Suppression of Hepatitis C Virus Replicon by RNA Interference Directed against the NS3 and NS5B Regions of the Viral Genome

Takigawa, Yuki; Nagano-Fujii, Motoko; Deng, Lin; Hidajat, Rachmat; Tanaka, Motofumi; Mizuta, Hiroyuki; Hotta, Hak

doi:10.1111/j.1348-0421.2004.tb03556.x

Cited by 64 publications

(55 citation statements)

References 53 publications

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“…This technology is potentially applicable to drug targeting for genes involved in the pathogenesis of human diseases. For example, siRNAs against Fas and TNFa have been reported to control fulminant hepatitis 23,24 and septic shock in mice, 25 respectively, while others have been reported to successfully inhibit replication of hepatitis B virus, 26,27 hepatitis C virus, 28,29 and human immunodeficiency virus type 1. 30,31 The application of an siRNA to cancer therapy appears to be more complicated, as recent molecular analyses have revealed that most oncogenes involved in carcinogenesis have no or only subtle sequence changes, such as point mutations and translocation.…”

Section: Effects Of E6 Sirna On the Growth Of Hpv18-related Cancer Cellsmentioning

confidence: 99%

Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA

et al. 2005

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Human cervical cancer is caused by high-risk types of human papillomavirus (HPV) such as HPV16 and HPV18, which possess the E6 and E7 oncogenes, whose concurrent expression is a prerequisite for cancer development and maintaining malignant phenotypes. Silencing these oncogenes is considered to be applicable in molecular therapies of human cervical cancer. However, it remains to be determined whether E6, E7, or both should be silenced to obtain most efficient antitumor activity by an HPV smallinterfering RNA (siRNA). Herein, we report two types of siRNAs targeting HPV18 E6, that exerted a negative growth effect on HPV18-positive cervical cancer cells (HeLa and SW756), in part, inducing cell death. One siRNA (Ex-18E6), designed to target both E6-E7 mRNA and its splicing variant, E6*I-E7 mRNA, efficiently knocked down both E6 and E7 expression. The other (Sp-18E6), designed to specifically target E6-E7 mRNA but not E6*I-E7 mRNA, suppressed E6 to a similar level as Ex-18E6; however, it less efficiently inhibited E7 as compared to Ex-18E6. Although both siRNAs induced cell death, Sp-18E6 siRNA induced more prominent cell death than Ex-18E6. Our results suggest that E6-specific suppression may induce more potent anticancer activity than simultaneous E6 and E7 suppression, and that E6-specific targeting is a promising strategy for siRNA-based therapy for HPVpositive cervical cancer.

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Section: Effects Of E6 Sirna On the Growth Of Hpv18-related Cancer Cellsmentioning

confidence: 99%

Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA

et al. 2005

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“…Immunofluorescence staining was performed essentially as described previously (Takigawa et al, 2004). Cells seeded on glass coverslips in a 24-well plate at a density of 4610 4 cells per well were infected with HCV.…”

Section: Methodsmentioning

confidence: 99%

Efficient production of infectious hepatitis C virus with adaptive mutations in cultured hepatoma cells

Bungyoku

Shoji

Makine

et al. 2009

Journal of General Virology

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Robust production of infectious hepatitis C virus (HCV) in cell culture was realized by using the JFH1 strain and the homologous chimeric J6/JFH1 strain in Huh-7.5 cells, a highly HCVpermissive subclone of Huh-7 cells. In this study, we aimed to establish a more efficient HCVproduction system and to gain some insight into the adaptation mechanisms of efficient HCV production. By serial passaging of J6/JFH1-infected Huh-7.5 cells, we obtained culture-adapted J6/JFH1 variants, designated P-27, P-38 and P-47. Sequence analyses revealed that the adaptive mutant viruses P-27, P-38 and P-47 possessed eight mutations [four in E2, two in NS2, one in NS5A and one in NS5B), 10 mutations [two additional mutations in the 59-untranslated region (59-UTR) and core] and 11 mutations (three additional mutations in 59-UTR, core and NS5B), respectively. We introduced amino acid substitutions into the wild-type J6/JFH1 clone, generated recombinant viruses with adaptive mutations and analysed their infectivity and ability to produce infectious viruses. The viruses with the adaptive mutations exhibited higher expression of HCV proteins than did the wild type in Huh-7.5 cells. Moreover, we provide evidence suggesting that the mutation N534H in the E2 glycoprotein of the mutant viruses conferred an advantage at the entry level. We thus demonstrate that an efficient HCV-production system could be obtained by introducing adaptive mutations into the J6/JFH1 genome. The J6/JFH1-derived mutant viruses presented here would be a good tool for producing HCV particles with enhanced infectivity and for studying the molecular mechanism of HCV entry. INTRODUCTIONHepatitis C virus (HCV) is the main cause of chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (Choo et al., 1989;Kuo et al., 1989;Saito et al., 1990). As more than 170 million people worldwide are infected chronically with HCV (Poynard et al., 2003) and because the current antiviral therapy, interferon and ribavirin, produces sustained virus clearance in ,50 % of treated patients (Manns et al., 2007), HCV infection is clearly a problem of major proportions. HCV is a single-stranded, positive-sense RNA virus that is classified in the genus Hepacivirus in the family Flaviviridae. The approximately 9.6 kb HCV genome encodes one large open reading frame (ORF) that is flanked at the 59 and 39 ends by untranslated regions (UTRs) (Choo et al., 1991). The HCV polyprotein is processed into at least 10 proteins by viral proteases and cellular signalases (Grakoui et al., 1993;Hijikata et al., 1993a;McLauchlan et al., 2002). The structural proteins core, E1 and E2 are located in the N terminus of the polyprotein, followed by p7 and the non-structural (NS) proteins NS2, NS3, NS4A, NS4B, NS5A and NS5B (Bartenschlager & Sparacio, 2007).Study of the HCV life cycle and virus-host interaction has been hampered severely by the lack of a robust in vitro cellculture system and small-animal models of HCV infection (Bartenschlager & Sparacio, 2007). The development of HCV replicon systems has made...

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“…Huh-7.5 cells stably harbouring an HCV subgenomic RNA replicon were prepared by using pFK5B/2884Gly, as described previously (Hidajat et al, 2005;Lohmann et al, 2001;Taguchi et al, 2004;Takigawa et al, 2004).…”

Section: Methodsmentioning

confidence: 99%

Hepatitis C virus NS5A protein interacts with and negatively regulates the non-receptor protein tyrosine kinase Syk

Inubushi

Nagano-Fujii

Kitayama

et al. 2008

Journal of General Virology

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Hepatitis C virus (HCV) is the major causative agent of hepatocellular carcinoma. However, the precise mechanism underlying the carcinogenesis is yet to be elucidated. It has recently been reported that Syk, a non-receptor protein tyrosine kinase, functions as a potent tumour suppressor in human breast carcinoma. This study first examined the possible effect of HCV infection on expression of Syk in vivo. Immunohistochemical analysis revealed that endogenous Syk, which otherwise was expressed diffusely in the cytoplasm of normal hepatocytes, was localized near the cell membrane with a patchy pattern in HCV-infected hepatocytes. The possible interaction between HCV proteins and Syk in human hepatoma-derived Huh-7 cells was then examined. Immunoprecipitation analysis revealed that NS5A interacted strongly with Syk. Deletion-mutation analysis revealed that an N-terminal portion of NS5A (aa 1-175) was involved in the physical interaction with Syk. An in vitro kinase assay demonstrated that NS5A inhibited the enzymic activity of Syk and that, in addition to the N-terminal 175 residues, a central portion of NS5A (aa 237-302) was required for inhibition of Syk. Moreover, Syk-mediated phosphorylation of phospholipase C-c1 was downregulated by NS5A. An interaction of NS5A with Syk was also detected in Huh-7.5 cells harbouring an HCV RNA replicon or infected with HCV. In conclusion, these results demonstrated that NS5A interacts with Syk resulting in negative regulation of its kinase activity. The results indicate that NS5A may be involved in the carcinogenesis of hepatocytes through the suppression of Syk kinase activities. INTRODUCTIONHepatitis C virus (HCV) is the major aetiological agent of viral hepatitis worldwide after hepatitis A and B viruses (Choo et al., 1989), with about 170 million people being infected. The majority of HCV-infected individuals develop chronic infection, which may progress to liver cirrhosis and hepatocellular carcinoma (HCC). HCV is a member of the family Flaviviridae and its genome consists of a single-stranded, positive-sense RNA of approximately 9600 nt, which encodes a polyprotein precursor of about 3010 aa. Currently, clinical HCV isolates are classified into six genotypes and more than 60 subtypes (Doi et al., 1996;Mellor et al., 1995;Robertson et al., 1998). The polyprotein is cleaved by signal peptidase, signal peptide peptidase and two virally encoded proteases to generate at least ten mature proteins: core, envelope glycoprotein 1 (E1), E2, p7, non-structural protein 2 (NS2), NS3, NS4A, NS4B, NS5A and NS5B Reed & Rice, 2000).HCV NS5A is part of the replication complex that catalyses replication of the viral genome. NS5A takes two forms, p56 and p58, with different degrees of phosphorylation, which may play distinct roles in the virus replication cycle (Evans 3These authors contributed equally to this work. YXX(L/I)X 6-8 YXX(L/I)] in the cytoplasmic tail of the Fc receptor c-chain or B-cell receptor subunit Iga to be activated after the engagement of immune receptors (Kurosaki et al...

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Suppression of Hepatitis C Virus Replicon by RNA Interference Directed against the NS3 and NS5B Regions of the Viral Genome

Cited by 64 publications

References 53 publications

Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA

Induction of cell death in human papillomavirus 18-positive cervical cancer cells by E6 siRNA

Efficient production of infectious hepatitis C virus with adaptive mutations in cultured hepatoma cells

Hepatitis C virus NS5A protein interacts with and negatively regulates the non-receptor protein tyrosine kinase Syk

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