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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

Coelectroporation of CpG/UpA-low and WT luciferase-containing replicons. Shown are luciferase expression levels in Huh7.5 cells (A) and 23 cells (B) after electroporation of CpG/UpA-low, WT, or both replicons; the y-axis scale is normalized to luciferase expression by 1b/l-GDD (100%). Error bars depict standard deviations.
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Figure 6: Coelectroporation of CpG/UpA-low and WT luciferase-containing replicons. Shown are luciferase expression levels in Huh7.5 cells (A) and 23 cells (B) after electroporation of CpG/UpA-low, WT, or both replicons; the y-axis scale is normalized to luciferase expression by 1b/l-GDD (100%). Error bars depict standard deviations.

Mentions: As the restriction in replication engendered by increased frequencies of CpG and UpA dinucleotides was not mediated by differences in translation efficiency or greater RNA instability, we next investigated whether the inhibition of replication in replicons expressing native (CpG/UpA-high) luciferase genes was mediated by the replicon containing the gene sequence (in cis) or induced a global change in the cell in permissivity to HCV replication (in trans). 1b/L-GDD and 1b/l-GDD (containing WT and CpG/UpA-low luciferase gene sequences, respectively) were coelectroporated into Huh7.5 cells (Fig. 6A). The presence of 1b/L-GDD RNA minimally reduced the replication of 1b/l-GDD (∼50%) compared to the expression levels of 1b/l-GDD electroporated alone. The experiment was repeated with the SEC14L2-expressing cell line, where there was no effect on the expression of luciferase compared to that with electroporation of 1b/l-GDD alone (Fig. 6B). These findings provide evidence that the effects of dinucleotide composition are mediated locally (in cis) on the RNA molecule possessing the altered dinucleotide composition rather than such a sequence inducing whole cellular restriction on replication (e.g., mediated through the induction of interferon beta).


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)

Coelectroporation of CpG/UpA-low and WT luciferase-containing replicons. Shown are luciferase expression levels in Huh7.5 cells (A) and 23 cells (B) after electroporation of CpG/UpA-low, WT, or both replicons; the y-axis scale is normalized to luciferase expression by 1b/l-GDD (100%). Error bars depict standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Coelectroporation of CpG/UpA-low and WT luciferase-containing replicons. Shown are luciferase expression levels in Huh7.5 cells (A) and 23 cells (B) after electroporation of CpG/UpA-low, WT, or both replicons; the y-axis scale is normalized to luciferase expression by 1b/l-GDD (100%). Error bars depict standard deviations.
Mentions: As the restriction in replication engendered by increased frequencies of CpG and UpA dinucleotides was not mediated by differences in translation efficiency or greater RNA instability, we next investigated whether the inhibition of replication in replicons expressing native (CpG/UpA-high) luciferase genes was mediated by the replicon containing the gene sequence (in cis) or induced a global change in the cell in permissivity to HCV replication (in trans). 1b/L-GDD and 1b/l-GDD (containing WT and CpG/UpA-low luciferase gene sequences, respectively) were coelectroporated into Huh7.5 cells (Fig. 6A). The presence of 1b/L-GDD RNA minimally reduced the replication of 1b/l-GDD (∼50%) compared to the expression levels of 1b/l-GDD electroporated alone. The experiment was repeated with the SEC14L2-expressing cell line, where there was no effect on the expression of luciferase compared to that with electroporation of 1b/l-GDD alone (Fig. 6B). These findings provide evidence that the effects of dinucleotide composition are mediated locally (in cis) on the RNA molecule possessing the altered dinucleotide composition rather than such a sequence inducing whole cellular restriction on replication (e.g., mediated through the induction of interferon beta).

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