Limits...
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.

Show MeSH

Related in: MedlinePlus

In vivo infectivity of Con1/wt, Con1/K1846T and Con1/NS3+K1846T genomes in uPA-SCID mice.Huh7-Lunet cells were transfected with either of these constructs, supernatants were collected 12 and 24 h post transfection, pooled for each construct and used for virus purification and concentration as described in Materials and Methods. Two mice were each inoculated with 2×108 IU HCV RNA per mouse and construct (100 µl inoculum size) and viral RNA loads in sera were determined at the indicated time points after inoculation by qRT-PCR. In case of Con1/K1846T inoculated mice, one died at week 2 (not shown) and the second shortly after week 6. While sera of Con1/wt and Con1/K1846T inoculated mice contained high viral loads already in the first blood sample, Con1/NS3+K1846T-inoculated mice remained HCV RNA negative throughout the 10 weeks observation period.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2691593&req=5

ppat-1000475-g007: In vivo infectivity of Con1/wt, Con1/K1846T and Con1/NS3+K1846T genomes in uPA-SCID mice.Huh7-Lunet cells were transfected with either of these constructs, supernatants were collected 12 and 24 h post transfection, pooled for each construct and used for virus purification and concentration as described in Materials and Methods. Two mice were each inoculated with 2×108 IU HCV RNA per mouse and construct (100 µl inoculum size) and viral RNA loads in sera were determined at the indicated time points after inoculation by qRT-PCR. In case of Con1/K1846T inoculated mice, one died at week 2 (not shown) and the second shortly after week 6. While sera of Con1/wt and Con1/K1846T inoculated mice contained high viral loads already in the first blood sample, Con1/NS3+K1846T-inoculated mice remained HCV RNA negative throughout the 10 weeks observation period.

Mentions: For each construct, two mice were inoculated with 100 µl of the concentrated stock and virus titers in serum were determined by qRT-PCR (Fig. 7). Unfortunately, one mouse inoculated with Con1/K1846T died spontaneously already at week 2 while the second mouse died at week 6, presumably a follow-up reaction of serum withdrawal. The time course of infection shown in Fig. 7 demonstrates that mice inoculated with Con1/wt or Con1/K1846T particles were readily infected and remained viremic throughout the observation period. In case of the wild type, peak viremia was observed at week 3 post inoculation and steadily declined thereafter, most likely due to a decreased survival of the engrafted human hepatocytes. In contrast, mice inoculated with supernatants from Con1/NS3+K1846T transfected cells remained negative and viral RNA was not detected in any of the serum samples. The fact that already one week post inoculation these animals were RNA negative also shows that input virus did not interfere with the read-out. The mouse inoculated with Con1/K1846T virus also was readily infected and viremia was well detectable in all available serum samples.


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 vivo infectivity of Con1/wt, Con1/K1846T and Con1/NS3+K1846T genomes in uPA-SCID mice.Huh7-Lunet cells were transfected with either of these constructs, supernatants were collected 12 and 24 h post transfection, pooled for each construct and used for virus purification and concentration as described in Materials and Methods. Two mice were each inoculated with 2×108 IU HCV RNA per mouse and construct (100 µl inoculum size) and viral RNA loads in sera were determined at the indicated time points after inoculation by qRT-PCR. In case of Con1/K1846T inoculated mice, one died at week 2 (not shown) and the second shortly after week 6. While sera of Con1/wt and Con1/K1846T inoculated mice contained high viral loads already in the first blood sample, Con1/NS3+K1846T-inoculated mice remained HCV RNA negative throughout the 10 weeks observation period.
© Copyright Policy
Related In: Results  -  Collection

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

ppat-1000475-g007: In vivo infectivity of Con1/wt, Con1/K1846T and Con1/NS3+K1846T genomes in uPA-SCID mice.Huh7-Lunet cells were transfected with either of these constructs, supernatants were collected 12 and 24 h post transfection, pooled for each construct and used for virus purification and concentration as described in Materials and Methods. Two mice were each inoculated with 2×108 IU HCV RNA per mouse and construct (100 µl inoculum size) and viral RNA loads in sera were determined at the indicated time points after inoculation by qRT-PCR. In case of Con1/K1846T inoculated mice, one died at week 2 (not shown) and the second shortly after week 6. While sera of Con1/wt and Con1/K1846T inoculated mice contained high viral loads already in the first blood sample, Con1/NS3+K1846T-inoculated mice remained HCV RNA negative throughout the 10 weeks observation period.
Mentions: For each construct, two mice were inoculated with 100 µl of the concentrated stock and virus titers in serum were determined by qRT-PCR (Fig. 7). Unfortunately, one mouse inoculated with Con1/K1846T died spontaneously already at week 2 while the second mouse died at week 6, presumably a follow-up reaction of serum withdrawal. The time course of infection shown in Fig. 7 demonstrates that mice inoculated with Con1/wt or Con1/K1846T particles were readily infected and remained viremic throughout the observation period. In case of the wild type, peak viremia was observed at week 3 post inoculation and steadily declined thereafter, most likely due to a decreased survival of the engrafted human hepatocytes. In contrast, mice inoculated with supernatants from Con1/NS3+K1846T transfected cells remained negative and viral RNA was not detected in any of the serum samples. The fact that already one week post inoculation these animals were RNA negative also shows that input virus did not interfere with the read-out. The mouse inoculated with Con1/K1846T virus also was readily infected and viremia was well detectable in all available serum samples.

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