Hepatitis C virus NS5A protein down-regulates the expression of spindle gene Aspm through PKR-p38 signaling pathway

Hepatitis C virus often causes persistent infection and hepatocellular carcinoma. Studies have demonstrated the roles of viral nonstructural protein 5A (NS5A) in the induction of chromosome aneuploidy, but the molecular mechanisms are not clear. In this study, hydrodynamics-based in vivo transfection was applied to a mouse system. Mouse hepatocytes that successfully expressed NS5A protein were isolated by laser capture microdissection. Gene expression profiles of the NS5A-expressing hepatocytes were examined by an Affymetrix oligonucleotide microarray system. Aspm (abnormal spindle-like, microcephaly associated), which encodes the mitotic spindle protein ASPM, was identified to be differentially expressed in the absence and the presence of NS5A. The down-regulation of Aspm mRNA and ASPM protein was confirmed by real time polymerase chain reaction and Western blot analysis, respectively, both in mouse model systems and in viral subgenomic replicon and in vitro transfection culturing systems. In addition, cultured cells that constitutively expressed NS5A protein showed G(2)/M cell cycle block and chromosome aneuploidy. Overexpression of ASPM relieved the G(2)/M cell cycle block. Furthermore, NS5A protein repressed the promoter activity of Aspm gene in a dose-dependent manner. The regulatory effect was abolished when amino acid substitutions P2209L, T2214A, and T2217G known to interrupt the NS5A-PKR interaction were introduced into the NS5A protein. This indicates that the down-regulation of Aspm expression is via the PKR-p38 signaling pathway. These results suggest that NS5A protein down-regulates the expression of the mitotic spindle protein ASPM and induces aberrant mitotic cell cycle associated with chromosome instability and hepatocellular carcinoma.     

The Hepatitis C virus NS5A protein activates a phosphoinositide 3-kinase-dependent survival signaling cascade

Hepatitis C virus (HCV) establishes a persistent infection, with up to 80% of infected individuals proceeding to chronic hepatitis, which in many cases may result in liver cirrhosis and hepatocellular carcinoma (HCC); indeed HCV infection is increasingly associated with the development of HCC. The long time period (up to 30 years) between primary infection and the onset of HCC implies that HCV is not directly oncogenic but in some way predisposes patients to develop tumors, though the mechanism for this is unclear as yet. We report here that NS5A binds directly to the Src homology 3 domain of the p85 regulatory subunit of phosphoinositide 3-kinase (PI3K), and this interaction is mediated by a novel (non-proline-rich) motif within NS5A. Coimmunoprecipitation analysis revealed that NS5A bound native heterodimeric PI3K and enhanced the phosphotransferase activity of the catalytic (p110) subunit both in vitro and in human cell lines harboring a subgenomic HCV replicon or expressing NS5A alone. NS5A-mediated activation of PI3K resulted in increased phosphorylation and activity of Akt/protein kinase B and concomitantly provided protection against the induction of apoptosis in both replicon-harboring cells and cells stably expressing NS5A alone. These data suggest that stimulation of PI3K by NS5A may represent an indirect mechanism for development of HCC in HCV-infected patients and further suggests potential therapeutic strategies to counteract the occurrence of HCV-related HCC.     

Modulation of the transforming growth factor-beta signal transduction pathway by hepatitis C virus nonstructural 5A protein

Transforming growth factor-beta (TGF-beta) is implicated in the pathogenesis of liver disease. TGF-beta is involved both in liver regeneration and in the fibrotic and cirrhotic transformation with hepatitis viral infection. Hepatitis C virus (HCV) infection often leads to cirrhosis and hepatocellular carcinoma. HCV nonstructural 5A (NS5A) protein is a multifunctional protein that modulates cytokine-mediated signal transduction pathways. To elucidate the molecular mechanism of HCV pathogenesis, we examined the effect of NS5A protein on TGF-beta-stimulated signaling cascades. We show that NS5A protein inhibited the TGF-beta-mediated signaling pathway in hepatoma cell lines as determined by reporter gene assay. To further investigate the role of NS5A, we examined the protein/protein interaction between NS5A and TGF-beta signal transducers. Both in vitro and in vivo binding data showed that NS5A protein directly interacted with TGF-beta receptor I (TbetaR-I) in hepatoma cell lines. This interaction was mapped to amino acids 148-238 of NS5A. We also found that NS5A protein co-localized with TbetaR-I in the cytoplasm of Huh7 cells and inhibited TGF-beta-mediated nuclear translocation of Smad2. Furthermore, we demonstrate that NS5A protein abrogated the phosphorylation of Smad2 and the heterodimerization of Smad3 and Smad4. To further explore the relevance to viral infection, we examined the effect of the HCV subgenomic replicon on the TGF-beta signaling pathway. We show that the HCV subgenomic replicon also inhibited TGF-beta-induced signaling cascades. These results indicate that HCV NS5A modulates TGF-beta signaling through interaction with TbetaR-I and that NS5A may be an important risk factor in HCV-associated liver pathogenesis.     

Epidermal Growth Factor Receptor-Dependent Mutual Amplification between Netrin-1 and the Hepatitis C Virus

Hepatitis C virus (HCV) is an oncogenic virus associated with the onset of hepatocellular carcinoma (HCC). The present study investigated the possible link between HCV infection and Netrin-1, a ligand for dependence receptors that sustains tumorigenesis, in particular in inflammation-associated tumors. We show that Netrin-1 expression is significantly elevated in HCV+ liver biopsies compared to hepatitis B virus (HBV+) and uninfected samples. Furthermore, Netrin-1 was upregulated in all histological stages of HCV+ hepatic lesions, from minimal liver fibrosis to cirrhosis and HCC, compared to histologically matched HCV- tissues. Both cirrhosis and HCV contributed to the induction of Netrin-1 expression, whereas anti-HCV treatment resulted in a reduction of Netrin-1 expression. In vitro, HCV increased the level and translation of Netrin-1 in a NS5A-La-related protein 1 (LARP1)-dependent fashion. Knockdown and forced expression experiments identified the receptor uncoordinated receptor-5 (UNC5A) as an antagonist of the Netrin-1 signal, though it did not affect the death of HCV-infected cells. Netrin-1 enhanced infectivity of HCV particles and promoted viral entry by increasing the activation and decreasing the recycling of the epidermal growth factor receptor (EGFR), a protein that is dysregulated in HCC. Netrin-1 and HCV are, therefore, reciprocal inducers in vitro and in patients, as seen from the increase in viral morphogenesis and viral entry, both phenomena converging toward an increase in the level of infectivity of HCV virions. This functional association involving a cancer-related virus and Netrin-1 argues for evaluating the implication of UNC5 receptor ligands in other oncogenic microbial species.     

Proteomic approach identifies HSP27 as an interacting partner of the hepatitis C virus NS5A protein

Chronic infection by HCV is closely correlated with liver diseases such as cirrhosis, steatosis, and hepatocellular carcinoma. To understand how long-term interaction between HCV and the host leads to pathogenesis, we identified cellular proteins that interact with NS5A and NS5B using a biochemical approach. Stable cell lines that express flag-NS5A or flag-NS5B under tetracycline induction were generated. The induced flag-tagged proteins were immunoprecipitated (IP'd) and associated proteins separated on 2D gels. Protein spots that specifically co-IP'd with NS5A or NS5B were identified by mass spectrometry. HSP27 was identified as a protein that specifically co-IP'd with NS5A but not with NS5B. The N-terminal regions of NS5A (a.a. 1-181) and HSP27 (a.a. 1-122) were defined to be the domains that interact with each other. HSP27 is generally distributed in the cytoplasm. When heat shocked, HSP27 is concentrated in the ER where NS5A is co-localized.     

Correlation between secondary structure of an amino-terminal portion of the nonstructural protein 3 (NS3) of hepatitis C virus and development of hepatocellular carcinoma

Correlation between sequence variation of hepatitis C virus (HCV) and development of hepatocellular carcinoma (HCC) has not yet been demonstrated. In the present study, we analyzed sequence diversity of the NS3 protein of HCV and its possible correlation with HCC. On the basis of secondary structure of an amino-terminal portion of NS3, HCV subtype lb (HCV-1b) isolates were classified into two groups, A and B. Group A isolates were found in 4 (11%) of 36 patients with HCC, and 22 (63%) of 35 patients without HCC. On the other hand, group B isolates were found in 32 (89%) of 36 patients with HCC, and 12 (34%) of 35 patients without HCC. The distribution patterns of those groups were significantly different between patients with and without HCC (P< 0.001). HCV isolates of group B were found in both tumor and adjacent non-tumor tissues obtained from patients with HCC, suggesting that the emergence of group B isolates was not a result of, but rather a possible causative factor for development of HCC. Taken together, our present results suggest that HCV-lb strains of group B are highly associated with HCC and that the secondary structure analysis of NS3 would be useful to predict high risk for development of HCC in HCV-lb-infected patients.     

RacGTPase-activating protein 1 interacts with hepatitis C virus polymerase NS5B to regulate viral replication

Hepatitis C virus (HCV) is a positive-strand RNA virus responsible for chronic liver disease and hepatocellular carcinoma (HCC). RacGTPase-activating protein 1 (RacGAP1) plays an important role during GTP hydrolysis to GDP in Rac1 and CDC42 protein and has been demonstrated to be upregulated in several cancers, including HCC. However, the molecular mechanism leading to the upregulation of RacGAP1 remains poorly understood. Here, we showed that RacGAP1 levels were enhanced in HCV cell-culture-derived (HCVcc) infection. More importantly, we illustrated that RacGAP1 interacts with the viral protein NS5B in mammalian cells. The small interfering RNA (siRNA)-mediated knockdown of RacGAP1 in human hepatoma cell lines inhibited replication of HCV RNA, protein, and production of infectious particles of HCV genotype 2a strain JFH1. Conversely, these were reversed by the expression of a siRNA-resistant RacGAP1 recombinant protein. In addition, viral protein NS5B polymerase activity was significantly reduced by silencing RacGAP1 and, vice versa, was increased by overexpression of RacGAP1 in a cell-based reporter assay. Our results suggest that RacGAP1 plays a crucial role in HCV replication by affecting viral protein NS5B polymerase activity and holds importance for antiviral drug development.     

四环素诱导的丙型肝炎病毒非结构蛋白5B稳定细胞株的构建和检测

丙型肝炎病毒(HCV)感染个体后在宿主细胞内长时间保持低水平复制,与慢性肝炎、肝硬化及肝细胞肝癌的发生密切相关.目前,HCV感染后肝细胞发生转化的具体机制还不清楚.非结构蛋白5B(NS5B)是HCV编码的非结构蛋白之一,具有RNA依赖的RNA聚合酶活性(RdRp),是病毒复制所需的关键酶.除参与病毒复制外,NS5B通过...     

HCV NS3/4A蛋白酶抑制剂细胞筛选模型

丙型肝炎病毒(hepatitis C virus,HCV)持续感染可导致慢性丙型肝炎,并可发展为肝硬化和肝细胞癌。HCV NS3/4A蛋白酶在HCV复制和致病机制中起着非常关键的作用,已成为HCV研究热点。由于候选药物分子必须具有易进入细胞、稳定性好等特征,建立一种细胞水平上酶活性的测定系统对于筛选抗NS3/4A药物无疑有着重要意义。目前已有多种NS3/4A蛋白酶筛选系统开发出来,本文将对此作一综述。     

Hepatitis C virus NS5A and subgenomic replicon activate NF-kappaB via tyrosine phosphorylation of IkappaBalpha and its degradation by calpain protease

Hepatitis C virus nonstructural protein 5A (NS5A) has been implicated in the HCV antiviral resistance, replication, and transactivation of cellular gene expression. We have recently shown that HCV NS5A activates NF-kappaB via oxidative stress (22). In this study, we investigate the molecular mechanism(s) of NF-kappaB activation in response to oxidative stress induced by NS5A protein. In contrast to the classic Ser32,36 phosphorylation of IkappaBalpha, we report here that tyrosine phosphorylation of IkappaBalpha at Tyr42 and Tyr305 residues is induced by the HCV NS5A and the subgenomic replicons in the NF-kappaB activation process. Use of IkappaBalpha-Tyr42,305 double mutant provided the evidence for their key role in the activation of NF-kappaB. Activation of NF-kappaB was blocked by a series of tyrosine kinase inhibitors but not by IkappaB kinase inhibitor BAY 11-7085. More specifically, a ZAP-70 knock-out cell line expressing NS5A and other nonstructural proteins respectively prevented the NF-kappaB activation, indicating the involvement of ZAP-70 as a probable tyrosine kinase in the activation process. Evidence is also presented for the possible role of calpain proteases in the NS5A-induced IkappaBalpha degradation. These studies collectively define an alternate pathway of NF-kappaB activation by NS5A alone or in the context of the HCV subgenomic replicon. Constitutive activation of NF-kappaB by HCV has implications in the chronic liver disease including hepatocellular carcinoma associated with HCV infection.     

Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly

Persistent infection with the hepatitis C virus (HCV) is a major risk factor for the development of liver cirrhosis and hepatocellular carcinoma. With an estimated about 3% of the world population infected with this virus, the lack of a prophylactic vaccine and a selective therapy, chronic hepatitis C currently is a main indication for liver transplantation. The establishment of cell-based replication and virus production systems has led to first insights into the functions of HCV proteins. However, the role of nonstructural protein 5A (NS5A) in the viral replication cycle is so far not known. NS5A is a membrane-associated RNA-binding protein assumed to be involved in HCV RNA replication. Its numerous interactions with the host cell suggest that NS5A is also an important determinant for pathogenesis and persistence. In this study we show that NS5A is a key factor for the assembly of infectious HCV particles. We specifically identify the C-terminal domain III as the primary determinant in NS5A for particle formation. We show that both core and NS5A colocalize on the surface of lipid droplets, a proposed site for HCV particle assembly. Deletions in domain III of NS5A disrupting this colocalization abrogate infectious particle formation and lead to an enhanced accumulation of core protein on the surface of lipid droplets. Finally, we show that mutations in NS5A causing an assembly defect can be rescued by trans-complementation. These data provide novel insights into the production of infectious HCV and identify NS5A as a major determinant for HCV assembly. Since domain III of NS5A is one of the most variable regions in the HCV genome, the results suggest that viral isolates may differ in their level of virion production and thus in their level of fitness and pathogenesis.     

Hepatocellular carcinoma: molecular interactions between hepatitis C virus and p53 in hepatocarcinogenesis

Hepatocellular carcinoma (HCC) is the most important primary hepatic cancer and is a common cancer type worldwide. Many aetiological factors have been related to HCC development, such as liver cirrhosis, hepatitis viruses and alcohol consumption. Inactivation of the p53 tumour suppressor gene is one of the most common abnormalities in many tumours, including HCC. p53 is of crucial importance for the regulation of the cell cycle and the maintenance of genomic integrity. In HCC, hepatitis B and C virus (HBV and HCV) effect carcinogenic pathways, independently leading to anomalies in p53 function. Several authors have reported that some HCV proteins, such as the core, NS5A and NS3 proteins, interact with p53 and prevent its correct function. The mechanisms of action of these HCV proteins in relation to p53 are not completely clear, but they might cause its cytoplasmic retention or accumulation in the perinuclear region where the protein is not functional. The identification of the interactions between p53 and HCV proteins is of great importance for therapeutic strategies aimed at reducing the chronicity and/or carcinogenicity of the virus.     

Antiapoptotic and Oncogenic Potentials of Hepatitis C Virus Are Linked to Interferon Resistance by Viral Repression of the PKR Protein Kinase

Hepatitis C virus (HCV) is prevalent worldwide and has become a major cause of liver dysfunction and hepatocellular carcinoma. The high prevalence of HCV reflects the persistent nature of infection and the large frequency of cases that resist the current interferon (IFN)-based anti-HCV therapeutic regimens. HCV resistance to IFN has been attributed, in part, to the function of the viral nonstructural 5A (NS5A) protein. NS5A from IFN-resistant strains of HCV can repress the PKR protein kinase, a mediator of the IFN-induced antiviral and apoptotic responses of the host cell and a tumor suppressor. Here we examined the relationship between HCV persistence and resistance to IFN therapy. When expressed in mammalian cells, NS5A from IFN-resistant HCV conferred IFN resistance to vesicular stomatitis virus (VSV), which normally is sensitive to the antiviral actions of IFN. NS5A blocked viral double-stranded RNA (dsRNA)-induced PKR activation and phosphorylation of eIF-2alpha in IFN-treated cells, resulting in high levels of VSV mRNA translation. Mutations within the PKR-binding domain of NS5A restored PKR function and the IFN-induced block to viral mRNA translation. The effects due to NS5A inhibition of PKR were not limited to the rescue of viral mRNA translation but also included a block in PKR-dependent host signaling pathways. Cells expressing NS5A exhibited defective PKR signaling and were refractory to apoptosis induced by exogenous dsRNA. Resistance to apoptosis was attributed to an NS5A-mediated block in eIF-2alpha phosphorylation. Moreover, cells expressing NS5A exhibited a transformed phenotype and formed solid tumors in vivo. Disruption of apoptosis and tumorogenesis required the PKR-binding function of NS5A, demonstrating that these properties may be linked to the IFN-resistant phenotype of HCV.     

Raf-1 kinase associates with Hepatitis C virus NS5A and regulates viral replication

Hepatitis C virus (HCV) is a positive-strand RNA virus that frequently causes persistent infection associated with severe liver disease. HCV nonstructural protein 5A (NS5A) is essential for viral replication. Here, the kinase Raf-1 was identified as a novel cellular binding partner of NS5A, binding to the C-terminal domain of NS5A. Raf-1 colocalizes with NS5A in the HCV replication complex. The interaction of NS5A with Raf-1 results in increased Raf-1 phosphorylation at serine 338. Integrity of Raf-1 is crucial for HCV replication: inhibition of Raf-1 by the small-molecule inhibitor BAY43-9006 or downregulation of Raf-1 by siRNA attenuates viral replication.     

The Hepatitis C Virus Non-structural NS5A Protein Impairs Both the Innate and Adaptive Hepatic Immune Response in Vivo

The role of hepatitis C virus (HCV) protein non-structural (NS) 5A in HCV-associated pathogenesis is still enigmatic. To investigate the in vivo role of NS5A for viral persistence and virus-associated pathogenesis a transgenic (Tg) mouse model was established. Mice with liver-targeted NS5A transgene expression were generated using the albumin promoter. Alterations in the hepatic immune response were determined by Western blot, infection by lymphocytic choriomeningitis virus (LCMV), and using transient NS3/4A Tg mice generated by hydrodynamic injection. Cytotoxic T lymphocyte (CTL) activity was investigated by the Cr-release assay. The stable NS5A Tg mice did not reveal signs of spontaneous liver disease. The intrahepatic immunity was disrupted in the NS5A Tg mice as determined by clearance of LCMV infection or transiently NS3/4A Tg hepatocytes in vivo. This impaired immunity was explained by a reduced induction of interferon β, 2′,5′-OAS, and PKR after LCMV infection and an impairment of the CTL-mediated elimination of NS3-expressing hepatocytes. In conclusion, these data indicate that in the present transgenic mouse model, NS5A does not cause spontaneous liver disease. However, we discovered that NS5A could impair both the innate and the adaptive immune response to promote chronic HCV infection.Chronic hepatitis C virus (HCV)⁴ infection is associated with an increased risk of liver cirrhosis and hepatocellular carcinoma (HCC). The HCV genome is a single-stranded positive-sense RNA molecule of ∼9600 bp (1). The viral RNA codes for one large polyprotein of ∼3100 amino acids that is post-translationally processed by cellular and viral proteases, leading to the structural proteins core, E1 and E2, the p7 protein, and the non-structural proteins NS2, NS3, NS4A, NS4B, NS5A, and NS5B (2). The mature NS5A protein is generated by the action of the NS3/NS4A serine protease. NS5A is a phosphoprotein that exists in a basal or in a hyperphosphorylated state (p56 and p58) (3). Through an amphipathic α-helix, NS5A is associated with the cytoplasmic face of the ER (4) and is an integral part of the replication complex (5). Mutations in NS5A affect the rate of HCV replication suggesting a role of NS5A in modulating viral expression and replication (6). Moreover, NS5A is able to interfere with a variety of cellular proteins. Some of these interaction partners, such as Grb2, PI3K, p53, or Raf-1 are important key players in host cell signal transduction, enabling NS5A to deregulate important cellular check points (7–10). Recent reports even suggest that NS5A may deregulate cell cycle progression by modulating the expression of cell cycle regulatory genes (11). In light of these observations and that it has been suggested to transform murine fibroblasts (12), it is speculated that NS5A could represent an important factor for the development of HCV-associated HCC (13).Infection of transgenic mice expressing the complete HCV polyprotein with lymphocytic choriomeningitis virus (LCMV) showed a reduced IFN response and a delayed viral elimination (14). Cell culture-based experiments have shown that NS5A interacts directly with the interferon-dependent induced protein kinase R (PKR), a key player in the cellular antiviral response and that this interaction results in an inhibition of PKR function (15). Therefore, a role of NS5A for the establishment of a chronic HCV infection by inhibiting the innate immunity is conceivable.To enable in vivo studies of NS5A-specific effects transgenic mice were generated with a liver-specific expression of NS5A. We used these mice to show that NS5A affects both the innate and the adaptive hepatic immunity.