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Enhancement of the Replication of Hepatitis C Virus Replicons of Genotypes 1 to 4 by Manipulation of CpG and UpA Dinucleotide Frequencies and Use of Cell Lines Expressing SECL14L2 for Antiviral Resistance Testing.

Witteveldt J, Martin-Gans M, Simmonds P - Antimicrob. Agents Chemother. (2016)

Bottom Line: Removal of CpG and UpA dinucleotides in the neomycin gene or deletion of the whole gene in replicons of genotype 3a (S52) and genotype 4a (ED43) enhanced replication, but phenotypic effects on altering luciferase gene composition were minimal.A further 10-fold replication enhancement of replicons from all four genotypes was achieved by using a transgenic Huh7.5 cell line expressing SECL14L2, whose expression showed a dose-dependent effect on HCV replication that was reversible by small interfering RNA (siRNA) knockdown of gene expression.These methods of replication enhancement provide new tools for monitoring the susceptibility and resistance of a wide range of HCV genotypes to DAAs.

View Article: PubMed Central - PubMed

Affiliation: Roslin Institute, University of Edinburgh, Easter Bush, Edinburgh, United Kingdom.

No MeSH data available.


Related in: MedlinePlus

Comparison of replication of replicons from genotypes 1 to 4 in the Huh7.5 and 23 cell lines. Shown are data for the replication of replicons from genotypes 1 to 4 generated in this study in the Huh7.5 and 23 cell lines; the right panels show luciferase expression from the replication-incompetent control. Error bars depict standard deviations.
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Figure 4: Comparison of replication of replicons from genotypes 1 to 4 in the Huh7.5 and 23 cell lines. Shown are data for the replication of replicons from genotypes 1 to 4 generated in this study in the Huh7.5 and 23 cell lines; the right panels show luciferase expression from the replication-incompetent control. Error bars depict standard deviations.

Mentions: Replicons with all four genotype backbones and the corresponding CpG/UpA-low luciferase mutants were electroporated into the SEC14L2-expressing cell line 23, and replication was compared to that in parental Huh7.5 cells (Fig. 4). Replication of the 2a/L-GDD replicon was increased ∼17-fold in the SEC14L2-expressing cell line at 72 h p.e.; a slightly lower enhancement was observed for the 2a/l-GDD (CpG/UpA-low) construct (Fig. 4A). Replication enhancement was synergistic, with a 340-fold increase in luciferase expression levels for 2a/l-GDD in 23 cells compared to that for 2a/L-GDD in Huh7.5 cells. Consistent with a role for SEC14L2 expression in enhancing replication, luciferase expression levels from the defective 2a/L-GND and 2a/l-GND replicons were comparable between cell lines (Fig. 4A).


Enhancement of the Replication of Hepatitis C Virus Replicons of Genotypes 1 to 4 by Manipulation of CpG and UpA Dinucleotide Frequencies and Use of Cell Lines Expressing SECL14L2 for Antiviral Resistance Testing.

Witteveldt J, Martin-Gans M, Simmonds P - Antimicrob. Agents Chemother. (2016)

Comparison of replication of replicons from genotypes 1 to 4 in the Huh7.5 and 23 cell lines. Shown are data for the replication of replicons from genotypes 1 to 4 generated in this study in the Huh7.5 and 23 cell lines; the right panels show luciferase expression from the replication-incompetent control. Error bars depict standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4862521&req=5

Figure 4: Comparison of replication of replicons from genotypes 1 to 4 in the Huh7.5 and 23 cell lines. Shown are data for the replication of replicons from genotypes 1 to 4 generated in this study in the Huh7.5 and 23 cell lines; the right panels show luciferase expression from the replication-incompetent control. Error bars depict standard deviations.
Mentions: Replicons with all four genotype backbones and the corresponding CpG/UpA-low luciferase mutants were electroporated into the SEC14L2-expressing cell line 23, and replication was compared to that in parental Huh7.5 cells (Fig. 4). Replication of the 2a/L-GDD replicon was increased ∼17-fold in the SEC14L2-expressing cell line at 72 h p.e.; a slightly lower enhancement was observed for the 2a/l-GDD (CpG/UpA-low) construct (Fig. 4A). Replication enhancement was synergistic, with a 340-fold increase in luciferase expression levels for 2a/l-GDD in 23 cells compared to that for 2a/L-GDD in Huh7.5 cells. Consistent with a role for SEC14L2 expression in enhancing replication, luciferase expression levels from the defective 2a/L-GND and 2a/l-GND replicons were comparable between cell lines (Fig. 4A).

Bottom Line: Removal of CpG and UpA dinucleotides in the neomycin gene or deletion of the whole gene in replicons of genotype 3a (S52) and genotype 4a (ED43) enhanced replication, but phenotypic effects on altering luciferase gene composition were minimal.A further 10-fold replication enhancement of replicons from all four genotypes was achieved by using a transgenic Huh7.5 cell line expressing SECL14L2, whose expression showed a dose-dependent effect on HCV replication that was reversible by small interfering RNA (siRNA) knockdown of gene expression.These methods of replication enhancement provide new tools for monitoring the susceptibility and resistance of a wide range of HCV genotypes to DAAs.

View Article: PubMed Central - PubMed

Affiliation: Roslin Institute, University of Edinburgh, Easter Bush, Edinburgh, United Kingdom.

No MeSH data available.


Related in: MedlinePlus