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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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Density profiles of HCV particles derived from Con1/wt, Con1/K1846T, JFH-1 and patient serum.(A) Approximately 22 ml of filtered cell culture fluid harvested 24 h after transfection of Huh-7 cells with Con1/wt RNA were concentrated via ultracentrifugation over a 60% iodixanol cushion. In case of JFH-1 approximately 26 ml supernatant harvested 96 h post transfection and concentrated in the same manner were used. The concentrates were overlaid with a linear iodixanol gradient (0%–60%) and spun for 20 h at 110,000 g at 4°C. In a similar experiment, 0.5 ml of high titer patient serum was resolved in an iodixanol density gradient. Twelve fractions à 1ml were harvested from the top, and the amount of HCV core protein contained in each fraction was determined by Trak C ELISA, respectively. HCV core content per fraction is plotted against the density of the respective fraction. (B) Result of an analogous experiment but using culture supernatant of Huh-7 cells after transfection with Con1/wt or Con1/K1846T.
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ppat-1000475-g005: Density profiles of HCV particles derived from Con1/wt, Con1/K1846T, JFH-1 and patient serum.(A) Approximately 22 ml of filtered cell culture fluid harvested 24 h after transfection of Huh-7 cells with Con1/wt RNA were concentrated via ultracentrifugation over a 60% iodixanol cushion. In case of JFH-1 approximately 26 ml supernatant harvested 96 h post transfection and concentrated in the same manner were used. The concentrates were overlaid with a linear iodixanol gradient (0%–60%) and spun for 20 h at 110,000 g at 4°C. In a similar experiment, 0.5 ml of high titer patient serum was resolved in an iodixanol density gradient. Twelve fractions à 1ml were harvested from the top, and the amount of HCV core protein contained in each fraction was determined by Trak C ELISA, respectively. HCV core content per fraction is plotted against the density of the respective fraction. (B) Result of an analogous experiment but using culture supernatant of Huh-7 cells after transfection with Con1/wt or Con1/K1846T.

Mentions: Densities of Con1-derived particles were determined by using density gradient centrifugation in comparison to virus particles produced from JFH-1 transfected Huh-7 cells and virus particles contained in a high titer patient serum. Gradient fractions were harvested from the top and core protein amounts contained in each fraction were determined by ELISA. We did not use qRT-PCR because of residual amounts of in vitro transcripts and plasmid DNA in virus preparations generated by RNA transfection precluding unambiguous measurements of particle associated HCV RNA. In the first set of experiments we compared particles derived from Con1/wt, JFH-1 and patient serum (Fig. 5A). Most particles present in patient serum had a density of about 1.04 g/ml probably representing viruses associated with very low density and low density lipoproteins [33]–[36]. The minor peak with a density of ca. 1.12 g/ml may correspond to virus that is less complexed with lipoproteins. Interestingly, cell culture-derived Con1/wt and JFH-1 particles exhibited an analogous density profile but with very different ratios when compared to the patient-derived particles. Most particles had a density of 1.14 g/ml, whereas only a minor and broad peak was found at very low density in the range of 1.03 to 1.08 g/ml. In this respect, density profiles of Con1/wt and JFH-1 derived virus particles were indistinguishable. Likewise, density profiles of Con1/wt and Con1/K1846T derived particles were also very similar (Fig. 5B) arguing that the genome with this REM in NS4B is capable of producing virus particles, too.


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)

Density profiles of HCV particles derived from Con1/wt, Con1/K1846T, JFH-1 and patient serum.(A) Approximately 22 ml of filtered cell culture fluid harvested 24 h after transfection of Huh-7 cells with Con1/wt RNA were concentrated via ultracentrifugation over a 60% iodixanol cushion. In case of JFH-1 approximately 26 ml supernatant harvested 96 h post transfection and concentrated in the same manner were used. The concentrates were overlaid with a linear iodixanol gradient (0%–60%) and spun for 20 h at 110,000 g at 4°C. In a similar experiment, 0.5 ml of high titer patient serum was resolved in an iodixanol density gradient. Twelve fractions à 1ml were harvested from the top, and the amount of HCV core protein contained in each fraction was determined by Trak C ELISA, respectively. HCV core content per fraction is plotted against the density of the respective fraction. (B) Result of an analogous experiment but using culture supernatant of Huh-7 cells after transfection with Con1/wt or Con1/K1846T.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2691593&req=5

ppat-1000475-g005: Density profiles of HCV particles derived from Con1/wt, Con1/K1846T, JFH-1 and patient serum.(A) Approximately 22 ml of filtered cell culture fluid harvested 24 h after transfection of Huh-7 cells with Con1/wt RNA were concentrated via ultracentrifugation over a 60% iodixanol cushion. In case of JFH-1 approximately 26 ml supernatant harvested 96 h post transfection and concentrated in the same manner were used. The concentrates were overlaid with a linear iodixanol gradient (0%–60%) and spun for 20 h at 110,000 g at 4°C. In a similar experiment, 0.5 ml of high titer patient serum was resolved in an iodixanol density gradient. Twelve fractions à 1ml were harvested from the top, and the amount of HCV core protein contained in each fraction was determined by Trak C ELISA, respectively. HCV core content per fraction is plotted against the density of the respective fraction. (B) Result of an analogous experiment but using culture supernatant of Huh-7 cells after transfection with Con1/wt or Con1/K1846T.
Mentions: Densities of Con1-derived particles were determined by using density gradient centrifugation in comparison to virus particles produced from JFH-1 transfected Huh-7 cells and virus particles contained in a high titer patient serum. Gradient fractions were harvested from the top and core protein amounts contained in each fraction were determined by ELISA. We did not use qRT-PCR because of residual amounts of in vitro transcripts and plasmid DNA in virus preparations generated by RNA transfection precluding unambiguous measurements of particle associated HCV RNA. In the first set of experiments we compared particles derived from Con1/wt, JFH-1 and patient serum (Fig. 5A). Most particles present in patient serum had a density of about 1.04 g/ml probably representing viruses associated with very low density and low density lipoproteins [33]–[36]. The minor peak with a density of ca. 1.12 g/ml may correspond to virus that is less complexed with lipoproteins. Interestingly, cell culture-derived Con1/wt and JFH-1 particles exhibited an analogous density profile but with very different ratios when compared to the patient-derived particles. Most particles had a density of 1.14 g/ml, whereas only a minor and broad peak was found at very low density in the range of 1.03 to 1.08 g/ml. In this respect, density profiles of Con1/wt and JFH-1 derived virus particles were indistinguishable. Likewise, density profiles of Con1/wt and Con1/K1846T derived particles were also very similar (Fig. 5B) arguing that the genome with this REM in NS4B is capable of producing virus particles, too.

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