STAT1, suggesting that this viral protein is associated with STAT1 degradation. STAT1 plays an indispensable role in innate antiviral immunity against HCV expression. In turn, HCV subverts the Jak-STAT kinase by selectively inducing STAT1 degradation.     

Inhibition of subgenomic hepatitis C virus RNA replication in HeLa cells

BACKGROUND/AIMS: Antiviral therapy such as combination interferon and ribavirin can eradicate hepatitis C virus (HCV) RNA by up to 40-50%. However, many patients still remain non-responders to this treatment for various reasons. The aim of this study was to evaluate the effect of interferon or ribavirin treatment on subgenomic HCV RNA replication in 'non-hepatic' HeLa cells. METHODOLOGY: Huh-7 or HeLa cells harboring HCV replicon were constructed by using cellular RNA of Huh-7 harboring HCV replicon RNAs, named as C13-3 cells. We also tested whether interferon or ribavirin can suppress HCV RNA in HeLa cells. RESULTS: Huh-7 or HeLa cells harboring HCV replicon RNAs were constructed by using cellular RNA of C13-3 cells than using in vitro-transcribed RNA. Ribavirin at 1 microg/mL or 10 microg/mL did not suppress colony formation in HeLa cells, but at 100 microg/mL suppression was observed. Interferon-alpha 2b suppressed HCV replication even at 1 U/mL. CONCLUSIONS: HeLa cells harboring HCV replicon RNAs also might be useful for the development of antiviral drugs.