Faculty Opinions recommendation of Adaptive mutations producing efficient replication of genotype 1a hepatitis C virus RNA in normal Huh7 cells.

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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Adaptive Mutations Producing Efficient Replication of Genotype 1a Hepatitis C Virus RNA in Normal Huh7 Cells

Journal of Virology ◽

10.1128/jvi.78.15.7904-7915.2004 ◽

2004 ◽

Vol 78 (15) ◽

pp. 7904-7915 ◽

Cited By ~ 106

Author(s):

MinKyung Yi ◽

Stanley M. Lemon

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Host Cell ◽

Nonstructural Protein ◽

Northern Analysis ◽

Adaptive Mutations ◽

Amino Terminal ◽

Genotype 1A ◽

Genotype 1B ◽

Huh7 Cells

ABSTRACT Despite recent successes in generating subgenomic RNA replicons derived from genotype 1b strains of hepatitis C virus (HCV) that replicate efficiently in cultured cells, it has proven difficult to generate efficiently replicating RNAs from any other genotype of HCV. This includes genotype 1a, even though it is closely related to genotype 1b. We show here that an important restriction to replication of the genotype 1a H77c strain RNA in normal Huh7 cells resides within the amino-terminal 75 residues of the NS3 protease. We identified adaptive mutations located within this NS3 domain and within NS4A, in close proximity to the essential protease cofactor sequence, that act cooperative to substantially enhance the replication of this genotype 1a RNA in Huh7 cells. These and additional adaptive mutations, identified through a series of iterative transfections and the selection of G418-resistant cell clones, form two groups associating with distinct nonstructural protein domains: the NS3/4A protease and NS5A. A combination of mutations from both groups led to robust replication of otherwise unmodified H77c genomic RNA that was readily detectable by northern analysis within 4 days of transfection into Huh7 cells. We speculate that these adaptive mutations favorably influence assembly of the replicase complex with host cell-specific proteins, or alternatively promote interactions of NS3/4A and/or NS5A with cellular proteins involved in host cell antiviral defenses.

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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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Mutations that permit efficient replication of hepatitis C virus RNA in Huh-7 cells prevent productive replication in chimpanzees

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.212532699 ◽

2002 ◽

Vol 99 (22) ◽

pp. 14416-14421 ◽

Cited By ~ 199

Author(s):

J. Bukh ◽

T. Pietschmann ◽

V. Lohmann ◽

N. Krieger ◽

K. Faulk ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Hepatitis C Virus Rna ◽

Virus Rna ◽

Efficient Replication ◽

Productive Replication

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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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