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Tumor-initiating stem-like cells (TICs) are defective in maintaining asymmetric cell division and responsible for tumor recurrence. Cell-fate-determinant molecule NUMB-interacting protein (TBC1D15) is overexpressed and contributes to p53 degradation in TICs. Here we identify TBC1D15-mediated oncogenic mechanisms and tested the tumorigenic roles of TBC1D15 in vivo. We examined hepatocellular carcinoma (HCC) development in alcohol Western diet-fed hepatitis C virus NS5A Tg mice with hepatocyte-specific TBC1D15 deficiency or expression of non-phosphorylatable NUMB mutations. Liver-specific TBC1D15 deficiency or non-p-NUMB expression reduced TIC numbers and HCC development. TBC1D15–NuMA1 association impaired asymmetric division machinery by hijacking NuMA from LGN binding, thereby favoring TIC self-renewal. TBC1D15–NOTCH1 interaction activated and stabilized NOTCH1 which upregulated transcription of NANOG essential for TIC expansion. TBC1D15 activated three novel oncogenic pathways to promote self-renewal, p53 loss, and Nanog transcription in TICs. Thus, this central regulator could serve as a potential therapeutic target for treatment of HCC. Normal stem cells are maintained by asymmetric cell division, but this process is dysregulated in tumour initiating stem-like cells (TICs). Here, the authors show that TBC1D15 impairs the asymmetric division machinery and activates NOTCH pathway for TIC self-renewal and expansion to promote liver tumorigenesis. [ABSTRACT FROM AUTHOR]

Significance: Neoplasms contain tumor-initiating stem-like cells (TICs) that drive malignant progression and tumor growth with drug resistance. TICs proliferate through a self-renewal process in which the two daughter cells differ in their proliferative potential, with one retaining the self-renewing phenotype and another displaying the differentiated phenotype. Recent Advances: Cancer traits (hepatocellular carcinoma) are triggered by alcoholism, obesity, and hepatitis B or C virus (HBV and HCV), including genetic changes, angiogenesis, defective tumor immunity, immortalization, metabolic reprogramming, excessive and prolonged inflammation, migration/invasion/metastasis, evasion of cell cycle arrest, anticell death, and compensatory regeneration/proliferation. Critical Issues: This review describes how metabolic reprogramming in mitochondria promotes self-renewal and oncogenicity of TICs. Pluripotency transcription factors (TFs), NANOG, OCT4, MYC, and SOX2, contribute to cancer progression by mitochondrial reprogramming, leading to the genesis of TICs and cancer. For example, oxidative phosphorylation (OXPHOS) and fatty acid metabolism are identified as major pathways contributing to pluripotency TF-mediated oncogenesis. Future Directions: Identification of novel metabolic pathways provides potential drug targets for neutralizing the activity of highly malignant TICs found in cancer patients. [ABSTRACT FROM AUTHOR]

Background: Various kinds of autoimmune diseases have been reported to have a significant relationship with persistent hepatitis c virus (HCV) infection and Th17 cells. Previously, our group reported that the existence of HCV in T lymphocytes could affect the development of CD4+ helper T cells and their proliferation, in addition to the induction of immunoglobulin hyper-mutation. Methods: Therefore, we analyzed the relationship between persistent infection of HCV and the mechanism of Th17 cell induction ex vivo and in vitro. Results: The prevalence of autoimmune-related diseases in chronic hepatitis c patients (CH-C) was significantly higher than in other types of chronic hepatitis (hepatitis B and NASH). A significantly higher frequency of IL6 and TGF-β double-high patients was detected in CH-C than in other liver diseases. Moreover, these double-high patients had significantly higher positivity of anti-nuclear antibody, cryoglobulinemia, and lymphotropic HCV and higher amounts of IL1-β, IL21, IL23. In addition to the previously reported lymphotropic SB-HCV strain, we found a novel, genotype 1b lymphotropic HCV (Ly-HCV), by deep sequencing analysis. Lymphotropic-HCV replication could be detected in the lymphoid cells with various kinds of cytokine-conditions including IL1β, IL23, IL6 and TGF-β in vitro. Infection by HCV could significantly enhance the development of Th17 cells. The HCV protein responsible for inducing the Th17 cells was HCV-Core protein, which could enhance the STAT-3 signaling and up-regulate the expression of RORγt as a Th17 master gene. Conclusion: Infection by lymphotropic HCV might enhance the Th17 development and contribute to understanding the pathogenesis of autoimmune-related diseases. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) RNA initiates its replication on a detergent- resistant membrane structure derived from the endoplasmic reticulum (ER) in the HCV replicon cells. By performing a pulse-chase study of BrU-labeled HCV RNA, we found that the newly-synthesized HCV RNA traveled along the anterograde-membrane traffic and moved away from the ER. Presumably, the RNA moved to the site of translation or virion assembly in the later steps of viral life cycle. In this study, we further addressed how HCV RNA translation was regulated by HCV RNA trafficking. When the movement of HCV RNA from the site of RNA synthesis to the Golgi complex was blocked by nocodazole, an inhibitor of ER-Golgi transport, HCV protein translation was surprisingly enhanced, suggesting that the translation of viral proteins occurred near the site of RNA synthesis. We also found that the translation of HCV proteins was dependent on active RNA synthesis: inhibition of viral RNA synthesis by an NS5B inhibitor resulted in decreased HCV viral protein synthesis even when the total amount of intracellular HCV RNA remained unchanged. Furthermore, the translation activity of the replication- defective HCV replicons or viral RNA with an NS5B mutation was greatly reduced as compared to that of the corresponding wildtype RNA. By performing live cell labeling of newly synthesized HCV RNA and proteins, we further showed that the newly synthesized HCV proteins colocalized with the newly synthesized viral RNA, suggesting that HCV RNA replication and protein translation take place at or near the same site. Our findings together indicate that the translation of HCV RNA is coupled to RNA replication and that the both processes may occur at the same subcellular membrane compartments, which we term the replicasome. [ABSTRACT FROM AUTHOR]

Cancer stem cells (tumor-initiating stem-like cells: TISCs) are resistant to chemotherapy and are associated with metastatic hepatocellular carcinoma (HCC), which is commonly observed in hepatitis C virus (HCV)-infected patients with obesity or alcohol abuse. However, it is unknown whether the TLR4-NANOG pathway serves as a universal oncogenic signaling in the genesis of TISCs and HCC. We aimed to determine whether Tlr4 is a putative proto-oncogene for TISCs in liver oncogenesis due to different etiologies and how Tlr4 is regulated at the transcriptional and epigenetic levels. CD133+/CD49f+ TISCs were isolated using FACS from HCC developed in HCV Core Tg mice fed alcohol, diethylnitrosamine-treated mice, and alcoholic patients with or without HCV infection. CD133+/CD49f+ cells isolated from the animal models and patients are tumorigenic both in vitro and in a xenograft model, and Tlr4 or Nanog silencing with shRNA attenuates their tumor initiating property. Functional oncogene screening of a cDNA library identified the organ size control pathway targets Yap1 and AKT activator Igf2bp3 as NANOG-dependent genes that inhibit transforming growth factor-β signaling in TISCs. Tlr4 expression is higher in TISCs compared with CD133-/CD49f+ cells. Taken together, Tlr4 may be a universal proto-oncogene responsible for the genesis of TLR4-NANOG dependent TISCs, and this pathway serves as a novel therapeutic target for HCC. [ABSTRACT FROM AUTHOR]

Background: Lymphotropic hepatitis C virus (HCV) infection of B and T cells might play an important role in the pathogenesis of hepatitis C. Recently, we showed that a lymphotropic HCV (SB strain) could infect established T-cell lines and B-cell lines. However, whether HCV replication interferes with cell proliferation and function in primary T lymphocytes is still unclear. Aim: The aim of this study was to analyze whether HCV replication in primary T lymphocytes affected their development, proliferation, and Th1 commitment. Methods: SB strain cell culture supernatant (2 × 10 copies/ml HCV) was used to infect several kinds of primary lymphocyte subsets. Mock, UV-irradiated SB-HCV, JFH-1 strain, and JFH-1 NS5B mutant, which could not replicate in T cells, were included as negative controls. Carboxyfluorescein succinimidyl ester (CFSE) and CD45RA double staining was used to evaluate the proliferative activity of CD4CD45RACD45RO naïve CD4 cells. Interferon (IFN)-γ and interleukin (IL)-10 secretion assays magnetic cell sorting (MACS) were carried out. Results: Negative strand HCV RNA was detected in CD4, CD14, and CD19 cells. Among CD4 cells, CD4CD45RARO cells (naïve CD4 cells) were most susceptible to replication of the SB strain. The levels of CFSE and CD45RA expression gradually declined during cell division in uninfected cells, while HCV-infected naïve CD4 cells expressed higher levels of CFSE and CD45RA than Mock or UV-SB infected naïve CD4 cells. Moreover, the production of IFN-γ was significantly suppressed in SB-infected naïve CD4 cells. Conclusions: Lymphotropic HCV replication suppressed proliferation and development, including that towards Th1 commitment, in human primary naïve CD4 cells. [ABSTRACT FROM AUTHOR]

B cell non-Hodgkin lymphoma is a typical extrahepatic manifestation frequently associated with hepatitis C virus (HCV) infection. The mechanism by which HCV infection leads to lymphoproliferative disorder remains unclear. Our group established HCV transgenic mice that expressed the full HCV genome in B cells (RzCD19Cre mice).We observed a 25.0% incidence of diffuse large B cell non-Hodgkin lymphomas (22.2% in male and 29.6% in female mice) within 600 days of birth. Interestingly, RzCD19Cre mice with substantially elevated serum-soluble interleukin-2 receptor α-subunit (sIL-2Rα) levels (>1000 pg/mL) developed B cell lymphomas. Another mouse model of lymphoproliferative disorder was established by persistent expression of HCV structural proteins through disruption of interferon regulatory factor-1 (irf-1-/- /CN2 mice). Irf-1-/- /CN2 mice showed extremely high incidences of lymphomas and lymphoproliferative disorders. Moreover, these mice showed increased levels of interleukin (IL)-2, IL-10, and Bcl-2 as well as increased Bcl-2 expression, which promoted oncogenic transformation of lymphocytes. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) infection is associated with the development of hepatocellular carcinoma and probably also non-Hodgkin's B-cell lymphoma. The molecular mechanisms of HCV-associated carcinogenesis are unknown. Here we demonstrated that peripheral blood mononuclear cells obtained from hepatitis C patients and hepatocytes infected with HCV in vitro showed frequent chromosomal polyploidy. HCV infection or the expression of viral core protein alone in hepatocyte culture or transgenic mice inhibited mitotic spindle checkpoint function because of reduced Rb transcription and enhanced E2F-1 and Mad2 expression. The silencing of E2F-1 by RNA interference technology restored the function of mitotic checkpoint in core-expressing cells. Taken together, these data suggest that HCV infection may inhibit the mitotic checkpoint to induce polyploidy, which likely contributes to neoplastic transformation. [ABSTRACT FROM AUTHOR]

Background. A lymphotropic hepatitis C virus strain (HCV, SB strain, hereafter "SB-HCV") has been shown to infect established T cell lines (Molt-4 and Jurkat) and primary human naive CD4+ T cells. During T cell development and activation, transient expression of CD44 splicing variant 6 (CD44v6) plays a significant role. Methods. SB-HCV was used to infect Molt-4 cells, and their cellular proliferation and CD44 expression was examined. Results. SB-HCV-infected Molt-4 cells expressed a significantly lower level of the CD44v6 isoform. The infected cells could be divided into 2 carboxyfluorescein succinimidyl ester (CFSE) groups, CFSE-high (indicating low proliferation activity; 34.2% of the cells) and CFSE-low (indicating high proliferation activity; 62.5% of the cells), whereas uninfected cells consisted of only a CFSE-low population. Of the CFSE-high cells, 82.4% were positive for the HCV protein NS5A , whereas only 1.2% of the CFSE-low cells were positive for this protein. Among the HCV proteins, NS5A alone caused the down-regulation of CD44v6 expression. After cells were stimulated with phorbol myristate acetate, the amount of phosphorylated mitogen-activated protein (MAP) kinase was significantly reduced in CFSE-high, SB-HCV-infected Molt-4 cells. After Fas ligand stimulation, SB-HCV-infected Molt-4 cells had increased cleavage of caspase 8 and 3 and enhanced apoptosis, compared with the rates of cleavage and apoptosis in control groups, indicating that SB-HCV infection increased Fas-mediated apoptosis. Conclusion. HCV replication in T cells suppresses cellular proliferation and enhances susceptibility to Fas signaling by inhibiting CD44v6 signaling and expression. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) induces inflammatory signals, leading to hepatitis, hepatocellular carcinomas, and lymphomas. The mechanism of HCV involvement in the host's innate immune responses has not been well characterized. In this study, we analyzed expression and regulation of the entire panel of toll-like receptors (TLRs) in human B cells following HCV infection in vitro. Among all of the TLRs (TLRs 1 to 10) examined, only TLR4 showed an altered expression (a three- to sevenfold up-regulation) after HCV infection. Peripheral blood mononuclear cells from HCV-infected individuals also showed a higher expression level of TLR4 compared with those of healthy individuals. HCV infection significantly increased beta interferon (IFN-β) and interleukin-6 (IL-6) secretion from B cells, particularly after lipopolysaccharide stimulation. The increased IFN-β and IL-6 production was mediated by TLR4 induction, since the introduction of the small interfering RNA against TLR4 specifically inhibited the HCV-induced cytokine production. Among all of the viral proteins, only NS5A caused TLR4 induction in hepatocytes and B cells. NS5A specifically activated the promoter of the TLR4 gene in both hepatocytes and B cells. In conclusion, HCV infection directly induces TLR4 expression and thereby activates B cells, which may contribute to the host's innate immune responses. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is one of the leading causes of chronic liver diseases and B-lymphocyte proliferative disorders, including mixed cryoglobulinemia and B-cell lymphoma. It has been suggested that HCV infects human cells through the interaction of its envelope glycoprotein E2 with a tetraspanin molecule CD81, the putative viral receptor. Here, we show that the engagement of B cells by purified E2 induced double-strand DNA breaks specifically in the variable region of immunoglobulin (VH) gene locus, leading to hypermutation in the VH genes of B cells. Other gene loci were not affected. Preincubation with the anti-CD81 monoclonal antibody blocked this effect. E2-CD81 interaction on B cells triggered the enhanced expression of activation-induced cytidine deaminase (AID) and also stimulated the production of tumor necrosis factor alpha. Knockdown of AID by the specific small interfering RNA blocked the E2-induced double-strand DNA breaks and hypermutation of the VH gene. These findings suggest that HCV infection, through E2-CD81 interaction, may modulate host's innate or adaptive immune response by activation of AID and hypermutation of immunoglobulin gene in B cells, leading to HCV-associated B-cell lymphoproliferative diseases. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) infection causes hepatitis, hepatocellular carcinoma, and B-cell lymphomas in a significant number of patients. Previously we have shown that HCV infection causes double-stranded DNA breaks and enhances the mutation frequency of cellular genes, including proto-oncogenes and immunoglobulin genes. To determine the mechanisms, we studied in vitro HCV infection of cell culture. Here we report that HCV infection activated the immunologic (type II) isoform of nitric oxide (NO) synthase (NOS), i.e., inducible NOS (iNOS), thereby inducing NO, which in turn induced DNA breaks and enhanced the mutation frequencies of cellular genes. Treatment of HCV-infected cells with NOS inhibitors or small interfering RNA specific for iNOS abolished most of these effects. Expression of the core protein or nonstructural protein 3 (NS3), but not the other viral proteins, in B cells or hepatocytes induced iNOS and DNA breaks, which could be blocked by NOS inhibitors. The core protein also enhanced the mutation frequency of cellular genes in hepatocytes derived from HCV core transgenic mice compared with that in control mice. The iNOS promoter was activated more than fivefold in HCV-infected cells, as revealed by a luciferase reporter assay driven by the iNOS promoter. Similarly, the core and NS3 proteins also induced the same effects. Therefore, we conclude that HCV infection can stimulate the production of NO through activation of the gene for iNOS by the viral core and NS3 proteins. NO causes DNA breaks and enhances DNA mutation. This sequence of events provides a mechanism for HCV pathogenesis and oncogenesis. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) is a nonretroviral oncogenic RNA virus, which is frequently associated with hepatocellular carcinoma (HCC) and B cell lymphoma. We demonstrated here that acute and chronic HCV infection caused a 5- to 10-fold increase in mutation frequency in Ig heavy chain. BCL-6, p53, and β-catenin genes of in vitro HCV-infected B cell lines and HCV-associated peripheral blood mononuclear cells, lymphomas, and HCCs. The nucleotide-substitution pattern of p53 and β-catenin was different from that of Ig heavy chain in HCV- infected cells, suggesting two different mechanisms of mutation. In addition, the mutated protooncogenes were amplified in HCV-associated Iymphomas and HCCs, but not in lymphomas of nonviral origin or HBV-associated HCC. HCV induced error-prone DNA polymerase ζ polymerase l, and activation-induced cytidine deaminase, which together, contributed to the enhancement of mutation frequency, as demonstrated by the RNA interference experiments. These results indicate that HCV induces a mutator phenotype and may transform cells by a hit-and-run mechanism. This finding provides a mechanism of oncogenesis for an RNA virus. [ABSTRACT FROM AUTHOR]

Background & Aims: The molecular mechanisms of lymphoproliferation associated with the disruption of interferon (IFN) signaling and chronic hepatitis C virus (HCV) infection are poorly understood. Lymphomas are extrahepatic manifestations of HCV infection; we sought to clarify the molecular mechanisms of these processes. Methods: We established interferon regulatory factor-1–null (irf-1−/−) mice with inducible and persistent expression of HCV structural proteins (irf-1/CN2 mice). All the mice (n = 900) were observed for at least 600 days after Cre/loxP switching. Histologic analyses, as well as analyses of lymphoproliferation, sensitivity to Fas-induced apoptosis, colony formation, and cytokine production, were performed. Proteins associated with these processes were also assessed. Results: Irf-1/CN2 mice had extremely high incidences of lymphomas and lymphoproliferative disorders and displayed increased mortality. Disruption of irf-1 reduced the sensitivity to Fas-induced apoptosis and decreased the levels of caspases-3/7 and caspase-9 messenger RNA species and enzymatic activities. Furthermore, the irf-1/CN2 mice showed decreased activation of caspases-3/7 and caspase-9 and increased levels of interleukin (IL)-2, IL-10, and Bcl-2, as well as increased Bcl-2 expression, which promoted oncogenic transformation of lymphocytes. IL-2 and IL-10 were induced by the HCV core protein in splenocytes. Conclusions: Disruption of IFN signaling resulted in development of lymphoma, indicating that differential signaling occurs in lymphocytes compared with liver. This mouse model, in which HCV expression and disruption of IFN signaling synergize to promote lymphoproliferation, will be an important tool for the development of therapeutic agents that target the lymphoproliferative pathway. [Copyright &y& Elsevier]

Hepatitis C virus (HCV) often causes persistent infection despite the presence of neutralizing antibodies against the virus in the sera of hepatitis C patients. HCV infects both hepatocytes and B cells through the binding of its envelope glycoprotein E2 to CD81, the putative viral receptor. Previously, we have shown that E2-CD81 interaction induces hypermutation of heavy-chain immunoglobulin (VH) in B cells. We hypothesize that if HCV infects antibody-producing B cells, the resultant hypermutation of VH may lower the affinity and specificity of the HCV-specific antibodies, enabling HCV to escape from immune surveillance. To test this hypothesis, we infected human hybridoma clones producing either neutralizing or non-neutralizing anti-E2 or anti-E1 antibodies with a lymphotropic HCV (SB strain). All of the hybridoma clones, except for a neutralizing antibody-producing hybridoma, could be infected with HCV and support virus replication for at least 8 weeks after infection. The VH sequences in the infected hybridomas had a significantly higher mutation frequency than those in the uninfected hybridomas, with mutations concentrating in complementarity-determining region 3. These mutations lowered the antibody affinity against the targeting protein and also lowered the virus-neutralizing activity of anti-E2 antibodies. Furthermore, antibody-mediated complement-dependent cytotoxicity with the antibodies secreted from the HCV-infected hybridomas was impaired. These results suggest that HCV infection could cause some anti-HCV-antibody-producing hybridoma B cells to make less-protective antibodies. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) infection is frequently associated with the development of hepatocellular carcinomas and non-Hodgkin's B-cell lymphomas. Previously, we reported that HCV infection causes cellular DNA damage and mutations, which are mediated by nitric oxide (NO). NO often damages mitochondria, leading to induction of double-stranded DNA breaks (DSBs) and accumulation of oxidative DNA damage. Here we report that HCV infection causes production of reactive oxygen species (ROS) and lowering of mitochondrial transmembrane potential (ΔΨm) in in vitro HCV-infected cell cultures. The changes in membrane potential could be inhibited by BCL-2. Furthermore, an inhibitor of ROS production, antioxidant N-acetyl-L-cysteine (NAC), or an inhibitor of NO, 1400W, prevented the alterations of ΔΨm. The HCV-induced DSB was also abolished by a combination of NO and ROS inhibitors. These results indicated that the mitochondrial damage and DSBs in HCV-infected cells were mediated by both NO and ROS. Among the HCV proteins, core, E1, and NS3 are potent ROS inducers: their expression led to DNA damage and activation of STAT3. Correspondingly, core-protein-transgenic mice showed elevated levels of lipid peroxidation and oxidatively damaged DNA. These HCV studies thus identified ROS, along with the previously identified NO, as the primary inducers of DSBs and mitochondrial damage in HCV-infected cells. [ABSTRACT FROM AUTHOR]

The PI3K-AKT signaling pathway plays an important role in cell growth and metabolism. Here we report that hepatitis C virus (HCV) transiently activates the PI3K-AKT pathway. This activation was observed as early as 15 min postinfection, peaked by 30 min, and became undetectable at 24 h postinfection. The activation of AKT could also be mediated by UV-inactivated HCV, HCV pseudoparticle, and the ectodomain of the HCV E2 envelope protein. Because antibodies directed againstCD81and claudin-1, but not antibodies directed against scavenger receptor class B type I or occludin, could also activate AKT, the interaction betweenHCVE2 and its two co-receptorsCD81and claudin- 1 probably triggered the activation of AKT. This activation of AKT by HCV was important for HCV infectivity, because the silencing of AKT by siRNA or the treatment of cells with its inhibitors or with the inhibitor of its upstream regulator PI3K significantly inhibitedHCVinfection, whereas the expression of constitutively active AKT enhanced HCV infection. The PI3KAKT pathway is probably involved in HCV entry, because the inhibition of this pathway could inhibit the entry of HCV pseudoparticle but not the VSV pseudoparticle into cells. Furthermore, the treatment of cells with the AKT inhibitor AKT-V prior to HCV infection inhibited HCV infection, whereas the treatment after HCV infection had no obvious effect. Taken together, our studies indicated that HCV transiently activates the PI3K-AKT pathway to facilitate its entry. These results provide important information for understanding HCV replication and pathogenesis and raised the possibility of targeting this cellular pathway to treat HCV patients. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) assembly is known to occur in juxtaposition to lipid droplets, but the mechanisms of nascent virion transport and release remain poorly understood. Here we demonstrate that HCV core protein targets to early and late endosomes but not to mitochondria or peroxisomes. Further, by employing inhibitors of early and late endosome motility in HCV-infected cells, we demonstrate that the movement of core protein to the early and late endosomes and virus production require an endosome-based secretory pathway. We also observed that this way is independent of that of the internalization of endocytosed virus particles during virus entry. [ABSTRACT FROM AUTHOR]

Abstract: Hepatitis C virus (HCV) infection is frequently associated with the development of hepatocellular carcinomas and non-Hodgkin''s B-cell lymphomas. Nonstructural protein 3 (NS3) of HCV possesses serine protease, nucleoside triphosphatase, and helicase activities, while NS4A functions as a cofactor for the NS3 serine protease. Here, we show that HCV NS3/4A interacts with the ATM (ataxia-telangiectasia mutated), a cellular protein essential for cellular response to irradiation. The expression of NS3/4A caused cytoplasmic translocation of either endogenous or exogenous ATM and delayed dephosphorylation of the phosphorylated ATM and γ-H2AX following ionizing irradiation. As a result, the irradiation-induced γ-H2AX foci persisted longer in the NS3/4A-expressing cells. Furthermore, these cells showed increased comet tail moment in single-cell electrophoresis assay, indicating increased double-strand DNA breaks. The cells harboring an HCV replicon also exhibited cytoplasmic localization of ATM and increased sensitivity to irradiation. These results demonstrate that NS3/4A impairs the efficiency of DNA repair by interacting with ATM and renders the cells more sensitive to DNA damage. This effect may contribute to HCV oncogenesis. [Copyright &y& Elsevier]

The hepatitis C virus (HCV) RNA replication complex (RC), which is composed of viral nonstructural (NS) proteins and host cellular proteins, replicates the viral RNA genome in association with intracellular membranes. Two viral NS proteins, NS3 and NS5A, are essential elements of the RC. Here, by using immunoprecipitation and fluorescence resonance energy transfer assays, we demonstrated that NS3 and NS5A interact with tubulin and actin. Furthermore, immunofluorescence microscopy and electron microscopy revealed that HCV RCs were aligned along microtubules and actin filaments in both HCV replicon cells and HCV-infected cells. In addition, the movement of RCs was inhibited when microtubules or actin filaments were depolymerized by colchicine and cytochalasin B, respectively. Based on our observations, we propose that microtubules and actin filaments provide the tracks for the movement of HCV RCs to other regions in the cell, and the molecular interactions between RCs and microtubules, or RCs and actin filaments, are mediated by NS3 and NS5A. [ABSTRACT FROM AUTHOR]

Previous studies indicated that hepatitis C virus (HCV) perturbs the autophagic pathway to induce the accumulation of autophagosomes in cells. To understand the role of autophagosomes in the HCV life cycle, we established a stable Huh7 hepatoma cell line that contained an HCV subgenomic RNA replicon and also expressed a GFP-LC3 fusion protein. The GFP-LC3 protein is localized to autophagosomes during autophagy and served as a convenient marker for autophagosomes. Our results indicate that the silencing of the expression of LC3 or Atg7, two protein factors critical for the formation of autophagosomes, suppresses the replication of HCV RNA. Confocal microscopy studies revealed the localization of HCV NS5A and NS5B proteins, which are two important components of the HCV RNA replication complex, and nascent HCV RNA to autophagosomes. The association of the HCV RNA replication complex with the autophagosomal membranes was further confirmed by co-immunoprecipitation and immunoelectron microscopy studies. Interestingly, inhibition of Class III PI3K activity had no effect on the autophagosomes induced by HCV. These results indicate that HCV induces autophagosomes via a Class III PI3Kindependent pathway and uses autophagosomal membranes as sites for its RNA replication. [ABSTRACT FROM AUTHOR]

Hepatitis C virus (HCV) replication involves many viral and host factors. Here, we employed a lentivirusbased RNA interference (RNAi) screening approach to search for possible cellular factors. By using a kinase-phosphatase RNAi library and an HCV replicon reporter system, we identified a serine-threonine kinase, Polo-like kinase 1 (Plk1), as a potential host factor regulating HCV replication. Knockdown of Plk1 reduced both HCV RNA replication and nonstructural (NS) protein production in both HCV replicon cells and HCV-infected cells while it did not significantly affect host cellular growth or cell cycle. Overexpression of Plk1 in the knockdown cells rescued HCV replication. Interestingly, the ratio between the hyperphosphorylated form (p58) and the basal phosphorylated form (p56) of NS5A was lower in the Plk1 knockdown cells and Plk1 kinase inhibitor-treated cells than in the control groups. Further studies showed that Plk1 could be immunoprecipitated together with NS5A. Both proteins partially colocalized in the perinuclear region. Furthermore, Plk1 could phosphorylate NS5A to both the p58 and p56 forms in an in vitro assay system; the phosphorylation efficiency was comparable to that of the reported casein kinase. Taken together, this study shows that Plk1 is an NS5A phosphokinase and thereby indirectly regulates HCV RNA replication. Because of the differential effects of Plk1 on HCV replication and host cell growth, Plk1 could potentially serve as a target for anti-HCV therapy. [ABSTRACT FROM AUTHOR]

Abstract: Hepatitis C virus (HCV) RNA replication requires viral nonstructural proteins as well as cellular factors. Recently, a cellular protein, synaptotagmin-binding, cytoplasmic RNA-interacting protein (SYNCRIP), also known as NSAP1, was found to bind HCV RNA and enhance HCV IRES-dependent translation. We investigate whether this protein is also involved in the HCV RNA replication. We found that SYNCRIP was associated with detergent-resistant membrane fractions and colocalized with newly-synthesized HCV RNA. Knock-down of SYNCRIP by siRNA significantly decreased the amount of HCV RNA in the cells containing a subgenomic replicon or a full-length viral RNA. Lastly, an in vitro replication assay after immunodepletion of SYNCRIP showed that SYNCRIP was directly involved in HCV RNA replication. These findings indicate that SYNCRIP has dual functions, participating in both RNA replication and translation in HCV life cycle. [Copyright &y& Elsevier]