丙型肝炎病毒高水平复制细胞株的构建及其机制初步研究

旨在探讨丙型肝炎病毒(hepatitis C virus, HCV)cured细胞株的易感机制。本研究将体外转录的HCV RNA电转入肝癌细胞系Huh 7细胞,建立HCV复制子细胞株,用 γ-干扰素(interferon,IFN)处理复制子细胞株,获得HCV cured Huh 7A和Huh 7B细胞株。用插入报告基因的HCV毒株Jc1-G感染上述细胞株,分别进行荧光素酶活性测定、蛋白质印迹法和荧光定量聚合酶链反应(polymerase chain reaction,PCR)检测以验证其易感性。收集Huh 7、Huh 7.5、Huh 7A和Huh 7B细胞并利用IFN-α处理,之后用蛋白质印迹法及荧光定量PCR进行检测,验证细胞株中IFN诱生信号通路中关键因子内源性表达及抗病毒活性ISGs的激活水平。结果显示,在Huh 7A和Huh 7B细胞中检测不到病毒RNA,与Huh 7细胞一致。病毒感染实验中,与Huh 7细胞相比,Huh 7A和Huh 7B细胞株中荧光素酶活性增高百倍,病毒蛋白表达和RNA水平亦显著上调,与Huh 7.5细胞株中的表达水平接近。IFN信号通路实验中,与Huh 7细胞相比,Huh 7A和Huh 7B细胞株中RIG-I/MDA5/MAVS内源性蛋白表达和mRNA水平无明显差异;IFN-α处理细胞后IFN刺激基因isg56,mx1,mx2,oax1,oax2,viperin,cxcl10,ifitm1和ifitm3激活水平亦无显著变化。结果提示,本研究制备的Huh 7A和Huh 7B细胞株可支持HCV高水平复制,将为研究病毒复制机制提供有力的支持。     

Subgenomic HCV RNA replication and its localization in the nucleus of the infected cells

Cell culture systems have been established, where a hepatitis C virus (HCV) subgenomic replicon was efficiently replicated and maintained for a long period. It is known that HCV contains proteins which interact with host cell proteins.To see whether a HCV RNA replicon can interact in the same way with host cell proteins, HCV RNA replicon was transfected in Huh7 cells. In most infected cells, HCV replicon is present in the cytoplasm; however, in a minority of HCV-infected cells, both the cytoplasm and the nucleus or the nucleus on its own is positive for NS3. The presence of NS3 in the nuclei of Huh7 cells indicates that the protein may play a role other than in virus replication, such as in persistence of HCV infection.Keyword: Hepatitis C Virus     

Replication of hepatitis C virus (HCV) RNA in mouse embryonic fibroblasts: protein kinase R (PKR)-dependent and PKR-independent mechanisms for controlling HCV RNA replication and mediating interferon activities

Hepatitis C virus (HCV) infection causes chronic hepatitis and is currently treated with alpha interferon (IFN-alpha)-based therapies. The underlying mechanisms of chronic HCV infection and IFN-based therapies, however, have not been defined. Protein kinase R (PKR) was implicated in the control of HCV replication and mediation of IFN-induced antiviral response. In this report, we demonstrate that a subgenomic RNA replicon of genotype 2a HCV replicated efficiently in mouse embryonic fibroblasts (MEFs), as determined by cell colony formation efficiency and the detection of HCV proteins and both positive- and negative-strand RNAs. Additionally, the subgenomic HCV RNA was found to replicate more efficiently in the PKR knockout (PKR(-/-)) MEF than in the wild-type (PKR(+/+)) MEF. The knockdown expression of PKR by specific small interfering RNAs significantly enhanced the level of HCV RNA replication, suggesting that PKR is involved in the control of HCV RNA replication. The level of ISG56 (p56) was induced by HCV RNA replication, indicating the activation of PKR-independent antiviral pathways. Furthermore, IFN-alpha/beta inhibited HCV RNA replication in PKR(-/-) MEFs as efficiently as in PKR(+/+) MEFs. These findings demonstrate that PKR-independent antiviral pathways play important roles in controlling HCV replication and mediating IFN-induced antiviral effect. Our findings also provide a foundation for the development of transgenic mouse models of HCV replication and set a stage to further define the roles of cellular genes in the establishment of chronic HCV infection and the mediation of intracellular innate antiviral response by using MEFs derived from diverse gene knockout animals.     

Alpha interferon induces distinct translational control programs to suppress hepatitis C virus RNA replication

Hepatitis C virus (HCV) infection is treated with interferon (IFN)-based therapy. The mechanisms by which IFN suppresses HCV replication are not known, and only limited efficacy is achieved with therapy because the virus directs mechanisms to resist the host IFN response. In the present study we characterized the effects of IFN action upon the replication of two distinct quasispecies of an HCV replicon whose encoded NS5A protein exhibited differential abilities to bind and inhibit protein kinase R (PKR). Metabolic labeling experiments revealed that IFN had little overall effect upon HCV protein stability or polyprotein processing but specifically blocked translation of the HCV RNA, such that the replication of both viral quasispecies was suppressed by IFN treatment of the Huh7 host cells. However, within cells expressing an NS5A variant that inhibited PKR, we observed a reduced level of eukaryotic initiation factor 2 alpha subunit (eIF2alpha) phosphorylation and a concomitant increase in HCV protein synthetic rates, enhancement of viral RNA replication, and a partial rescue of viral internal ribosome entry site (IRES) function from IFN suppression. Assessment of the ribosome distribution of the HCV replicon RNA demonstrated that the NS5A-mediated block in eIF2alpha phosphorylation resulted in enhanced recruitment of the HCV RNA into polyribosome complexes in vivo but only partially rescued the RNA from polyribosome dissociation induced by IFN treatment. Examination of cellular proteins associated with HCV-translation complexes in IFN-treated cells identified the P56 protein as an eIF3-associated factor that fractionated with the initiator ribosome-HCV RNA complex. Importantly, we found that P56 could independently suppress HCV IRES function both in vitro and in vivo, but a mutant P56 that was unable to bind eIF3 had no suppressive action. We conclude that IFN blocks HCV replication through translational control programs involving PKR and P56 to, respectively, target eIF2- and eIF3-dependent steps in the viral RNA translation initiation process.     

Interleukin-1 inhibits hepatitis C virus subgenomic RNA replication by activation of extracellular regulated kinase pathway

Interleukin-1 (IL-1) plays an important role in the inflammatory process. Some studies have demonstrated that IL-1 production was impaired in patients with chronic infections of hepatitis C virus (HCV), implying that IL-1 may play a role in viral clearance. Using an HCV subgenomic replicon cell line, we demonstrate that IL-1 can effectively inhibit HCV subgenomic RNA replication and viral protein expression, suggesting that IL-1 has direct antiviral activity. The inhibitory effect is associated with the extracellular regulatory kinase (ERK) activation. In addition, we also show that IL-1 can induce one of the interferon-stimulated genes (ISGs), 1-8U, which exhibits antiviral activity. However, it has no effect on the other ISG, 6-16, suggesting that IL-1 induces novel antiviral pathways within a cell.     

Replication studies using genotype 1a subgenomic hepatitis C virus replicons

Recently, cell-based replicon systems for hepatitis C virus (HCV), in which the nonstructural proteins stably replicate subgenomic viral RNA in Huh7 cells, were developed. To date, one limitation of using these replicon systems to advance drug discovery is the inability of other genotypic derivatives, beyond those of two distinct strains of genotype 1b (HCV-N and Con1), to stably replicate in Huh7 cells. In this report, we evaluated a series of replicon genotype 1a-1b chimeras, as well as a complete genotype 1a replicon clone. A subgenomic replicon construct containing only type 1a sequences failed to generate stable colonies in Huh7 cells even after repeated attempts. Furthermore, addition of an NS5A adaptive mutation (S2204I) which enhances type 1b replicon efficiency was insufficient to confer replication to the wild-type 1a replicon. This subgenomic replicon was subsequently found to be inefficiently translated in Huh7 cells compared to a type 1b replicon, and the attenuation of translation mapped to the N-terminal region of NS3. Therefore, to ensure efficient translation and thereby support replication of the 1a genome, the coding sequence for first 75 residues from type 1a were replaced with the type 1b (strain Con 1) NS3 coding sequence. Although nonstructural proteins were expressed at lower levels with this replicon than with type 1b and although the amount of viral RNA was also severalfold lower (150 copies of positive-strand RNA per cell), the replicon stably replicated in Huh7 cells. Notwithstanding this difference, the ratio of positive- to negative-strand RNA of 26 was similar to that found with the type 1b replicon. Similar results were found for a 1b replicon expressing the type 1a RNA-dependent RNA polymerase. These 1a hybrid replicons maintained sensitivity to alpha interferon (IFN-alpha), albeit with an eightfold-higher 50% inhibitory concentration than type 1b replicons. Evidence is provided herein to confirm that this differential response to IFN-alpha may be attributed directly to the type 1a polymerase.     

Effect of alpha interferon on the hepatitis C virus replicon

Chronic hepatitis C virus (HCV) infections can be cured only in a fraction of patients treated with alpha interferon (IFN-alpha) and ribavirin combination therapy. The mechanism of the IFN-alpha response against HCV is not understood, but evidence for a role for viral nonstructural protein 5A (NS5A) in IFN resistance has been provided. To elucidate the mechanism by which NS5A and possibly other viral proteins inhibit the cellular antiviral program, we have constructed a subgenomic replicon from a known infectious HCV clone and demonstrated that it has an approximately 1,000-fold-higher transduction efficiency than previously used subgenomes. We found that IFN-alpha reduced replication of HCV subgenomic replicons approximately 10-fold. The estimated half-life of viral RNA in the presence of the cytokine was about 12 h. HCV replication was sensitive to IFN-alpha independently of whether the replicon expressed an NS5A protein associated with sensitivity or resistance to the cytokine. Furthermore, our results indicated that HCV replicons can persist in Huh7 cells in the presence of high concentrations of IFN-alpha. Finally, under our conditions, selection for IFN-alpha-resistant variants did not occur.     

Relationships between hepatitis C virus replication and CXCL-8 production in vitro

The chemokine CXCL-8 (interleukin-8) is induced by many viruses, including hepatitis C virus (HCV). In the current study, we examined CXCL-8 levels in the context of acute and chronic HCV replication in vitro. Two different small interfering RNAs were used to silence CXCL-8 mRNA and protein expression in Huh7 and BB7 replicon cells. HCV RNA synthesis in BB7 cells was inhibited by CXCL-8 knockdown. Furthermore, antibody neutralization of endogenous CXCL-8 activity inhibited HCV replication, while addition of recombinant human CXCL-8 stimulated NS5A protein expression. Moreover, CXCL-8 protein levels correlated positively with HCV RNA levels in four independent subgenomic and genomic replicon lines (R = 0.41, P = 0.0013). However, CXCL-8 mRNA levels correlated inversely with CXCL-8 protein and HCV RNA levels in all replicon lines and in Huh7 cells. Transient replication assays with strongly permissive and weakly permissive Huh7 cells and three independent subgenomic replicons with various replicative capacities revealed that CXCL-8 protein levels were higher in weakly than in strongly permissive cells. The JFH-1 subgenomic replicon, which replicated to high levels in both strongly and weakly permissive Huh7 cells, induced CXCL-8 protein to high levels in both cell types. The data indicate that in the replicon system, CXCL-8 protein levels are positively associated with chronic HCV replication and that CXCL-8 removal inhibits HCV replication. During acute HCV replication, CXCL-8 production may be inhibitory to viruses with low replicative capacity. The data underscore the complex regulation of CXCL-8 mRNA and protein expression and further suggest that in addition to contributing to HCV pathology via proinflammatory actions, CXCL-8 may have opposing antiviral and proviral effects depending on the level of HCV replication, the cellular context, and whether the infection is acute or chronic.     

Production of infectious hepatitis C virus in cell culture

Hepatitis C virus (HCV) is a major public health problem, infecting an estimated 170 million people worldwide. Current therapy for HCV-related chronic hepatitis is based on the use of interferon. However, virus clearance rates are insufficient. Investigations to develop the anti-viral therapy or to understand the life cycle of this virus have been hampered by the lack of viral culture systems. We isolated the JFH-1 strain from a patient with fulminant hepatitis, and the JFH-1 subgenomic replicon could replicate efficiently in culture cell without adaptive mutation. Recently, we developed the HCV infection system in culture cells with this JFH-1 strain. The full-length JFH-1 RNA was transfected into Huh7 cells. Subsequently, viral RNA efficiently replicated in transfected cells and viral particles were secreted. Furthermore, secreted virus displayed infectivity for naive Huh7 cells. This system provides a powerful tool for studying the viral life cycle and constructing anti-viral strategies.     

Establishment of a subgenomic replicon for bovine viral diarrhea virus in Huh-7 cells and modulation of interferon-regulated factor 3-mediated antiviral response

We describe the development of a selectable, bi-cistronic subgenomic replicon for bovine viral diarrhea virus (BVDV) in Huh-7 cells, similar to that established for hepatitis C virus (HCV). The selection marker and reporter (Luc-Ubi-Neo) in the BVDV replicon was fused with the amino-terminal protease N(pro), and expression of the nonstructural proteins (NS3 to NS5B) was driven by an encephalomyocarditis virus internal ribosome entry site. This BVDV replicon allows us to compare RNA replication of these two related viruses in a similar cellular background and to identify antiviral molecules specific for HCV RNA replication. The BVDV replicon showed similar sensitivity as the HCV replicon to interferons (alpha, beta, and gamma) and 2'-beta-C-methyl ribonucleoside inhibitors. Known nonnucleoside inhibitor molecules specific for either HCV or BVDV can be easily distinguished by using the parallel replicon systems. The HCV replicon has been shown to block, via the NS3/4A serine protease, Sendai virus-induced activation of interferon regulatory factor 3 (IRF-3), a key antiviral signaling molecule. Similar suppression of IRF-3-mediated responses was also observed with the Huh-7-BVDV replicon but was independent of NS3/4A protease activity. Instead, the amino-terminal cysteine protease N(pro) of BVDV appears to be, at least partly, responsible for suppressing IRF-3 activation induced by Sendai virus infection. This result suggests that different viruses, including those closely related, may have developed unique mechanisms for evading host antiviral responses. The parallel BVDV and HCV replicon systems provide robust counterscreens to distinguish viral specificity of small-molecule inhibitors of viral replication and to study the interactions of the viral replication machinery with the host cell innate immune system.     

Lipid Droplet-Binding Protein TIP47 Regulates Hepatitis C Virus RNA Replication through Interaction with the Viral NS5A Protein

The nonstructural protein NS5A has emerged as a new drug target in antiviral therapies for Hepatitis C Virus (HCV) infection. NS5A is critically involved in viral RNA replication that takes place at newly formed membranes within the endoplasmic reticulum (membranous web) and assists viral assembly in the close vicinity of lipid droplets (LDs). To identify host proteins that interact with NS5A, we performed a yeast two-hybrid screen with the N-terminus of NS5A (amino acids 1–31), a well-studied α-helical domain important for the membrane tethering of NS5A. Our studies identified the LD-associated host protein, Tail-Interacting Protein 47 (TIP47) as a novel NS5A interaction partner. Coimmunoprecipitation experiments in Huh7 hepatoma cells confirmed the interaction of TIP47 with full-length NS5A. shRNA-mediated knockdown of TIP47 caused a more than 10-fold decrease in the propagation of full-length infectious HCV in Huh7.5 hepatoma cells. A similar reduction was observed when TIP47 was knocked down in cells harboring an autonomously replicating HCV RNA (subgenomic replicon), indicating that TIP47 is required for efficient HCV RNA replication. A single point mutation (W9A) in NS5A that disrupts the interaction with TIP47 but preserves proper subcellular localization severely decreased HCV RNA replication. In biochemical membrane flotation assays, TIP47 cofractionated with HCV NS3, NS5A, NS5B proteins, and viral RNA, and together with nonstructural viral proteins was uniquely distributed to lower-density LD-rich membrane fractions in cells actively replicating HCV RNA. Collectively, our data support a model where TIP47—via its interaction with NS5A—serves as a novel cofactor for HCV infection possibly by integrating LD membranes into the membranous web.     

Hemin potentiates the anti-hepatitis C virus activity of the antimalarial drug artemisinin

We report that the antimalarial drug artemisinin inhibits hepatitis C virus (HCV) replicon replication in a dose-dependent manner in two replicon constructs at concentrations that have no effect on the proliferation of the exponentially growing host cells. The 50% effective concentration (EC(50)) for inhibition of HCV subgenomic replicon replication in Huh 5-2 cells (luciferase assay) by artemisinin was 78+/-21 microM. Hemin, an iron donor, was recently reported to inhibit HCV replicon replication [mediated by inhibition of the viral polymerase (C. Fillebeen, A.M. Rivas-Estilla, M. Bisaillon, P. Ponka, M. Muckenthaler, M.W. Hentze, A.E. Koromilas, K. Pantopoulos, Iron inactivates the RNA polymerase NS5B and suppresses subgenomic replication of hepatitis C virus, J. Biol. Chem. 280 (2005) 9049-9057.)] at a concentration that had no adverse effect on the host cells. When combined, artemisinin and hemin resulted, over a broad concentration range, in a pronounced synergistic antiviral activity. Also at a concentration (2 microM) that alone had no effect on HCV replication, hemin still potentiated the anti-HCV activity of artemisinin.     

Subproteomic study of hepatitis C virus replicon reveals Ras-GTPase-activating protein binding protein 1 as potential HCV RC component

Hepatitis C virus (HCV) RNA synthesis takes place on a detergent resistant membrane (DRM) structure. To identify potential cellular proteins related to HCV replication complexes (RC), we purified DRMs from HCV subgenomic replicon cells and its parental Huh7 cells. The proteins of DRM fractions were separated by two-dimensional gel electrophoresis and identified by mass spectrometry. Comparing with parental Huh7 cells, 60 proteins were up-regulated while 14 proteins were down-regulated in HCV replicon cells. Ras-GTPase-activating protein binding protein 1 (G3BP1), one of the elevated proteins, was found to be associated with HCV NS5B and knockdown of G3BP1 by siRNA in HCV replicon cells significantly reduced HCV replication, which may indicate it a potential component of HCV RC. These results suggest that HCV viral gene and proteins may regulate the presence of host cellular proteins in DRM, ensure appropriate concentrations of replication components, and hence control the rates or efficiencies of HCV replication.     

Azasugar-Containing Phosphorothioate Oligonucleotide (AZPSON) DBM-2198 Inhibits Human Immunodeficiency Virus Type 1 (HIV-1) Replication by Blocking HIV-1 gp120 without Affecting the V3 Region

DBM-2198, a six-membered azasugar nucleotide (6-AZN)-containing phosphorothioate (P = S) oligonucleotide (AZPSON), was described in our previous publication [Lee et al. (2005)] with regard to its antiviral activity against a broad spectrum of HIV-1 variants. This report describes the mechanisms underlying the anti-HIV-1 properties of DBM-2198. The LTR-mediated reporter assay indicated that the anti-HIV-1 activity of DBM-2198 is attributed to an extracellular mode of action rather than intracellular sequence-specific antisense activity. Nevertheless, the antiviral properties of DBM-2198 and other AZPSONs were highly restricted to HIV-1. Unlike other P = S oligonucleo-tides, DBM-2198 caused no host cell activation upon administration to cultures. HIV-1 that was pre-incubated with DBM-2198 did not show any infectivity towards host cells whereas host cells pre-incubated with DBM-2198 remained susceptible to HIV-1 infection, suggesting that DBM-2198 acts on the virus particle rather than cell surface molecules in the inhibition of HIV-1 infection. Competition assays for binding to HIV-1 envelope protein with anti-gp120 and anti-V3 antibodies revealed that DBM-2198 acts on the viral attachment site of HIV-1 gp120, but not on the V3 region. This report provides a better understanding of the antiviral mechanism of DBM-2198 and may contribute to the development of a potential therapeutic drug against a broad spectrum of HIV-1 variants.     

The level of CD81 cell surface expression is a key determinant for productive entry of hepatitis C virus into host cells

Recently a cell culture model supporting the complete life cycle of the hepatitis C virus (HCV) was developed. Searching for host cell determinants involved in the HCV replication cycle, we evaluated the efficiency of virus propagation in different Huh-7-derived cell clones. We found that Huh-7.5 cells and Huh7-Lunet cells, two former replicon cell clones that had been generated by removal of an HCV replicon by inhibitor treatment, supported comparable levels of RNA replication and particle production, whereas virus spread was severely impaired in the latter cells. Analysis of cell surface expression of CD81 and scavenger receptor class B type I (SR-BI), two molecules previously implicated in HCV entry, revealed similar expression levels for SR-BI, while CD81 surface expression was much higher on Huh-7.5 cells than on Huh7-Lunet cells. Ectopic expression of CD81 in Huh7-Lunet cells conferred permissiveness for HCV infection to a level comparable to that for Huh-7.5 cells. Modulation of CD81 cell surface density in Huh-7.5 cells by RNA interference indicated that a certain amount of this molecule (approximately 7 x 10(4) molecules per cell) is required for productive infection with a low dose of HCV. Consistent with this, we show that susceptibility to HCV infection depends on a critical quantity of CD81 molecules. While infection is restricted in cells expressing very small amounts of CD81, susceptibility rapidly rises within a narrow range of CD81 levels, reaching a plateau where higher expression does not further increase the efficiency of infection. Together these data indicate that a high density of cell surface-exposed CD81 is a key determinant for productive HCV entry into host cells.