Hepatitis C virus inhibits intracellular interferon alpha expression in human hepatic cell lines

The chronicity of hepatitis C virus (HCV) infection raises the question of how HCV is able to persist in hepatic cells. We show that human primary hepatocytes and human hepatic cell lines (Huh7 and HepG2) spontaneously produce interferon (IFN)-alpha that is inhibited in the HCV replicon cells (Huh.8 and FCA-1). Silencing IFN-alpha gene expression by IFN-alpha small interfering RNA (siRNA) in the HCV replicon cells resulted in increased HCV replicon expression. The activation of IFN-alpha expression by interferon regulatory factor (IRF-7) led to the inhibition of HCV replicon expression, whereas the anti-IFN-alpha receptor antibody could partially block IRF-7-mediated HCV replicon inhibition. In addition, the blockade of IFN-alpha receptor by anti-IFN-alpha receptor antibody on the replicon cells increased HCV replicon expression. Among the HCV nonstructural (NS) proteins tested, NS5A is the most potent inhibitor of IFN-alpha expression by the hepatic cells. Investigation of the mechanism of HCV action on IFN-alpha showed that IRF-7-induced IFN-alpha promoter activation was inhibited in the HCV replicon cells. Furthermore, IRF-7 expression was restricted in the HCV replicon cells. In conclusion, we provide direct evidence that HCV undermines the intracellular innate immunity of the target cells, which may account for HCV persistence in hepatic cells.     

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.     

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.     

CD8+ T cell depletion amplifies hepatitis C virus replication in peripheral blood mononuclear cells

We investigated the ability of CD8+ T cells to inhibit hepatitis C virus (HCV) replication in peripheral blood mononuclear cells (PBMCs). PBMCs isolated from 11 of 20 HCV-infected subjects had no detectable HCV RNA. Removal of CD8+ T cells from these PBMCs resulted in detection of HCV RNA, and depletion of CD8+ T cells from PBMCs that had detectable HCV RNA amplified HCV replication. Reconstitution of CD8- PBMCs with autologous CD8+ T cells led to inhibition of HCV replication. Interferon-gamma produced by CD8+ T cells was partially responsible for CD8+ T cell-mediated noncytotoxic anti-HCV activity in PBMCs. This noncytotoxic anti-HCV activity was confirmed in HCV replicon cells. Supernatants from CD8+ T cell cultures inhibited HCV RNA expression in the replicon cells. These findings may have important implications for the immunopathogenesis of HCV in both immune and hepatic cells and are relevant to the development of host innate immunity-based anti-HCV interventions.     

Cyclosporin A suppresses replication of hepatitis C virus genome in cultured hepatocytes

Persistent infection of hepatitis C virus (HCV) is a major cause of liver diseases such as chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma. Searching for a substance with anti-HCV potential, we examined the effects of a variety of compounds on HCV replication using a HCV subgenomic replicon cell culture system. Consequently, the immunosuppressant cyclosporin A (CsA) was found to have a suppressive effect on the HCV replicon RNA level and HCV protein expression in these cells. CsA also inhibited multiplication of the HCV genome in a cultured human hepatocyte cell line infected with HCV using HCV-positive plasma. This anti-HCV activity of CsA appeared to be independent of its immunosuppressive function. In conclusion, our results suggest that CsA may represent a new approach for the development of anti-HCV therapy.     

Acetylsalicylic acid inhibits hepatitis C virus RNA and protein expression through cyclooxygenase 2 signaling pathways

It has been reported that salicylates (sodium salicylate and aspirin) inhibit the replication of flaviviruses, such as Japanese encephalitis virus and dengue virus. Therefore, we considered it important to test whether acetylsalicylic acid (ASA) had anti-hepatitis C virus (HCV) activity. To this end, we examined the effects of ASA on viral replication and protein expression, using an HCV subgenomic replicon cell culture system. We incubated Huh7 replicon cells with 2-8 mM ASA for different times and measured HCV-RNA and protein levels by northern blot, real-time polymerase chain reaction, and western analysis, respectively. We found that ASA had a suppressive effect on HCV-RNA and protein levels (nearly 58%). ASA-dependent inhibition of HCV expression was not mediated by the 5'-internal ribosome entry site or 3'-untranslated regions, as determined by transfection assays using bicistronic constructs containing these regulatory regions. However, we found that HCV-induced cyclooxygenase 2 (COX-2) messenger RNA and protein levels and activity and these effects were down-regulated by ASA, possibly by a nuclear factor kappa B-independent mechanism. We also observed that the ASA-dependent inhibition of viral replication was due in part to inhibition of COX-2 and activation of p38 and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2) mitogen-activated protein kinases (MAPKs). Inhibition of these kinases by SB203580 and U0126, respectively, and by short interfering RNA silencing of p38 and MEK1 MAPK prevented the antiviral effect of ASA. Taken together, our findings suggest that the anti-HCV effect of ASA in the Huh7 replicon cells is due to its inhibitory effect on COX-2 expression, which is mediated in part by the activation of MEK1/2/p38 MAPK. CONCLUSION: These findings suggest the possibility that ASA could be an excellent adjuvant in the treatment of chronic HCV infection.     

Hepatitis C and Alcohol

BACKGROUND: Alcohol abuse and hepatitis C virus (HCV) infection frequently coexist in patients with chronic liver disease. It is widely believed that alcohol and HCV act synergistically in these patients to promote the development and progression of liver damage. METHODS: A review of the relevant medical literature, identified by computer assisted literature search, was conducted. RESULTS: It has been established that alcohol consumption is associated with the accelerated progression of liver injury, higher frequency of cirrhosis, and higher incidence of hepatocellular carcinoma. Alcohol abuse is also associated with decreased response to interferon treatment, and there are reports to suggest that patients with HCV cirrhosis, who abuse alcohol, have higher mortality than those who do not. Abstinence may reverse some of these deleterious effects of alcohol, and may even improve the ultimate response to treatment. The mechanism for the synergistic effect of alcohol and HCV is not fully understood, but has been attributed to alcohol's effect on viral replication, or to its effect on the immune system, hepatic iron content, or hepatic regeneration. CONCLUSIONS: Alcohol has a deleterious effect on HCV associated liver disease. It is recommended that patients with HCV infection abstain from alcohol consumption.     

Hepatitis C virus infection in patients with clinically diagnosed alcoholic liver diseases

Summary To determine the incidence of hepatitis C virus (HCV) infection in patients with alcoholic liver disease (AID), serum samples from 252 patients with AID were tested for anti-HCV and HCV RNA. Serial sera of these patients were collected and stored under optimal conditions to allow exact quantification of HCV RNA. Fifteen patients who visited our hospital during the same period of time with chronic HCV infections served as controls. In those with AID, anti-HCV and HCV RNA were positive in 55.5% and 41.2%, respectively. Patients with histologically diagnosed chronic hepatitis and hepatocellular carcinoma had much higher prevalence rates of HCV RNA (84% and 100%, respectively) compared to those with fatty liver (4.3%), hepatic fibrosis (10.1%) and alcoholic hepatitis (22.2%) ( P < 0.01). Although no difference in serum HCV RNA levels was observed between the patients with both AID and chronic HCV infection and those with chronic HCV infection alone, HCV RNA levels significantly (10-fold) dropped after abstinence in nearly half of the patients ( P < 0.01). These data indicate that HCV infection in patients with AID promotes progression of liver disease, and abstinence from alcohol is associated with a reduction in serum HCV RNA levels.     

干扰素γ对丙型肝炎病毒复制子的抑制作用

目的研究干扰素（IFN）γ直接抑制丙型肝炎病毒（HCV）复制子的作用及其与IFN α联合用药的疗效，并对可能介导IFN γ抗HCV作用的干扰素刺激基因（ISG）的表达水平进行探讨。方法建立HCV复制子细胞模型，用IFN γ或IFN γ联合IFN α处理HCV复制子细胞，通过半定量逆转录聚合酶链反应（RT-PCR）及实时定量PCR检测细胞中HCV RNA水平；western blot检测细胞中HCV非结构蛋白5A（NS5A）。结果IFN γ对HCV RNA的复制及NS5A的表达有明显抑制作用，10U／ml IFN γ可使HCV RNA较对照组减少36％，25U／ml减少80％；IFN γ对HCV RNA及NS5A蛋白的抑制作用与其剂量及作用时间呈正相关。用IFN γ预处理的细胞对IFN α抗HCV的作用更敏感，且细胞内干扰素调节因子-1（IRF-1）、干扰素刺激基因因子3 γ（ISGF3 γ）和2’5’-寡腺苷酸合成酶（2’5’-OAS）呈明显的诱导性表达。结论IFN γ能有效抑制HCV RNA的复制，并与IFN α有协同抗HCV的作用。IRF-1、ISGF3 γ和2’5’-OAS可能参与介导IFN γ抗HCV的作用。     

Toll-like receptor 4 mediates synergism between alcohol and HCV in hepatic oncogenesis involving stem cell marker Nanog

Alcohol synergistically enhances the progression of liver disease and the risk for liver cancer caused by hepatitis C virus (HCV). However, the molecular mechanism of this synergy remains unclear. Here, we provide the first evidence that Toll-like receptor 4 (TLR4) is induced by hepatocyte-specific transgenic (Tg) expression of the HCV nonstructural protein NS5A, and this induction mediates synergistic liver damage and tumor formation by alcohol-induced endotoxemia. We also identify Nanog, the stem/progenitor cell marker, as a novel downstream gene up-regulated by TLR4 activation and the presence of CD133/Nanog-positive cells in liver tumors of alcohol-fed NS5A Tg mice. Transplantation of p53-deficient hepatic progenitor cells transduced with TLR4 results in liver tumor development in mice following repetitive LPS injection, but concomitant transduction of Nanog short-hairpin RNA abrogates this outcome. Taken together, our study demonstrates a TLR4-dependent mechanism of synergistic liver disease by HCV and alcohol and an obligatory role for Nanog, a TLR4 downstream gene, in HCV-induced liver oncogenesis enhanced by alcohol.     

Activation of anti-hepatitis C virus responses via Toll-like receptor 7

IFN-alpha is used to suppress the replication of hepatitis C virus (HCV) in chronically infected patients with partial success. Here we present evidence showing that a ligand of Toll-like receptor 7 (TLR7) can induce anti-HCV immunity not only by IFN induction, but also through an IFN-independent mechanism. Human hepatocyte line Huh-7 carrying an HCV replicon expressed TLR7, and activation of the receptor induced several antiviral genes including IFN regulatory factor-7. Inhibitors of the enzyme inosine monophosphate dehydrogenase augmented both IFN-dependent and -independent antiviral effect. Prolonged exposure of Huh-7 cells to a TLR7 ligand [SM360320 (9-benzyl-8-hydroxy-2-(2-methoxyethoxy)adenine)], alone or in combination with an inosine monophosphate dehydrogenase inhibitor, reduced HCV levels dose dependently. Immunohistochemical analysis of livers shows that TLR7 is expressed in hepatocytes of normal or HCV-infected people. Because TLR7 agonists can impede HCV infection both via type I IFN and independently of IFN, they may be considered as an alternative treatment of chronic HCV infection, especially in IFN-alpha-resistant patients.     

The impact of steatosis and alcohol on hepatitis C

Alcohol consumption and hepatic steatosis interact synergistically with hepatitis C virus (HCV) to accelerate fibrosis progression and reduce the efficacy of standard antiviral therapies. Research aimed at delineating the viral and host interactions involved in the pathogenesis of steatosis in HCV infection may provide novel therapeutic strategies that can modify disease progression and improve treatment response. This review discusses the clinical aspects of HCV fibrosis progression and treatment response in the setting of alcohol use or hepatic steatosis.     

Alcohol and hepatitis C

Alcohol abuse and hepatitis C virus (HCV) infection coexist with chronic liver disease in many patients. The mechanism of injury in these patients is probably multifactorial and involves, but is not limited to, a combination of diminished immune clearance of HCV, oxidative stress, emergence of HCV quasi-species, hepatic steatosis, increased iron stores, and increased rate of hepatocyte apoptosis. In patients with HCV infection, alcohol consumption is known to cause accelerated progression of liver fibrosis, higher frequency of cirrhosis, and increased incidence of hepatocellular carcinoma (HCC). These patients also have decreased survival as compared with patients with either alcohol abuse or HCV liver injury alone. Alcohol abuse causes decreased response to interferon treatment in HCV patients. It is therefore necessary for patients with HCV infection to abstain from alcohol consumption.     

Alcohol metabolism increases the replication of hepatitis C virus and attenuates the antiviral action of interferon

The interactions between hepatitis C virus (HCV) and alcohol metabolism are not well understood. To determine the effect that alcohol metabolism has on HCV replication and the antiviral action of interferon (IFN), Huh-7 cells that harbor HCV replication and metabolize ethanol via the introduced expression of cytochrome P450 2E1 (Cyp2e1) were treated with ethanol and IFN-alpha. Treatment of these cells with ethanol (0-100 mmol/L) significantly increased HCV replication. This effect was dependent on Cyp2e1 expression and alcohol-metabolized oxidative stress (OS), because the antioxidant N-acetylcysteine blocked this effect. Furthermore, the anti-HCV action of IFN-alpha was attenuated in the presence of ethanol metabolism, most likely via attenuation of Stat1 tyrosine-701 phosphorylation. These in vitro results mimic what is often noted clinically, and further dissection of this model system will aid in our understanding of interactions between HCV and alcohol metabolism.     

Alcohol and hepatitis C virus core protein additively increase lipid peroxidation and synergistically trigger hepatic cytokine expression in a transgenic mouse model

BACKGROUND/AIMS: Alcohol consumption accelerates the appearance of liver fibrosis and hepatocellular carcinoma in patients with chronic hepatitis C virus (HCV) infection, but the mechanisms of these interactions are unknown. We therefore investigated the effects of chronic ethanol consumption in HCV core protein-expressing transgenic mice. METHODS: Ethanol was progressively added (up to 20%) to the drinking water that was given ad libidum. RESULTS: In vivo fatty acid oxidation was not inhibited by ethanol consumption and/or HCV core expression. Both chronic ethanol consumption and HCV core expression decreased hepatic lipoprotein secretion and caused steatosis, but had no additive effects on lipoprotein secretion or steatosis. However, chronic ethanol consumption and HCV core protein additively increased lipid peroxidation and acted synergistically to increase the hepatic expression of transforming growth factor-beta (TGF-beta) and, to a less extent, tumor necrosis factor-alpha (TNF-alpha). CONCLUSIONS: HCV core protein expression and chronic alcohol consumption have no effects on in vivo fatty acid oxidation and do not additively impair hepatic lipoprotein secretion, but additively increase hepatic lipid peroxidation and synergistically increase hepatic TNF-alpha and TGF-beta expression. These effects may be involved in the activation of fibrogenesis and the development of hepatocellular carcinoma in patients cumulating alcohol abuse and HCV infection.