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1.  Amphipathic α-Helices in Apolipoproteins Are Crucial to the Formation of Infectious Hepatitis C Virus Particles 
PLoS Pathogens  2014;10(12):e1004534.
Apolipoprotein B (ApoB) and ApoE have been shown to participate in the particle formation and the tissue tropism of hepatitis C virus (HCV), but their precise roles remain uncertain. Here we show that amphipathic α-helices in the apolipoproteins participate in the HCV particle formation by using zinc finger nucleases-mediated apolipoprotein B (ApoB) and/or ApoE gene knockout Huh7 cells. Although Huh7 cells deficient in either ApoB or ApoE gene exhibited slight reduction of particles formation, knockout of both ApoB and ApoE genes in Huh7 (DKO) cells severely impaired the formation of infectious HCV particles, suggesting that ApoB and ApoE have redundant roles in the formation of infectious HCV particles. cDNA microarray analyses revealed that ApoB and ApoE are dominantly expressed in Huh7 cells, in contrast to the high level expression of all of the exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3 in human liver tissues. The exogenous expression of not only ApoE, but also other exchangeable apolipoproteins rescued the infectious particle formation of HCV in DKO cells. In addition, expression of these apolipoproteins facilitated the formation of infectious particles of genotype 1b and 3a chimeric viruses. Furthermore, expression of amphipathic α-helices in the exchangeable apolipoproteins facilitated the particle formation in DKO cells through an interaction with viral particles. These results suggest that amphipathic α-helices in the exchangeable apolipoproteins play crucial roles in the infectious particle formation of HCV and provide clues to the understanding of life cycle of HCV and the development of novel anti-HCV therapeutics targeting for viral assembly.
Author Summary
In vitro systems have been developed for the study of hepatitis C virus (HCV) infection and have revealed many details of the life cycle of HCV. Apolipoprotein B (ApoB) and ApoE have been shown to play crucial roles in the particle formation of HCV, based on data obtained by siRNA-mediated gene knockdown and overexpression of the proteins. However, precise roles of the apolipoproteins in HCV assembly have not been elucidated yet. In this study, we show that infectious particle formation of HCV in Huh7 cells was severely impaired by the knockout of both ApoB and ApoE genes by artificial nucleases, and this reduction was cancelled by the expression of not only ApoE, but also other exchangeable apolipoproteins, including ApoA1, ApoA2, ApoC1, ApoC2 and ApoC3. In addition, expression of amphipathic α-helices in the exchangeable apolipoproteins restored the infectious particle formation in the double-knockout cells through an interaction with viral particles. These results provide clues to the understanding of life cycle of HCV and the development of novel antivirals to HCV.
PMCID: PMC4263759  PMID: 25502789
2.  Novel Permissive Cell Lines for Complete Propagation of Hepatitis C Virus 
Journal of Virology  2014;88(10):5578-5594.
Hepatitis C virus (HCV) is a major etiologic agent of chronic liver diseases. Although the HCV life cycle has been clarified by studying laboratory strains of HCV derived from the genotype 2a JFH-1 strain (cell culture-adapted HCV [HCVcc]), the mechanisms of particle formation have not been elucidated. Recently, we showed that exogenous expression of a liver-specific microRNA, miR-122, in nonhepatic cell lines facilitates efficient replication but not particle production of HCVcc, suggesting that liver-specific host factors are required for infectious particle formation. In this study, we screened human cancer cell lines for expression of the liver-specific α-fetoprotein by using a cDNA array database and identified liver-derived JHH-4 cells and stomach-derived FU97 cells, which express liver-specific host factors comparable to Huh7 cells. These cell lines permit not only replication of HCV RNA but also particle formation upon infection with HCVcc, suggesting that hepatic differentiation participates in the expression of liver-specific host factors required for HCV propagation. HCV inhibitors targeting host and viral factors exhibited different antiviral efficacies between Huh7 and FU97 cells. Furthermore, FU97 cells exhibited higher susceptibility for propagation of HCVcc derived from the JFH-2 strain than Huh7 cells. These results suggest that hepatic differentiation participates in the expression of liver-specific host factors required for complete propagation of HCV.
IMPORTANCE Previous studies have shown that liver-specific host factors are required for efficient replication of HCV RNA and formation of infectious particles. In this study, we screened human cancer cell lines for expression of the liver-specific α-fetoprotein by using a cDNA array database and identified novel permissive cell lines for complete propagation of HCVcc without any artificial manipulation. In particular, gastric cancer-derived FU97 cells exhibited a much higher susceptibility to HCVcc/JFH-2 infection than observed in Huh7 cells, suggesting that FU97 cells would be useful for further investigation of the HCV life cycle, as well as the development of therapeutic agents for chronic hepatitis C.
PMCID: PMC4019116  PMID: 24599999
3.  Innate Immune Response Induced by Baculovirus Attenuates Transgene Expression in Mammalian Cells 
Journal of Virology  2014;88(4):2157-2167.
The baculovirus Autographa californica nucleopolyhedrovirus (AcNPV) has been widely used to achieve a high level of foreign gene expression in insect cells, as well as for efficient gene transduction into mammalian cells without any replication. In addition to permitting efficient gene delivery, baculovirus has been shown to induce host innate immune responses in various mammalian cells and in mice. In this study, we examined the effects of the innate immune responses on gene expression by recombinant baculoviruses in cultured cells. The reporter gene expression in IRF3-deficient mouse embryonic fibroblasts (MEFs) infected with the recombinant baculovirus was shown to be enhanced in accordance with the suppression of beta interferon (IFN-β) production. Furthermore, efficient gene transduction by the recombinant baculovirus was achieved in MEFs deficient for stimulator of interferon genes (STING), TANK binding kinase 1 (TBK1), IFN regulatory factor 3 (IRF3), or IFN-β promoter stimulator 1 (IPS-1), but not in those deficient for IRF7, MyD88, or Z-DNA binding protein 1 (ZBP1)/DAI. Enhancement of gene expression by the recombinant baculovirus was also observed in human hepatoma cell lines replicating hepatitis C virus (HCV), in which innate immunity was impaired by the cleavage of IPS-1 by the viral protease. In addition, infection with the recombinant baculovirus expressing the BH3-only protein, BIMS, a potent inducer of apoptosis, resulted in a selective cell death in the HCV replicon cells. These results indicate that innate immune responses induced by infection with baculovirus attenuate transgene expression, and this characteristic might be useful for a selective gene transduction into cells with impaired innate immunity arising from infection with various viruses.
PMCID: PMC3911546  PMID: 24335288
4.  Japanese Encephalitis Virus Core Protein Inhibits Stress Granule Formation through an Interaction with Caprin-1 and Facilitates Viral Propagation 
Journal of Virology  2013;87(1):489-502.
Stress granules (SGs) are cytoplasmic foci composed of stalled translation preinitiation complexes induced by environmental stress stimuli, including viral infection. Since viral propagation completely depends on the host translational machinery, many viruses have evolved to circumvent the induction of SGs or co-opt SG components. In this study, we found that expression of Japanese encephalitis virus (JEV) core protein inhibits SG formation. Caprin-1 was identified as a binding partner of the core protein by an affinity capture mass spectrometry analysis. Alanine scanning mutagenesis revealed that Lys97 and Arg98 in the α-helix of the JEV core protein play a crucial role in the interaction with Caprin-1. In cells infected with a mutant JEV in which Lys97 and Arg98 were replaced with alanines in the core protein, the inhibition of SG formation was abrogated, and viral propagation was impaired. Furthermore, the mutant JEV exhibited attenuated virulence in mice. These results suggest that the JEV core protein circumvents translational shutoff by inhibiting SG formation through an interaction with Caprin-1 and facilitates viral propagation in vitro and in vivo.
PMCID: PMC3536427  PMID: 23097442
5.  Expression of MicroRNA miR-122 Facilitates an Efficient Replication in Nonhepatic Cells upon Infection with Hepatitis C Virus 
Journal of Virology  2012;86(15):7918-7933.
Hepatitis C virus (HCV) is one of the most common etiologic agents of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. In addition, HCV infection is often associated with extrahepatic manifestations (EHM), including mixed cryoglobulinemia and non-Hodgkin's lymphoma. However, the mechanisms of cell tropism of HCV and HCV-induced EHM remain elusive, because in vitro propagation of HCV has been limited in the combination of cell culture-adapted HCV (HCVcc) and several hepatic cell lines. Recently, a liver-specific microRNA called miR-122 was shown to facilitate the efficient propagation of HCVcc in several hepatic cell lines. In this study, we evaluated the importance of miR-122 on the replication of HCV in nonhepatic cells. Among the nonhepatic cell lines expressing functional HCV entry receptors, Hec1B cells derived from human uterus exhibited a low level of replication of the HCV genome upon infection with HCVcc. Exogenous expression of miR-122 in several cells facilitates efficient viral replication but not production of infectious particles, probably due to the lack of hepatocytic lipid metabolism. Furthermore, expression of mutant miR-122 carrying a substitution in a seed domain was required for efficient replication of mutant HCVcc carrying complementary substitutions in miR-122-binding sites, suggesting that specific interaction between miR-122 and HCV RNA is essential for the enhancement of viral replication. In conclusion, although miR-122 facilitates efficient viral replication in nonhepatic cells, factors other than miR-122, which are most likely specific to hepatocytes, are required for HCV assembly.
PMCID: PMC3421686  PMID: 22593164
6.  CD44 Participates in IP-10 Induction in Cells in Which Hepatitis C Virus RNA Is Replicating, through an Interaction with Toll-Like Receptor 2 and Hyaluronan 
Journal of Virology  2012;86(11):6159-6170.
The mechanisms of induction of liver injury during chronic infection with hepatitis C virus (HCV) are not well understood. Gamma interferon (IFN-γ)-inducible protein 10 (IP-10), a member of the CXC chemokine family, is expressed in the liver of chronic hepatitis C (CHC) patients and selectively recruits activated T cells to the sites of inflammation. Recently, it was shown that a low plasma concentration of IP-10 in CHC patients was closely associated with the outcome of antiviral therapy. In this study, we examined the role of the Toll-like receptor (TLR) pathway on IP-10 production in cells replicating HCV. Among the CXC chemokines, the expression of IP-10 was specifically increased in cells replicating HCV upon stimulation with conventional TLR2 ligands. The enhancement of IP-10 production upon stimulation with TLR2 ligands in cells replicating HCV induced CD44 expression. CD44 is a broadly distributed type I transmembrane glycoprotein and a receptor for the glycosaminoglycan hyaluronan (HA). In CHC patients, the expression of HA in serum has been shown to increase in accord with the progression of liver fibrosis, and HA also works as a ligand for TLR2. In the present study, IP-10 production upon HA stimulation was dependent on the expression of TLR2 and CD44, and a direct association between TLR2 and CD44 was observed. These results suggest that endogenous expression of HA in hepatocytes in CHC patients participates in IP-10 production through an engagement of TLR2 and CD44.
PMCID: PMC3372182  PMID: 22491449
7.  Effect of laparoscopic splenectomy in patients with Hepatitis C and cirrhosis carrying IL28B minor genotype 
BMC Gastroenterology  2012;12:158.
IL28B and ITPA genetic variants are associated with the outcome of pegylated-interferon and ribavirin (PEG-IFN/RBV) therapy. However, the significance of these genetic variants in cirrhotic patients following splenectomy has not been determined.
Thirty-seven patients with HCV-induced cirrhosis who underwent laparoscopic splenectomy (Spx group) and 90 who did not (non-Spx group) were genotyped for IL28B and ITPA. The outcome or adverse effects were compared in each group. Interferon-stimulated gene 15 (ISG15) and protein kinase R expression in the spleen was measured using total RNA extracted from exenterate spleen.
Sustained virological response (SVR) rate was higher in patients carrying IL28B major genotype following splenectomy (50% vs 27.3%) and in patients carrying minor genotype in the Spx group compared to non-Spx group (27.3% vs 3.6%, P < 0.05). Pretreatment splenic ISG expression was higher in patients carrying IL28B major. There was no difference in progression of anemia or thrombocytopenia between patients carrying each ITPA genotype in the Spx group. Although splenectomy did not increase hemoglobin (Hb) level, Hb decline tended to be greater in the non-Spx group. In contrast, splenectomy significantly increased platelet count (61.1 × 103/μl vs 168.7 × 103/μl, P < 0.01), which was maintained during the course of PEG-IFN/RBV therapy.
IL28B genetic variants correlated with response to PEG-IFN/RBV following splenectomy. Splenectomy improved SVR rate among patients carrying IL28B minor genotype and protected against anemia and thrombocytopenia during the course of PEG-IFN/RBV therapy regardless of ITPA genotype.
PMCID: PMC3503804  PMID: 23145809
IL28B; ITPA; Splenectomy; Liver cirrhosis
8.  Neither MICA Nor DEPDC5 Genetic Polymorphisms Correlate with Hepatocellular Carcinoma Recurrence following Hepatectomy 
HPB Surgery  2012;2012:185496.
Purpose. Genetic polymorphisms of MICA and DEPDC5 have been reported to correlate with progression to hepatocellular carcinoma (HCC) in chronic hepatitis C patients. However, correlation of these genetic variants with HCC recurrence following hepatectomy has not yet been clarified. Methods. Ninety-six consecutive HCC patients who underwent hepatectomy, including 64 patients who were hepatitis C virus (HCV) positive, were genotyped for MICA (rs2596542) and DEPDC5 (rs1012068). Recurrence-free survival rates (RFS) were compared for each genotype. Results. Five-year HCC recurrence-free survival (RFS) rates following hepatectomy were 20.7% in MICA GG allele carriers, 38.7% in GA, and 20.8% in AA, respectively (P = 0.72). The five-year RFS rate was 23.8% in DEPDC5 TT allele carriers and 31.8% in TG/GG, respectively (P = 0.47). The survival rates in all (including HCV-negative) patients were also similar among each MICA and DEPDC5 genotype following hepatectomy. Among HCV-positive patients carrying the DEPDC5 TG/GG allele, low fibrosis stage (F0-2) occurred more often compared with TT carriers (P < 0.05). Conclusions. Neither MICA nor DEPDC5 genetic polymorphism correlates with HCC recurrence following hepatectomy. DEPDC5 minor genotype data suggest a high susceptibility for HCC development in livers, even those with low fibrosis stages.
PMCID: PMC3485991  PMID: 23132957
9.  Role of miR-122 and lipid metabolism in HCV infection 
Journal of Gastroenterology  2012;48(2):169-176.
Hepatitis C virus (HCV) exhibits a narrow host range and a specific tissue tropism. Mice expressing major entry receptors for HCV permit viral entry, and therefore the species tropism of HCV infection is considered to be reliant on the expression of the entry receptors. However, HCV receptor candidates are expressed and replication of HCV-RNA can be detected in several nonhepatic cell lines, suggesting that nonhepatic cells are also susceptible to HCV infection. Recently it was shown that the exogenous expression of a liver-specific microRNA, miR-122, facilitated the efficient replication of HCV not only in hepatic cell lines, including Hep3B and HepG2 cells, but also in nonhepatic cell lines, including Hec1B and HEK-293T cells, suggesting that miR-122 is required for the efficient replication of HCV in cultured cells. However, no infectious particle was detected in the nonhepatic cell lines, in spite of the efficient replication of HCV-RNA. In the nonhepatic cells, only small numbers of lipid droplets and low levels of very-low-density lipoprotein-associated proteins were observed compared with findings in the hepatic cell lines, suggesting that functional lipid metabolism participates in the assembly of HCV. Taken together, these findings indicate that miR-122 and functional lipid metabolism are involved in the tissue tropism of HCV infection. In this review, we would like to focus on the role of miR-122 and lipid metabolism in the cell tropism of HCV.
PMCID: PMC3698423  PMID: 22965312
HCV; miR-122; Lipid metabolism
10.  Baculovirus GP64-Mediated Entry into Mammalian Cells 
Journal of Virology  2012;86(5):2610-2620.
The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) serves as an efficient viral vector, not only for abundant gene expression in insect cells, but also for gene delivery into mammalian cells. Lentivirus vectors pseudotyped with the baculovirus envelope glycoprotein GP64 have been shown to acquire more potent gene transduction than those with vesicular stomatitis virus (VSV) envelope glycoprotein G. However, there are conflicting hypotheses about the molecular mechanisms of the entry of AcMNPV. Moreover, the mechanisms of the entry of pseudotyped viruses bearing GP64 into mammalian cells are not well characterized. Determination of the entry mechanisms of AcMNPV and the pseudotyped viruses bearing GP64 is important for future development of viral vectors that can deliver genes into mammalian cells with greater efficiency and specificity. In this study, we generated three pseudotyped VSVs, NPVpv, VSVpv, and MLVpv, bearing envelope proteins of AcMNPV, VSV, and murine leukemia virus, respectively. Depletion of membrane cholesterol by treatment with methyl-β-cyclodextrin, which removes cholesterol from cellular membranes, inhibited GP64-mediated internalization in a dose-dependent manner but did not inhibit attachment to the cell surface. Treatment of cells with inhibitors or the expression of dominant-negative mutants for dynamin- and clathrin-mediated endocytosis abrogated the internalization of AcMNPV and NPVpv into mammalian cells, whereas inhibition of caveolin-mediated endocytosis did not. Furthermore, inhibition of macropinocytosis reduced GP64-mediated internalization. These results suggest that cholesterol in the plasma membrane, dynamin- and clathrin-dependent endocytosis, and macropinocytosis play crucial roles in the entry of viruses bearing baculovirus GP64 into mammalian cells.
PMCID: PMC3302255  PMID: 22190715
11.  Establishment of a Novel Permissive Cell Line for the Propagation of Hepatitis C Virus by Expression of MicroRNA miR122 
Journal of Virology  2012;86(3):1382-1393.
The robust cell culture systems for hepatitis C virus (HCV) are limited to those using cell culture-adapted clones (HCV in cell culture [HCVcc]) and cells derived from the human hepatoma cell line Huh7. However, accumulating data suggest that host factors, including innate immunity and gene polymorphisms, contribute to the variation in host response to HCV infection. Therefore, the existing in vitro systems for HCV propagation are not sufficient to elucidate the life cycle of HCV. A liver-specific microRNA, miR122, has been shown to participate in the efficient replication of HCV. In this study, we examined the possibility of establishing a new permissive cell line for HCV propagation by the expression of miR122. A high level of miR122 was expressed by a lentiviral vector placed into human liver cell lines at a level comparable to the endogenous level in Huh7 cells. Among the cell lines that we examined, Hep3B cells stably expressing miR122 (Hep3B/miR122) exhibited a significant enhancement of HCVcc propagation. Surprisingly, the levels of production of infectious particles in Hep3B/miR122 cells upon infection with HCVcc were comparable to those in Huh7 cells. Furthermore, a line of “cured” cells, established by elimination of HCV RNA from the Hep3B/miR122 replicon cells, exhibited an enhanced expression of miR122 and a continuous increase of infectious titers of HCVcc in every passage. The establishment of the new permissive cell line for HCVcc will have significant implications not only for basic HCV research but also for the development of new therapeutics.
PMCID: PMC3264374  PMID: 22114337
12.  Heterogeneous Nuclear Ribonucleoprotein A2 Participates in the Replication of Japanese Encephalitis Virus through an Interaction with Viral Proteins and RNA ▿  
Journal of Virology  2011;85(21):10976-10988.
Japanese encephalitis virus (JEV) is a mosquito-borne flavivirus that is kept in a zoonotic transmission cycle between pigs and mosquitoes. JEV causes infection of the central nervous system with a high mortality rate in dead-end hosts, including humans. Many studies have suggested that the flavivirus core protein is not only a component of nucleocapsids but also an important pathogenic determinant. In this study, we identified heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) as a binding partner of the JEV core protein by pulldown purification and mass spectrometry. Reciprocal coimmunoprecipitation analyses in transfected and infected cells confirmed a specific interaction between the JEV core protein and hnRNP A2. Expression of the JEV core protein induced cytoplasmic retention of hnRNP A2 in JEV subgenomic replicon cells. Small interfering RNA (siRNA)-mediated knockdown of hnRNP A2 resulted in a 90% reduction of viral RNA replication in cells infected with JEV, and the reduction was cancelled by the expression of an siRNA-resistant hnRNP A2 mutant. In addition to the core protein, hnRNP A2 also associated with JEV nonstructural protein 5, which has both methyltransferase and RNA-dependent RNA polymerase activities, and with the 5′-untranslated region of the negative-sense JEV RNA. During one-step growth, synthesis of both positive- and negative-strand JEV RNAs was delayed by the knockdown of hnRNP A2. These results suggest that hnRNP A2 plays an important role in the replication of JEV RNA through the interaction with viral proteins and RNA.
PMCID: PMC3194950  PMID: 21865391
13.  Solitary Asymptomatic Thyroid Metastasis from Hepatocellular Carcinoma Detected by FDG-PET/CT 
Case Reports in Gastroenterology  2010;4(2):279-285.
Thyroid metastases from hepatocellular carcinoma (HCC) seldom occur and are often difficult to diagnose because of their asymptomatic clinical course. We evaluated a very rare case of solitary thyroid metastasis from HCC that showed high uptake of fluorine-18 fluorodeoxyglucose (FDG), when imaged using fluorine-18 fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/CT). The patient was a 74-year-old man and presented with a remarkably elevated des-gamma-carboxy prothrombin level of 1,157 mAU/ml 22 months after hepatic lobectomy. FDG-PET/CT imaging revealed a hypodense tumor with high FDG uptake, with a maximum standardized uptake value of 5.2 in the thyroid left lobe. Solitary thyroid metastasis from HCC was suspected and subsequent fine needle aspiration did indeed reveal HCC. The patient received left thyroidectomy with left regional lymph node dissection. Two months after left thyroidectomy, contrast-enhanced computed tomography showed local recurrence, and the patient received ongoing radiotherapy treatment. To our knowledge, the present study is the first to demonstrate the feasibility of FDG-PET/CT in the diagnosis and management of clinically diagnosed, asymptomatic, solitary thyroid metastasis from HCC.
PMCID: PMC3047758  PMID: 21383951
Thyroid metastasis; Hepatocellular carcinoma; FDG-PET/CT
14.  Human VAP-C Negatively Regulates Hepatitis C Virus Propagation▿  
Journal of Virology  2009;83(16):7959-7969.
Human vesicle-associated membrane protein-associated protein (VAP) subtype A (VAP-A) and subtype B (VAP-B) are involved in the regulation of membrane trafficking, lipid transport and metabolism, and the unfolded protein response. VAP-A and VAP-B consist of the major sperm protein (MSP) domain, the coiled-coil motif, and the C-terminal transmembrane anchor and form homo- and heterodimers through the transmembrane domain. VAP-A and VAP-B interact with NS5B and NS5A of hepatitis C virus (HCV) through the MSP domain and the coiled-coil motif, respectively, and participate in the replication of HCV. VAP-C is a splicing variant of VAP-B consisting of the N-terminal half of the MSP domain of VAP-B followed by the subtype-specific frameshift sequences, and its biological function has not been well characterized. In this study, we have examined the biological functions of VAP-C in the propagation of HCV. VAP-C interacted with NS5B but not with VAP-A, VAP-B, or NS5A in immunoprecipitation analyses, and the expression of VAP-C inhibited the interaction of NS5B with VAP-A or VAP-B. Overexpression of VAP-C impaired the RNA replication of the HCV replicon and the propagation of the HCV JFH1 strain, whereas overexpression of VAP-A and VAP-B enhanced the replication. Furthermore, the expression of VAP-C was observed in various tissues, whereas it was barely detected in the liver. These results suggest that VAP-C acts as a negative regulator of HCV propagation and that the expression of VAP-C may participate in the determination of tissue tropism of HCV propagation.
PMCID: PMC2715746  PMID: 19515777

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