Efficient replication of a full-length hepatitis C virus genome, strain O, in cell culture, and development of a luciferase reporter system

Since the first report of frameshifting in HCV-1, its sequence has been the paradigm for examining the mechanism that directs alternative translation of the hepatitis C virus (HCV) genome. The region encoding the core protein from this strain contains a cluster of 10 adenines at codons 8–11, which is thought to direct programmed ribosomal frameshifting (PRF), but formal evidence for this process has not been established unequivocally. To identify the mechanisms of frameshifting, this study used a bicistronic dual luciferase reporter system in a coupled transcription/translation in vitro assay. This approach revealed +1 as well as –1 frameshifting, whereas point mutations, selectively introduced between codons 8 and 11, demonstrated that PRF did not readily account for frameshifting in strain HCV-1. Sequence analysis of cDNAs derived from RNA transcribed by T7 RNA polymerase in the dual luciferase reporter system, as well as in both a subgenomic replicon and an infectious clone derived from strain JFH1, identified additions and deletions of adenines between codons 8 and 11 due to transcriptional slippage (TS). Moreover, RNA isolated from cells infected with virus generated by JFH1 containing the A-rich tract also contained heterogeneity in the adenine sequence, strongly suggesting TS by the NS5B viral polymerase. These findings have important implications for insight into frameshifting events in HCV-1 and demonstrate for the first time the involvement of transcriptional slippage in this recoding event.

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Development of NS3/4A Protease-Based Reporter Assay Suitable for Efficiently Assessing Hepatitis C Virus Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00601-09 ◽

2009 ◽

Vol 53 (11) ◽

pp. 4825-4834 ◽

Cited By ~ 20

Author(s):

Kao-Lu Pan ◽

Jin-Ching Lee ◽

Hsing-Wen Sung ◽

Teng-Yuang Chang ◽

John T.-A. Hsu

Keyword(s):

Cell Culture ◽

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

High Throughput ◽

High Throughput Screening ◽

Reporter System ◽

Viral Protease ◽

Peptide Cleavage

ABSTRACT A cell culture system for the production of hepatitis C virus (HCV) whole virions has greatly accelerated studies of the virus life cycle and the discovery of anti-HCV agents. However, the quantification of the HCV titers in a whole-virus infection/replication system currently relies mostly on reverse transcription-PCR or immunofluorescence assay, which would be cumbersome for high-throughput drug screening. To overcome this problem, this study has generated a novel cell line, Huh7.5-EG(Δ4B5A)SEAP, that carries a dual reporter, EG(Δ4B5A)SEAP. The EG(Δ4B5A)SEAP reporter is a viral protease-cleavable fusion protein in which the enhanced green fluorescence protein is linked to secreted alkaline phosphatase (SEAP) in frame via Δ4B5A, a short peptide cleavage substrate for NS3/4A viral protease. This study demonstrates that virus replication/infection in the Huh7.5-EG(Δ4B5A)SEAP cells can be quantitatively indicated by measuring the SEAP activity in cell culture medium. The levels of SEAP released from HCV-infected Huh7.5-EG(Δ4B5A)SEAP cells correlated closely with the amounts of HCV in the inocula. The Huh7.5-EG(Δ4B5A)SEAP cells were also shown to be a suitable host for the discovery of anti-HCV inhibitors by using known compounds that target multiple stages of the HCV life cycle. The Z′-factor of this assay ranged from 0.64 to 0.74 in 96-well plates, indicating that this reporter system is suitable for high-throughput screening of prospective anti-HCV agents.

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Involvement of Creatine Kinase B in Hepatitis C Virus Genome Replication through Interaction with the Viral NS4A Protein

Journal of Virology ◽

10.1128/jvi.02179-08 ◽

2009 ◽

Vol 83 (10) ◽

pp. 5137-5147 ◽

Cited By ~ 30

Author(s):

Hiromichi Hara ◽

Hideki Aizaki ◽

Mami Matsuda ◽

Fumiko Shinkai-Ouchi ◽

Yasushi Inoue ◽

...

Keyword(s):

Creatine Kinase ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Viral Genome ◽

Proteome Analysis ◽

Virus Genome ◽

Genome Replication ◽

Creatine Kinase B ◽

Efficient Replication

ABSTRACT Persistent infection with hepatitis C virus (HCV) is a major cause of chronic liver diseases. The aim of this study was to identify host cell factor(s) participating in the HCV replication complex (RC) and to clarify the regulatory mechanisms of viral genome replication dependent on the host-derived factor(s) identified. By comparative proteome analysis of RC-rich membrane fractions and subsequent gene silencing mediated by RNA interference, we identified several candidates for RC components involved in HCV replication. We found that one of these candidates, creatine kinase B (CKB), a key ATP-generating enzyme that regulates ATP in subcellular compartments of nonmuscle cells, is important for efficient replication of the HCV genome and propagation of infectious virus. CKB interacts with HCV NS4A protein and forms a complex with NS3-4A, which possesses multiple enzyme activities. CKB upregulates both NS3-4A-mediated unwinding of RNA and DNA in vitro and replicase activity in permeabilized HCV replicating cells. Our results support a model in which recruitment of CKB to the HCV RC compartment, which has high and fluctuating energy demands, through its interaction with NS4A is important for efficient replication of the viral genome. The CKB-NS4A association is a potential target for the development of a new type of antiviral therapeutic strategy.

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Generation of Infectious Hepatitis C Virus in Immortalized Human Hepatocytes

Journal of Virology ◽

10.1128/jvi.80.9.4633-4639.2006 ◽

2006 ◽

Vol 80 (9) ◽

pp. 4633-4639 ◽

Cited By ~ 71

Author(s):

Tatsuo Kanda ◽

Arnab Basu ◽

Robert Steele ◽

Takaji Wakita ◽

Jan S. Ryerse ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Protein Expression ◽

Culture Media ◽

Human Hepatocytes ◽

Full Length ◽

Dependent Manner ◽

Virus Growth ◽

Hcv Genotype

ABSTRACT Progress in understanding hepatitis C virus (HCV) biology has remained a challenge due to the lack of an efficient cell culture system for virus growth. In this study, we examined HCV core protein-mediated immortalized human hepatocytes (IHH) for growth of HCV. In vitro-transcribed full-length RNA from HCV genotype 1a (clone H77) was introduced into IHH by electroporation. Reverse transcription-PCR of cellular RNA isolated from HCV genome-transfected IHH suggested that viral RNA replication occurred. IHH transfected with the full-length HCV genome also displayed viral protein expression by indirect immunofluorescence. In contrast, cells transfected with polymerase-defective HCV (H77/GND) RNA as a negative control did not exhibit expression of the viral genome. Immunogold labeling demonstrated localization of E1 protein in the rough endoplasmic reticulum of RNA-transfected IHH. Virus-like particles of ∼50 nm were observed in the cytoplasm. After being inoculated with culture media of cells transfected with the full-length HCV genome, naïve IHH displayed NS5a protein expression in a dilution-dependent manner, but expression of NS5a was inhibited by prior incubation of culture medium with HCV-infected patient sera. NS5a-positive immunofluorescence of cell culture media of IHH transfected with full-length H77 RNA yielded ∼4.5 × 104 to 1 × 105 focus-forming units/ml. A similar level of virus growth was observed upon transfection of RNA from HCV genotype 2a (JFH1) into IHH. Taken together, our results suggest that IHH support HCV genome replication and virus assembly.

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Virion-like structures in HeLa G cells transfected with the full-length sequence of the hepatitis C virus genome

European Journal of Gastroenterology & Hepatology ◽

10.1097/00042737-199604000-00028 ◽

1996 ◽

Vol 8 (4) ◽

pp. 410

Author(s):

M. Mizuno ◽

G. Yamada ◽

T. Tanaka ◽

K. Shimotohno ◽

M. Takatani ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Genome ◽

Full Length ◽

G Cells ◽

Full Length Sequence

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A comparative cell biological analysis reveals only limited functional homology between the NS5A proteins of hepatitis C virus and GB virus B

Journal of General Virology ◽

10.1099/vir.0.2008/001131-0 ◽

2008 ◽

Vol 89 (8) ◽

pp. 1911-1920 ◽

Cited By ~ 4

Author(s):

Jamel Mankouri ◽

Andrew Milward ◽

Kenneth R. Pryde ◽

Lucile Warter ◽

Annette Martin ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Signalling ◽

Pi3k Pathway ◽

Luciferase Reporter ◽

Signalling Pathways ◽

New World Monkeys ◽

Structural Protein ◽

Reporter System ◽

Early Endosomes

GB virus B (GBV-B) is the closest relative to hepatitis C virus (HCV) with which it shares a common genome organization, however, unlike HCV in humans, it generally causes an acute resolving hepatitis in New World monkeys. It is important to understand the factors regulating the different disease profiles of the two viruses and in this regard, as well as playing a key role in viral RNA replication, the HCV NS5A non-structural protein modulates a variety of host-cell signalling pathways. We have shown previously that HCV NS5A, expressed either alone, or in the context of the complete polyprotein, inhibits the Ras-extracellular-signal-regulated kinase (Erk) pathway and activates the phosphoinositide 3-kinase (PI3K) pathway. In this report, we investigate whether these functions are shared by GBV-B NS5A. Immunofluorescence analysis revealed that a C-terminally FLAG-tagged GBV-B NS5A exhibited a punctate cytoplasmic distribution. However, unlike HCV NS5A, the GBV-B protein did not partially co-localize with early endosomes. Utilizing a transient luciferase reporter system, we observed that GBV-B NS5A failed to inhibit Ras–Erk signalling, however GBV-B NS5A expression did result in the elevation of β-catenin-dependent transcription via activation of the PI3K pathway. These effects of GBV-B and HCV NS5A on the PI3K and Ras–Erk pathways were confirmed in cells harbouring subgenomic replicons derived from the two viruses. Based on these data we speculate that the differential effects of the two NS5A proteins on cellular signalling pathways may contribute to the differences in the natural history of the two viruses.

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Reduction of Hepatitis C Virus NS5A Hyperphosphorylation by Selective Inhibition of Cellular Kinases Activates Viral RNA Replication in Cell Culture

Journal of Virology ◽

10.1128/jvi.78.23.13306-13314.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13306-13314 ◽

Cited By ~ 102

Author(s):

Petra Neddermann ◽

Manuela Quintavalle ◽

Chiara Di Pietro ◽

Angelica Clementi ◽

Mauro Cerretani ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Kinase Inhibitors ◽

Rna Replication ◽

Hcv Rna ◽

Adaptive Mutations ◽

Direct Demonstration ◽

Huh7 Cells ◽

Efficient Replication

ABSTRACT Efficient replication of hepatitis C virus (HCV) subgenomic RNA in cell culture requires the introduction of adaptive mutations. In this report we describe a system which enables efficient replication of the Con1 subgenomic replicon in Huh7 cells without the introduction of adaptive mutations. The starting hypothesis was that high amounts of the NS5A hyperphosphorylated form, p58, inhibit replication and that reduction of p58 by inhibition of specific kinase(s) below a certain threshold enables HCV replication. Upon screening of a panel of kinase inhibitors, we selected three compounds which inhibited NS5A phosphorylation in vitro and the formation of NS5A p58 in cell culture. Cells, transfected with the HCV Con1 wild-type sequence, support HCV RNA replication upon addition of any of the three compounds. The effect of the kinase inhibitors was found to be synergistic with coadaptive mutations in NS3. This is the first direct demonstration that the presence of high amounts of NS5A-p58 causes inhibition of HCV RNA replication in cell culture and that this inhibition can be relieved by kinase inhibitors.

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Full-length complementary DNA of hepatitis C virus genome from an infectious blood sample

Hepatology ◽

10.1002/hep.510270242 ◽

1998 ◽

Vol 27 (2) ◽

pp. 621-627 ◽

Cited By ~ 49

Author(s):

Hideki Aizaki ◽

Yoichiro Aoki ◽

Takashi Harada ◽

Koji Ishii ◽

Tetsuro Suzuki ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Blood Sample ◽

Virus Genome ◽

Full Length ◽

Complementary Dna

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Near full length hepatitis C virus genome reconstruction by next generation sequencing based on genotype-independent amplification

Digestive and Liver Disease ◽

10.1016/j.dld.2015.03.015 ◽

2015 ◽

Vol 47 (7) ◽

pp. 608-612 ◽

Cited By ~ 4

Author(s):

Barbara Bartolini ◽

Emanuela Giombini ◽

Isabella Abbate ◽

Marina Selleri ◽

Gabriella Rozera ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Next Generation Sequencing ◽

Virus Genome ◽

Full Length ◽

Next Generation ◽

Genome Reconstruction ◽

Generation Sequencing

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Selectable Subgenomic and Genome-Length Dicistronic RNAs Derived from an Infectious Molecular Clone of the HCV-N Strain of Hepatitis C Virus Replicate Efficiently in Cultured Huh7 Cells

Journal of Virology ◽

10.1128/jvi.76.6.2997-3006.2002 ◽

2002 ◽

Vol 76 (6) ◽

pp. 2997-3006 ◽

Cited By ~ 310

Author(s):

Masanori Ikeda ◽

MinKyung Yi ◽

Kui Li ◽

Stanley M. Lemon

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Northern Analysis ◽

Infectious Molecular Clone ◽

Wild Type ◽

Molecular Clone ◽

Adaptive Mutations ◽

Huh7 Cells ◽

Efficient Replication

ABSTRACT Dicistronic, selectable subgenomic replicons derived from the Con1 strain of hepatitis C virus (HCV) are capable of autonomous replication in cultured Huh7 cells (Lohmann et al., Science 285:110-113, 1999). However, adaptive mutations in the NS3, NS5A, and/or NS5B proteins are required for efficient replication of these RNAs and increase by orders of magnitude the numbers of G418-resistant colonies selected following transfection of Huh7 cells. Here, we demonstrate that a subgenomic replicon (NNeo/3-5B) derived from an infectious molecular clone of a second genotype 1b virus, HCV-N (Beard et al., Hepatology 30:316-324, 1999) is also capable of efficient replication in Huh7 cells. G418-resistant cells selected following transfection with NNeo/3-5B RNA contained abundant NS5A antigen and HCV RNA detectable by Northern analysis. Replicon RNA in one of three clonally isolated cell lines contained no mutations in the NS3-NS5B polyprotein, confirming that adaptive mutations are not required for efficient replication in these cells. However, the deletion of a unique 4-amino-acid insertion that is present within the interferon sensitivity-determining region (ISDR) of the NS5A protein in wild-type HCV-N drastically decreased the number of G418-resistant colonies obtained following transfection of Huh7 cells. This effect could be reversed by inclusion of a previously described Con1 cell culture-adaptive mutation (S2005→I), confirming that this natural insertion has a controlling role in determining the replication capacity of wild-type HCV-N RNA in Huh7 cells. Additional selectable, dicistronic RNAs encoding NS2-NS5B, E1-NS5B, or the full-length HCV polyprotein were also capable of replication and gave rise to G418-resistant cell clones following transfection of Huh7 cells. We conclude that RNA derived from this documented infectious molecular clone has a unique capacity for replication in Huh7 cells in the absence of additional cell culture-adaptive mutations.

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