Efficient Replication of Hepatitis C Virus Genotype 1a RNAs in Cell Culture

ABSTRACT Hepatitis C virus (HCV) genotype 1 (subtypes 1a and 1b) is responsible for the majority of treatment-resistant liver disease worldwide. Thus far, efficient HCV RNA replication has been observed only for subgenomic and full-length RNAs derived from genotype 1b isolates. Here, we report the establishment of efficient RNA replication systems for genotype 1a strain H77. Replication of subgenomic and full-length H77 1a RNAs required the highly permissive Huh-7.5 hepatoma subline and adaptive amino acid substitutions in both NS3 and NS5A. Replication could be detected by RNA quantification, fluorescence-activated cell sorting, and metabolic labeling of HCV-specific proteins. Replication efficiencies were similar for subgenomic and full-length RNAs and were most efficient for HCV RNAs lacking heterologous RNA elements. Interestingly, both subtype 1a and 1b NS3 adaptive mutations are surface exposed and present on only one face of the NS3 structure. The cell culture-adapted subtype 1a replicons should be useful for basic replication studies and for antiviral development. These results are also encouraging for the development of adapted replicons for the remaining HCV genotypes.

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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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Development of hepatitis C virus production reporter-assay systems using two different hepatoma cell lines

Journal of General Virology ◽

10.1099/vir.0.040725-0 ◽

2012 ◽

Vol 93 (7) ◽

pp. 1422-1431 ◽

Cited By ~ 8

Author(s):

Midori Takeda ◽

Masanori Ikeda ◽

Yasuo Ariumi ◽

Takaji Wakita ◽

Nobuyuki Kato

Keyword(s):

Cell Culture ◽

Life Cycle ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Lines ◽

Hepatoma Cell ◽

Rna Replication ◽

Hcv Rna ◽

Reporter Assay ◽

Hepatoma Cell Lines

A hepatitis C virus (HCV) infection system was developed previously using the HCV JFH-1 strain (genotype 2a) and HuH-7 cells, and this cell culture is so far the only robust production system for HCV. In patients with chronic hepatitis C, the virological effects of pegylated interferon and ribavirin therapy differ depending on the HCV strain and the genetic background of the host. Recently, we reported the hepatoma-derived Li23 cell line, in which the JFH-1 life cycle is reproduced at a level almost equal to that in HuH-7-derived RSc cells. To monitor the HCV life cycle more easily, we here developed JFH-1 reporter-assay systems using both HuH-7- and Li23-derived cell lines. To identify any genetic mutations by long-term cell culture, HCV RNAs in HuH-7 cells were amplified 130 days after infection and subjected to sequence analysis to find adaptive mutation(s) for robust virus replication. We identified two mutations, H2505Q and V2995L, in the NS5B region. V2995L but not H2505Q enhanced JFH-1 RNA replication. However, we found that H2505Q but not V2995L enhanced HCV RNA replication of strain O (genotype 1b). We also selected highly permissive D7 cells by serial subcloning of Li23 cells. The expression levels of claudin-1 and Niemann–Pick C1-like 1 in D7 cells are higher than those in parental Li23 cells. In this study, we developed HCV JFH-1 reporter-assay systems using two distinct hepatoma cell lines, HuH-7 and Li23. The mutations in NS5B resulted in different effects on strains O and JFH-1 HCV RNA replication.

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Enhancement of Hepatitis C Virus RNA Replication by Cell Culture-Adaptive Mutations

Journal of Virology ◽

10.1128/jvi.75.10.4614-4624.2001 ◽

2001 ◽

Vol 75 (10) ◽

pp. 4614-4624 ◽

Cited By ~ 412

Author(s):

Nicole Krieger ◽

Volker Lohmann ◽

Ralf Bartenschlager

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Colony Formation ◽

Marker Gene ◽

Rna Replication ◽

Clear Correlation ◽

Transfected Cells ◽

Adaptive Mutations ◽

Selection Of

ABSTRACT Studies of the Hepatitis C virus (HCV) replication cycle have been made possible with the development of subgenomic selectable RNAs that replicate autonomously in cultured cells. In these replicons the region encoding the HCV structural proteins was replaced by the neomycin phosphotransferase gene, allowing the selection of transfected cells that support high-level replication of these RNAs. Subsequent analyses revealed that, within selected cells, HCV RNAs had acquired adaptive mutations that increased the efficiency of colony formation by an unknown mechanism. Using a panel of replicons that differed in their degrees of cell culture adaptation, in this study we show that adaptive mutations enhance RNA replication. Transient-transfection assays that did not require selection of transfected cells demonstrated a clear correlation between the level of adaptation and RNA replication. The highest replication level was found with an adapted replicon carrying two amino acid substitutions located in NS3 and one in NS5A that acted synergistically. In contrast, the nonadapted RNA replicated only transiently and at a low level. The correlation between the efficiency of colony formation and RNA replication was corroborated with replicons in which the selectable marker gene was replaced by the gene encoding firefly luciferase. Upon transfection of naive Huh-7 cells, the levels of luciferase activity directly reflected the replication efficiencies of the various replicon RNAs. These results show that cell culture-adaptive mutations enhance HCV RNA replication.

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Comprehensive Analysis of the Effects of Ordinary Nutrients on Hepatitis C Virus RNA Replication in Cell Culture

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01426-06 ◽

2007 ◽

Vol 51 (6) ◽

pp. 2016-2027 ◽

Cited By ~ 57

Author(s):

Masahiko Yano ◽

Masanori Ikeda ◽

Ken-ichi Abe ◽

Hiromichi Dansako ◽

Shogo Ohkoshi ◽

...

Keyword(s):

Cell Culture ◽

Vitamin D ◽

Hepatitis C Virus ◽

Linoleic Acid ◽

Hepatitis C ◽

Combined Treatment ◽

Rna Replication ◽

Assay System ◽

Hcv Rna ◽

Β Carotene

ABSTRACT To date, only a limited number of studies have reported finding an influence of ordinary nutrients on hepatitis C virus (HCV) RNA replication. However, the effects of other nutrients on HCV RNA replication remain largely unknown. We recently developed a reporter assay system for genome-length HCV RNA replication in hepatoma-derived HuH-7 cells (OR6). Here, using this OR6 assay system, we comprehensively examined 46 nutrients from four nutrient groups: vitamins, amino acids, fatty acids, and salts. We found that three nutrients—β-carotene, vitamin D2, and linoleic acid—inhibited HCV RNA replication and that their combination caused additive and/or synergistic effects on HCV RNA replication. In addition, combined treatment with each of the three nutrients and interferon alpha or beta or fluvastatin inhibited HCV RNA replication in an additive manner, while combined treatment with cyclosporine synergistically inhibited HCV RNA replication. In contrast, we found that vitamin E enhanced HCV RNA replication and negated the effects of the three anti-HCV nutrients and cyclosporine but not those of interferon or fluvastatin. These results will provide useful information for the treatment of chronic hepatitis C patients who also take anti-HCV nutrients as an adjunctive therapy in combination with interferon. In conclusion, among the ordinary nutrients tested, β-carotene, vitamin D2, and linoleic acid possessed anti-HCV activity in a cell culture system, and these nutrients are therefore considered to be potential candidates for enhancing the effects of interferon therapy.

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Hepatitis C Virus Subgenomic Replicons in the Human Embryonic Kidney 293 Cell Line

Journal of Virology ◽

10.1128/jvi.78.1.491-501.2004 ◽

2004 ◽

Vol 78 (1) ◽

pp. 491-501 ◽

Cited By ~ 56

Author(s):

Samir Ali ◽

Charles Pellerin ◽

Daniel Lamarre ◽

George Kukolj

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Lines ◽

Rna Replication ◽

Northern Blotting ◽

Hcv Rna ◽

Metabolic Labeling ◽

Human Embryonic Kidney ◽

Western Blots ◽

293 Cells

ABSTRACT Hepatitis C virus (HCV) infects liver cells and its replication in other cells is incompletely defined. Human hepatoma Huh-7 cells harboring subgenomic HCV replicons were used in somatic cell fusion experiments with human embryonic kidney 293 cells as a means of examining the permissiveness of 293 cells for HCV subgenomic RNA replication. 293 cells were generally not permissive for replication of Huh-7 cell-adapted replicons. However, upon coculturing of the two cell lines, we selected rare replicon-containing cells, termed 293Rep cells, that resembled parental 293 cells. Direct metabolic labeling of cells with 33P in the presence of actinomycin D and Northern blotting to detect the negative strand of the replicon demonstrated functional RNA replicons in 293Rep cells. Furthermore, Western blots revealed that 293Rep cells expressed the HCV nonstructural proteins as well as markers of the naïve 293 cells but not Huh-7 cells. Propidium iodide staining and fluorescence-activated cell sorting analysis of 293Rep cells revealed that clone 293Rep17 closely resembled naïve 293 cells. Transfection of total RNA from 293Rep17 into naïve 293 cells produced replicon-containing 293 cell lines with characteristics distinct from those of Huh-7-derived replicon cell lines. Relative to Huh-7 replicons, the 293 cell replicons were less sensitive to inhibition by alpha interferon and substantially more sensitive to inhibition by poly(I)-poly(C) double-stranded RNA. This study established HCV subgenomic replicons in nonhepatic 293 cells and demonstrated their utility in expanding the study of cellular HCV RNA replication.

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Hepatitis C Virus Genotype 5a Subgenomic Replicons for Evaluation of Direct-Acting Antiviral Agents

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03534-14 ◽

2014 ◽

Vol 58 (9) ◽

pp. 5386-5394 ◽

Cited By ~ 27

Author(s):

Constance N. Wose Kinge ◽

Christine Espiritu ◽

Nishi Prabdial-Sing ◽

Nomathamsaqa Patricia Sithebe ◽

Mohsan Saeed ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Nonstructural Protein ◽

Antiviral Agents ◽

Rna Replication ◽

Firefly Luciferase ◽

Hcv Rna ◽

Virus Genotype ◽

Subgenomic Replicon

ABSTRACTHepatitis C virus (HCV) exists as six major genotypes that differ in geographical distribution, pathogenesis, and response to antiviral therapy.In vitroreplication systems for all HCV genotypes except genotype 5 have been reported. In this study, we recovered genotype 5a full-length genomes from four infected voluntary blood donors in South Africa and established a G418-selectable subgenomic replicon system using one of these strains. The replicon derived from the wild-type sequence failed to replicate in Huh-7.5 cells. However, the inclusion of the S2205I amino acid substitution, a cell culture-adaptive change originally described for a genotype 1b replicon, resulted in a small number of G418-resistant cell colonies. HCV RNA replication in these cells was confirmed by quantification of viral RNA and detection of the nonstructural protein NS5A. Sequence analysis of the viral RNAs isolated from multiple independent cell clones revealed the presence of several nonsynonymous mutations, which were localized mainly in the NS3 protein. These mutations, when introduced back into the parental backbone, significantly increased colony formation. To facilitate convenient monitoring of HCV RNA replication levels, the mutant with the highest replication level was further modified to express a fusion protein of firefly luciferase and neomycin phosphotransferase. Using such replicons from genotypes 1a, 1b, 2a, 3a, 4a, and 5a, we compared the effects of various HCV inhibitors on their replication. In conclusion, we have established anin vitroreplication system for HCV genotype 5a, which will be useful for the development of pan-genotype anti-HCV compounds.

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Viral and Cellular Determinants of Hepatitis C Virus RNA Replication in Cell Culture

Journal of Virology ◽

10.1128/jvi.77.5.3007-3019.2003 ◽

2003 ◽

Vol 77 (5) ◽

pp. 3007-3019 ◽

Cited By ~ 304

Author(s):

Volker Lohmann ◽

Sandra Hoffmann ◽

Ulrike Herian ◽

Francois Penin ◽

Ralf Bartenschlager

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Host Cell ◽

Cell Line ◽

Rna Replication ◽

Strong Increase ◽

Hepatoma Cell Line ◽

Replication Efficiency ◽

Adaptive Mutations

ABSTRACT Studies on the replication of hepatitis C virus (HCV) have been facilitated by the development of selectable subgenomic replicons replicating in the human hepatoma cell line Huh-7 at a surprisingly high level. Analysis of the replicon population in selected cells revealed the occurrence of cell culture-adaptive mutations that enhance RNA replication substantially. To gain a better understanding of HCV cell culture adaptation, we characterized conserved mutations identified by sequence analysis of 26 independent replicon cell clones for their effect on RNA replication. Mutations enhancing replication were found in nearly every nonstructural (NS) protein, and they could be subdivided into at least two groups by their effect on replication efficiency and cooperativity: (i) mutations in NS3 with a low impact on replication but that enhanced replication cooperatively when combined with highly adaptive mutations and (ii) mutations in NS4B, -5A, and -5B, causing a strong increase in replication but being incompatible with each other. In addition to adaptive mutations, we found that the host cell plays an equally important role for efficient RNA replication. We tested several passages of the same Huh-7 cell line and found up to 100-fold differences in their ability to support replicon amplification. These differences were not due to variations in internal ribosome entry site-dependent translation or RNA degradation. In a search for cellular factor(s) that might be responsible for the different levels of permissiveness of Huh-7 cells, we found that replication efficiency decreased with increasing amounts of transfected replicon RNA, indicating that viral RNA or proteins are cytopathic or that host cell factors in Huh-7 cells limit RNA amplification. In summary, these data show that the efficiency of HCV replication in cell culture is determined both by adaptation of the viral sequence and by the host cell itself.

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Effect of Interaction between Hepatitis C Virus NS5A and NS5B on Hepatitis C Virus RNA Replication with the Hepatitis C Virus Replicon

Journal of Virology ◽

10.1128/jvi.78.6.2738-2748.2004 ◽

2004 ◽

Vol 78 (6) ◽

pp. 2738-2748 ◽

Cited By ~ 66

Author(s):

Tetsuro Shimakami ◽

Makoto Hijikata ◽

Hong Luo ◽

Yuan Yuan Ma ◽

Shuichi Kaneko ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Point Mutations ◽

Biological Significance ◽

Rna Replication ◽

Hcv Rna ◽

Internal Deletion ◽

Hepatitis C Virus Rna ◽

Adaptive Mutations ◽

Virus Rna

ABSTRACT Hepatitis C virus (HCV) NS5A has been reported to be important for the establishment of replication by adaptive mutations or localization, although its role in viral replication remains unclear. It was previously reported that NS5A interacts with NS5B via two regions of NS5A in the isolate JK-1 and modulates the activity of NS5B RdRp (Y. Shirota et al., J. Biol. Chem., 277:11149-11155, 2002), but the biological significance of this interaction has not been determined. In this study, we addressed the effect of this interaction on HCV RNA replication with an HCV replicon system derived from the isolate M1LE (H. Kishine et al., Biochem. Biophys. Res. Commun., 293:993-999, 2002). We constructed three internal deletion mutants, M1LE/5Adel-1 and M1LE/5Adel-2, each encoding NS5A which cannot bind NS5B, and M1LE/5Adel-3, encoding NS5A that can bind NS5B. After transfection into Huh-7 cells, M1LE/5Adel-3 was replication competent, but both M1LE/5Adel-1 and M1LE/5Adel-2 were not. Next we prepared 20 alanine-substituted clustered mutants within both NS5B-binding regions and examined the effect of these mutants on HCV RNA replication. Only 5 of the 20 mutants were replication competent. Subsequently, we introduced a point mutation, S225P, a deletion of S229, or S232I into NS5A and prepared cured Huh-7 cells that were cured of RNA replication by alpha interferon. Finally, with these point mutations and cured cells, we established a highly improved replicon system. In this system, only the same five mutants were replication competent. These results strongly suggest that the interaction between NS5A and NS5B is critical for HCV RNA replication in the HCV replicon system.

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Dominant negative effect of wild-type NS5A on NS5A-adapted subgenomic hepatitis C virus RNA replicon

Journal of General Virology ◽

10.1099/vir.0.80006-0 ◽

2004 ◽

Vol 85 (7) ◽

pp. 1867-1875 ◽

Cited By ~ 10

Author(s):

Rita Graziani ◽

Giacomo Paonessa

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Dominant Negative ◽

Dominant Negative Effect ◽

Hcv Rna ◽

Wild Type ◽

Subgenomic Replicon ◽

Adaptive Mutations ◽

Negative Effect

An efficient model is currently used to study hepatitis C virus (HCV) replication in cell culture. It involves transfection in Huh7, a hepatoma-derived cell line, of an antibiotic (neomycin) selectable HCV subgenomic replicon encoding the non-structural (NS) proteins from NS3 to NS5B. However, strong and sustained replication is achieved only on the appearance of adaptive mutations in viral proteins. The most effective of these adaptive mutations are concentrated mainly in NS5A, not only into the original Con1 but also in the recently established HCV-BK and HCV-H77 isolate-derived replicons. This suggests that the expression of wild-type (wt) NS5A may not allow efficient HCV RNA replication in cell culture. With the use of a β-lactamase reporter gene as a marker for HCV replication and TaqMan RNA analysis, the replication of different HCV replicons in cotransfection experiments was investigated. Comparing wt with NS5A-adapted replicons, the strong evidence accumulated showed that the expression of wt NS5A was actually able to inhibit the replication of NS5A-adapted replicons. This feature was characterized as a dominant negative effect. Interestingly, an NS5B (R2884G)-adapted replicon, containing a wt NS5A, was dominant negative on an NS5A-adapted replicon but was not inhibited by the original Con1 replicon. In conclusion, these studies revealed that the original wt Con1 replicon is not only incompetent for replication in cell culture, but is also able to interfere with NS5A-adapted replicons.

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Persistent and Transient Replication of Full-Length Hepatitis C Virus Genomes in Cell Culture

Journal of Virology ◽

10.1128/jvi.76.8.4008-4021.2002 ◽

2002 ◽

Vol 76 (8) ◽

pp. 4008-4021 ◽

Cited By ~ 277

Author(s):

Thomas Pietschmann ◽

Volker Lohmann ◽

Artur Kaul ◽

Nicole Krieger ◽

Gabriele Rinck ◽

...

Keyword(s):

Cell Culture ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Hepatoma Cell ◽

Buoyant Density ◽

Full Length ◽

Structural Proteins ◽

Hepatoma Cell Line ◽

Particle Assembly ◽

Adaptive Mutations

ABSTRACT The recently developed subgenomic hepatitis C virus (HCV) replicons were limited by the fact that the sequence encoding the structural proteins was missing. Therefore, important information about a possible influence of these proteins on replication and pathogenesis and about the mechanism of virus formation could not be obtained. Taking advantage of three cell culture-adaptive mutations that enhance RNA replication synergistically, we generated selectable full-length HCV genomes that amplify to high levels in the human hepatoma cell line Huh-7 and can be stably propagated for more than 6 months. The structural proteins are efficiently expressed, with the viral glycoproteins E1 and E2 forming heterodimers which are stable under nondenaturing conditions. No disulfide-linked glycoprotein aggregates were observed, suggesting that the envelope proteins fold productively. Electron microscopy studies indicate that cell lines harboring these full-length HCV RNAs contain lipid droplets. The majority of the core protein was found on the surfaces of these structures, whereas the glycoproteins appear to localize to the endoplasmic reticulum and cis-Golgi compartments. In agreement with this distribution, no endoglycosidase H-resistant forms of these proteins were detectable. In a search for the production of viral particles, we noticed that these cells release substantial amounts of nuclease-resistant HCV RNA-containing structures with a buoyant density of 1.04 to 1.1 g/ml in iodixanol gradients. The same observation was made in transient-replication assays using an authentic highly adapted full-length HCV genome that lacks heterologous sequences. However, the fact that comparable amounts of such RNA-containing structures were found in the supernatant of cells carrying subgenomic replicons demonstrates a nonspecific release independent of the presence of the structural proteins. These results suggest that Huh-7 cells lack host cell factors that are important for virus particle assembly and/or release.

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