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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

Replication of unmodified replicons and mutants with low-CpG/UpA luciferase. Shown are data for the replication of genotype 2a (A), 1b (B), 3a (C), and 4a (D) replicons after electroporation into Huh7.5 cells. Luciferase activity was measured at four time points (x axis) and plotted as absolute values (y axis). Error bars represent standard deviations. RLU, relative light units.
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Figure 2: Replication of unmodified replicons and mutants with low-CpG/UpA luciferase. Shown are data for the replication of genotype 2a (A), 1b (B), 3a (C), and 4a (D) replicons after electroporation into Huh7.5 cells. Luciferase activity was measured at four time points (x axis) and plotted as absolute values (y axis). Error bars represent standard deviations. RLU, relative light units.

Mentions: Genotype 2a replicons with WT and CpG/UpA-low luciferase reporter genes (replicons 2a/l-GDD and 2a/L-GDD, respectively) were transfected into Huh7.5 cells, and luciferase expression was quantified at different time points postelectroporation (Fig. 2A). To ensure that equal amounts of RNA were delivered in the cells, an aliquot of cells was taken 15 min after electroporation and treated with RNase to remove extracellular RNA, and the internalized luciferase RNA was quantified by qRT-PCR. No significant differences in the amounts of RNA were found between the different mutants used (data not shown). Despite this equal delivery of RNA, the replication kinetics of 2a/L-GDD (original) and 2a/l-GDD (CpG/UpA minimized) replicons were distinct, with approximately a hundredfold (2-log) difference in luciferase expression levels at 48 h and 96 h. This difference was greater than the 5-fold difference in luciferase gene expression levels at 4 h posttransfection and at earlier time points before significant replication of HCV had taken place (see Fig. S1 in the supplemental material).


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)

Replication of unmodified replicons and mutants with low-CpG/UpA luciferase. Shown are data for the replication of genotype 2a (A), 1b (B), 3a (C), and 4a (D) replicons after electroporation into Huh7.5 cells. Luciferase activity was measured at four time points (x axis) and plotted as absolute values (y axis). Error bars represent standard deviations. RLU, relative light units.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Replication of unmodified replicons and mutants with low-CpG/UpA luciferase. Shown are data for the replication of genotype 2a (A), 1b (B), 3a (C), and 4a (D) replicons after electroporation into Huh7.5 cells. Luciferase activity was measured at four time points (x axis) and plotted as absolute values (y axis). Error bars represent standard deviations. RLU, relative light units.
Mentions: Genotype 2a replicons with WT and CpG/UpA-low luciferase reporter genes (replicons 2a/l-GDD and 2a/L-GDD, respectively) were transfected into Huh7.5 cells, and luciferase expression was quantified at different time points postelectroporation (Fig. 2A). To ensure that equal amounts of RNA were delivered in the cells, an aliquot of cells was taken 15 min after electroporation and treated with RNase to remove extracellular RNA, and the internalized luciferase RNA was quantified by qRT-PCR. No significant differences in the amounts of RNA were found between the different mutants used (data not shown). Despite this equal delivery of RNA, the replication kinetics of 2a/L-GDD (original) and 2a/l-GDD (CpG/UpA minimized) replicons were distinct, with approximately a hundredfold (2-log) difference in luciferase expression levels at 48 h and 96 h. This difference was greater than the 5-fold difference in luciferase gene expression levels at 4 h posttransfection and at earlier time points before significant replication of HCV had taken place (see Fig. S1 in the supplemental material).

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