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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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Related in: MedlinePlus

Transient replication of HCV Con1-derived constructs in Huh-7 cells and release of core protein from transfected cells.(A) Schematic representation of transfected RNA genomes. The polyprotein is indicated by an open box, the individual functional proteins are separated by vertical lines. Non-translated regions are depicted as shaded bars, REMs and the position of the mutation destroying the active site of the NS5B RdRp (D318N) are specified above the respective positions in the coding region. (B) Transient RNA replication of full length Con1-derived genomes. Ten µg of in vitro transcribed RNA of the constructs specified in the top were transfected into Huh-7 cells that were harvested at given time points. Total cellular RNA was prepared and HCV RNA and beta-actin RNA were detected by Northern hybridization. (C) The amount of HCV RNA was determined by phospho imaging and is expressed relative to the input determined 4 h post transfection. Values were normalized for equal RNA loading as determined with the beta-actin specific signal. (D) Time course of accumulation of HCV core in cell culture supernatant of transfected Huh7 cells. Cells were transfected and seeded as described in (A). At given time points, culture medium was harvested, filtered through 0.45 µm pore-size filters, and analysed for core protein by ELISA. Duplicate measurements, mean value of duplicates and standard errors of the means are given. (E) Efficiency of core release from cells transfected with Con1/wt or the adapted genome Con1/NS3+S2197P [27]. Amounts of core protein accumulated intracellularly or in cell culture medium were determined by ELISA and used to calculate the percentage of intracellular core protein released into the supernatant of transfected cells for each given time point. Mean values of two independent electroporations including standard errors of the means are shown.
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ppat-1000475-g001: Transient replication of HCV Con1-derived constructs in Huh-7 cells and release of core protein from transfected cells.(A) Schematic representation of transfected RNA genomes. The polyprotein is indicated by an open box, the individual functional proteins are separated by vertical lines. Non-translated regions are depicted as shaded bars, REMs and the position of the mutation destroying the active site of the NS5B RdRp (D318N) are specified above the respective positions in the coding region. (B) Transient RNA replication of full length Con1-derived genomes. Ten µg of in vitro transcribed RNA of the constructs specified in the top were transfected into Huh-7 cells that were harvested at given time points. Total cellular RNA was prepared and HCV RNA and beta-actin RNA were detected by Northern hybridization. (C) The amount of HCV RNA was determined by phospho imaging and is expressed relative to the input determined 4 h post transfection. Values were normalized for equal RNA loading as determined with the beta-actin specific signal. (D) Time course of accumulation of HCV core in cell culture supernatant of transfected Huh7 cells. Cells were transfected and seeded as described in (A). At given time points, culture medium was harvested, filtered through 0.45 µm pore-size filters, and analysed for core protein by ELISA. Duplicate measurements, mean value of duplicates and standard errors of the means are given. (E) Efficiency of core release from cells transfected with Con1/wt or the adapted genome Con1/NS3+S2197P [27]. Amounts of core protein accumulated intracellularly or in cell culture medium were determined by ELISA and used to calculate the percentage of intracellular core protein released into the supernatant of transfected cells for each given time point. Mean values of two independent electroporations including standard errors of the means are shown.

Mentions: HCV is a positive strand RNA virus which belongs to the family Flaviviridae [1]. Its genome of about 9.6 kb is composed of the 5′non-translated region (NTR), an open reading frame encoding a large polyprotein, and the 3′NTR [2] (Fig. 1A). In the N-terminal region, the polyprotein is processed by cellular proteases to yield the structural proteins Core (C), envelope proteins 1 and 2 (E1, E2), and p7. Cleavage of the non-structural (NS) proteins is accomplished by NS2 at the NS2/3 site and by the NS3 protease at all remaining sites [2]. NS4B induces cellular membrane alterations thought to provide a scaffold for the viral replication machinery [3],[4]. NS5B is the viral RNA-dependent RNA polymerase whereas NS5A is an RNA binding phosphoprotein involved in RNA replication and virus assembly [5]–[8]. Two NS5A phospho variants have been described assumed to correspond to a basal and a hyper phosphorylated form (p56 and p58, respectively) [9]. Phosphorylation of NS5A appears to be mediated by casein kinase I and II [7],[10]. Interestingly, interference with NS5A hyperphosphorylation by inhibitors of casein kinase I such as H479 enhances viral RNA replication by more than 10-fold arguing that this modification is of disadvantage for high level replication [11].


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)

Transient replication of HCV Con1-derived constructs in Huh-7 cells and release of core protein from transfected cells.(A) Schematic representation of transfected RNA genomes. The polyprotein is indicated by an open box, the individual functional proteins are separated by vertical lines. Non-translated regions are depicted as shaded bars, REMs and the position of the mutation destroying the active site of the NS5B RdRp (D318N) are specified above the respective positions in the coding region. (B) Transient RNA replication of full length Con1-derived genomes. Ten µg of in vitro transcribed RNA of the constructs specified in the top were transfected into Huh-7 cells that were harvested at given time points. Total cellular RNA was prepared and HCV RNA and beta-actin RNA were detected by Northern hybridization. (C) The amount of HCV RNA was determined by phospho imaging and is expressed relative to the input determined 4 h post transfection. Values were normalized for equal RNA loading as determined with the beta-actin specific signal. (D) Time course of accumulation of HCV core in cell culture supernatant of transfected Huh7 cells. Cells were transfected and seeded as described in (A). At given time points, culture medium was harvested, filtered through 0.45 µm pore-size filters, and analysed for core protein by ELISA. Duplicate measurements, mean value of duplicates and standard errors of the means are given. (E) Efficiency of core release from cells transfected with Con1/wt or the adapted genome Con1/NS3+S2197P [27]. Amounts of core protein accumulated intracellularly or in cell culture medium were determined by ELISA and used to calculate the percentage of intracellular core protein released into the supernatant of transfected cells for each given time point. Mean values of two independent electroporations including standard errors of the means are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2691593&req=5

ppat-1000475-g001: Transient replication of HCV Con1-derived constructs in Huh-7 cells and release of core protein from transfected cells.(A) Schematic representation of transfected RNA genomes. The polyprotein is indicated by an open box, the individual functional proteins are separated by vertical lines. Non-translated regions are depicted as shaded bars, REMs and the position of the mutation destroying the active site of the NS5B RdRp (D318N) are specified above the respective positions in the coding region. (B) Transient RNA replication of full length Con1-derived genomes. Ten µg of in vitro transcribed RNA of the constructs specified in the top were transfected into Huh-7 cells that were harvested at given time points. Total cellular RNA was prepared and HCV RNA and beta-actin RNA were detected by Northern hybridization. (C) The amount of HCV RNA was determined by phospho imaging and is expressed relative to the input determined 4 h post transfection. Values were normalized for equal RNA loading as determined with the beta-actin specific signal. (D) Time course of accumulation of HCV core in cell culture supernatant of transfected Huh7 cells. Cells were transfected and seeded as described in (A). At given time points, culture medium was harvested, filtered through 0.45 µm pore-size filters, and analysed for core protein by ELISA. Duplicate measurements, mean value of duplicates and standard errors of the means are given. (E) Efficiency of core release from cells transfected with Con1/wt or the adapted genome Con1/NS3+S2197P [27]. Amounts of core protein accumulated intracellularly or in cell culture medium were determined by ELISA and used to calculate the percentage of intracellular core protein released into the supernatant of transfected cells for each given time point. Mean values of two independent electroporations including standard errors of the means are shown.
Mentions: HCV is a positive strand RNA virus which belongs to the family Flaviviridae [1]. Its genome of about 9.6 kb is composed of the 5′non-translated region (NTR), an open reading frame encoding a large polyprotein, and the 3′NTR [2] (Fig. 1A). In the N-terminal region, the polyprotein is processed by cellular proteases to yield the structural proteins Core (C), envelope proteins 1 and 2 (E1, E2), and p7. Cleavage of the non-structural (NS) proteins is accomplished by NS2 at the NS2/3 site and by the NS3 protease at all remaining sites [2]. NS4B induces cellular membrane alterations thought to provide a scaffold for the viral replication machinery [3],[4]. NS5B is the viral RNA-dependent RNA polymerase whereas NS5A is an RNA binding phosphoprotein involved in RNA replication and virus assembly [5]–[8]. Two NS5A phospho variants have been described assumed to correspond to a basal and a hyper phosphorylated form (p56 and p58, respectively) [9]. Phosphorylation of NS5A appears to be mediated by casein kinase I and II [7],[10]. Interestingly, interference with NS5A hyperphosphorylation by inhibitors of casein kinase I such as H479 enhances viral RNA replication by more than 10-fold arguing that this modification is of disadvantage for high level replication [11].

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