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Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations.

Pietschmann T, Zayas M, Meuleman P, Long G, Appel N, Koutsoudakis G, Kallis S, Leroux-Roels G, Lohmann V, Bartenschlager R - PLoS Pathog. (2009)

Bottom Line: Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A.We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo.Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Virology, University of Heidelberg, Heidelberg, Germany.

ABSTRACT
With the advent of subgenomic hepatitis C virus (HCV) replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs), previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A), but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I) as well as NS5A (S2204R), whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes.

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In vitro infectivity of Con1/wt particles released from transfected Huh7.5 cells.(A) Enhancement of HCV RNA replication by kinase inhibitor H479. Subgenomic Con1 luciferase replicons were transfected into Huh7.5 cells that were seeded into medium containing H479 at concentrations specified in the right. Cell lysates were prepared at 4 h and 48 h after transfection and luciferase activities were determined. The replication defective replicon Con1/D318N served as negative control. Cells treated with DMSO only were used as reference. For each construct, values were normalized to the luciferase activity of the respective DMSO control in order to determine the fold induction or reduction of replication. Data (mean±S.D.; n = 3) were analyzed using two-way ANOVA test. (B) Experimental approach used to detect in vitro infectivity of Con1 virus. (C) Immunofluorescence analysis of Huh7.5 cells 72 h after inoculation with supernatant from cells transfected with the Con1/wt genome (upper panels) or mock transfected cells (lower panels). Cells were treated either with DMSO only (Mock; left panels), or with H479 (middle panels) or with H479 and ConcanamycinA (right panels) as specified in panel (B). Cells were fixed 72 h after inoculation and NS5A was detected by immunofluorescence microscopy. (D) Detection of NS3 and NS5A expression in Huh7.5 cells inoculated with cell-free concentrated supernatant containing Con1/K1846T particles. Cells were fixed 48 h after inoculation and processed for indirect immunofluorescence. Nuclei were counterstained with DAPI.
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ppat-1000475-g006: In vitro infectivity of Con1/wt particles released from transfected Huh7.5 cells.(A) Enhancement of HCV RNA replication by kinase inhibitor H479. Subgenomic Con1 luciferase replicons were transfected into Huh7.5 cells that were seeded into medium containing H479 at concentrations specified in the right. Cell lysates were prepared at 4 h and 48 h after transfection and luciferase activities were determined. The replication defective replicon Con1/D318N served as negative control. Cells treated with DMSO only were used as reference. For each construct, values were normalized to the luciferase activity of the respective DMSO control in order to determine the fold induction or reduction of replication. Data (mean±S.D.; n = 3) were analyzed using two-way ANOVA test. (B) Experimental approach used to detect in vitro infectivity of Con1 virus. (C) Immunofluorescence analysis of Huh7.5 cells 72 h after inoculation with supernatant from cells transfected with the Con1/wt genome (upper panels) or mock transfected cells (lower panels). Cells were treated either with DMSO only (Mock; left panels), or with H479 (middle panels) or with H479 and ConcanamycinA (right panels) as specified in panel (B). Cells were fixed 72 h after inoculation and NS5A was detected by immunofluorescence microscopy. (D) Detection of NS3 and NS5A expression in Huh7.5 cells inoculated with cell-free concentrated supernatant containing Con1/K1846T particles. Cells were fixed 48 h after inoculation and processed for indirect immunofluorescence. Nuclei were counterstained with DAPI.

Mentions: Attempts to directly demonstrate infectivity of Con1/wt or Con1/K1846T derived particles in cell culture were complicated by the low replicative capacity of both genomes. However, recently Neddermann and colleagues demonstrated that inhibition of casein kinase I that appears to be responsible for hyperphosphorylation of NS5A, with compound H479 results in substantial enhancement of RNA replication of a non-adapted genome whereas replication of a genome containing a REM in NS5A was blocked [10],[11],[38]. Assuming that enhancing replication of Con1/wt and possibly also Con1/K1846T with H479 would facilitate detection of viral proteins in infected cells, we first established the optimal concentration of this kinase inhibitor required to stimulate replication of Con1/wt and Con1/K1846T in Huh7.5 cells. In agreement with the report by Neddermann and colleagues [11] we observed an about 5-fold increase of replication of Con1/wt at 48 h post transfection when Huh7.5 cells were treated with 10 µM of H479 whereas replication of Con1/K1846T was not enhanced (Fig. 6A). Nevertheless, even under optimal conditions replication was about 10–100-fold below the level achieved with the highly cell culture adapted replicon Con1/NS3+K1846T (data not shown).


Production of infectious genotype 1b virus particles in cell culture and impairment by replication enhancing mutations.

Pietschmann T, Zayas M, Meuleman P, Long G, Appel N, Koutsoudakis G, Kallis S, Leroux-Roels G, Lohmann V, Bartenschlager R - PLoS Pathog. (2009)

In vitro infectivity of Con1/wt particles released from transfected Huh7.5 cells.(A) Enhancement of HCV RNA replication by kinase inhibitor H479. Subgenomic Con1 luciferase replicons were transfected into Huh7.5 cells that were seeded into medium containing H479 at concentrations specified in the right. Cell lysates were prepared at 4 h and 48 h after transfection and luciferase activities were determined. The replication defective replicon Con1/D318N served as negative control. Cells treated with DMSO only were used as reference. For each construct, values were normalized to the luciferase activity of the respective DMSO control in order to determine the fold induction or reduction of replication. Data (mean±S.D.; n = 3) were analyzed using two-way ANOVA test. (B) Experimental approach used to detect in vitro infectivity of Con1 virus. (C) Immunofluorescence analysis of Huh7.5 cells 72 h after inoculation with supernatant from cells transfected with the Con1/wt genome (upper panels) or mock transfected cells (lower panels). Cells were treated either with DMSO only (Mock; left panels), or with H479 (middle panels) or with H479 and ConcanamycinA (right panels) as specified in panel (B). Cells were fixed 72 h after inoculation and NS5A was detected by immunofluorescence microscopy. (D) Detection of NS3 and NS5A expression in Huh7.5 cells inoculated with cell-free concentrated supernatant containing Con1/K1846T particles. Cells were fixed 48 h after inoculation and processed for indirect immunofluorescence. Nuclei were counterstained with DAPI.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2691593&req=5

ppat-1000475-g006: In vitro infectivity of Con1/wt particles released from transfected Huh7.5 cells.(A) Enhancement of HCV RNA replication by kinase inhibitor H479. Subgenomic Con1 luciferase replicons were transfected into Huh7.5 cells that were seeded into medium containing H479 at concentrations specified in the right. Cell lysates were prepared at 4 h and 48 h after transfection and luciferase activities were determined. The replication defective replicon Con1/D318N served as negative control. Cells treated with DMSO only were used as reference. For each construct, values were normalized to the luciferase activity of the respective DMSO control in order to determine the fold induction or reduction of replication. Data (mean±S.D.; n = 3) were analyzed using two-way ANOVA test. (B) Experimental approach used to detect in vitro infectivity of Con1 virus. (C) Immunofluorescence analysis of Huh7.5 cells 72 h after inoculation with supernatant from cells transfected with the Con1/wt genome (upper panels) or mock transfected cells (lower panels). Cells were treated either with DMSO only (Mock; left panels), or with H479 (middle panels) or with H479 and ConcanamycinA (right panels) as specified in panel (B). Cells were fixed 72 h after inoculation and NS5A was detected by immunofluorescence microscopy. (D) Detection of NS3 and NS5A expression in Huh7.5 cells inoculated with cell-free concentrated supernatant containing Con1/K1846T particles. Cells were fixed 48 h after inoculation and processed for indirect immunofluorescence. Nuclei were counterstained with DAPI.
Mentions: Attempts to directly demonstrate infectivity of Con1/wt or Con1/K1846T derived particles in cell culture were complicated by the low replicative capacity of both genomes. However, recently Neddermann and colleagues demonstrated that inhibition of casein kinase I that appears to be responsible for hyperphosphorylation of NS5A, with compound H479 results in substantial enhancement of RNA replication of a non-adapted genome whereas replication of a genome containing a REM in NS5A was blocked [10],[11],[38]. Assuming that enhancing replication of Con1/wt and possibly also Con1/K1846T with H479 would facilitate detection of viral proteins in infected cells, we first established the optimal concentration of this kinase inhibitor required to stimulate replication of Con1/wt and Con1/K1846T in Huh7.5 cells. In agreement with the report by Neddermann and colleagues [11] we observed an about 5-fold increase of replication of Con1/wt at 48 h post transfection when Huh7.5 cells were treated with 10 µM of H479 whereas replication of Con1/K1846T was not enhanced (Fig. 6A). Nevertheless, even under optimal conditions replication was about 10–100-fold below the level achieved with the highly cell culture adapted replicon Con1/NS3+K1846T (data not shown).

Bottom Line: Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A.We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo.Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Virology, University of Heidelberg, Heidelberg, Germany.

ABSTRACT
With the advent of subgenomic hepatitis C virus (HCV) replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs), previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A), but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I) as well as NS5A (S2204R), whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes.

Show MeSH
Related in: MedlinePlus