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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

Expression of SEC14L2 in different cell lines and effect of HCV replication. (A) Quantification of SEC14L2 expression by Western blotting of cell lysates from different cell lines. Samples were normalized to the total amount of protein loaded; a control immunoblot using β-tubulin is shown at the bottom. P, parental Huh7.5 cell line. (B) Luciferase expression in different cell lines at different time points after electroporation of 1b/l-GDD. The values are normalized to luciferase expression of the 1b/l-GDD replicon in the parental Huh7.5 cell line at each time point. (C) Effect of transfection of SEC14L2-specific siRNA in cell line 23 on SEC14L2 expression levels as determined by Western blotting. (D) Replication of 1b/l-GDD in siRNA-treated and irrelevant siRNA-treated 23 cells. Error bars depict standard deviations.
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Figure 3: Expression of SEC14L2 in different cell lines and effect of HCV replication. (A) Quantification of SEC14L2 expression by Western blotting of cell lysates from different cell lines. Samples were normalized to the total amount of protein loaded; a control immunoblot using β-tubulin is shown at the bottom. P, parental Huh7.5 cell line. (B) Luciferase expression in different cell lines at different time points after electroporation of 1b/l-GDD. The values are normalized to luciferase expression of the 1b/l-GDD replicon in the parental Huh7.5 cell line at each time point. (C) Effect of transfection of SEC14L2-specific siRNA in cell line 23 on SEC14L2 expression levels as determined by Western blotting. (D) Replication of 1b/l-GDD in siRNA-treated and irrelevant siRNA-treated 23 cells. Error bars depict standard deviations.

Mentions: To investigate whether the reported enhancement of replication by the expression of SEC14L2 (19) could also be achieved in a transient-transfection replication assay, a number of clonal cell lines stably expressing SEC14L2 were made. Based on SEC14L2 expression levels measured by RT-qPCR, several lines were selected and tested for SEC14L2 protein expression levels by Western blotting (Fig. 3A). All five cell lines selected constitutively expressed detectable but variable levels of SEC14L2 protein, while it was undetectable in the parental Huh7.5 cell line (labeled “P”).


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)

Expression of SEC14L2 in different cell lines and effect of HCV replication. (A) Quantification of SEC14L2 expression by Western blotting of cell lysates from different cell lines. Samples were normalized to the total amount of protein loaded; a control immunoblot using β-tubulin is shown at the bottom. P, parental Huh7.5 cell line. (B) Luciferase expression in different cell lines at different time points after electroporation of 1b/l-GDD. The values are normalized to luciferase expression of the 1b/l-GDD replicon in the parental Huh7.5 cell line at each time point. (C) Effect of transfection of SEC14L2-specific siRNA in cell line 23 on SEC14L2 expression levels as determined by Western blotting. (D) Replication of 1b/l-GDD in siRNA-treated and irrelevant siRNA-treated 23 cells. Error bars depict standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4862521&req=5

Figure 3: Expression of SEC14L2 in different cell lines and effect of HCV replication. (A) Quantification of SEC14L2 expression by Western blotting of cell lysates from different cell lines. Samples were normalized to the total amount of protein loaded; a control immunoblot using β-tubulin is shown at the bottom. P, parental Huh7.5 cell line. (B) Luciferase expression in different cell lines at different time points after electroporation of 1b/l-GDD. The values are normalized to luciferase expression of the 1b/l-GDD replicon in the parental Huh7.5 cell line at each time point. (C) Effect of transfection of SEC14L2-specific siRNA in cell line 23 on SEC14L2 expression levels as determined by Western blotting. (D) Replication of 1b/l-GDD in siRNA-treated and irrelevant siRNA-treated 23 cells. Error bars depict standard deviations.
Mentions: To investigate whether the reported enhancement of replication by the expression of SEC14L2 (19) could also be achieved in a transient-transfection replication assay, a number of clonal cell lines stably expressing SEC14L2 were made. Based on SEC14L2 expression levels measured by RT-qPCR, several lines were selected and tested for SEC14L2 protein expression levels by Western blotting (Fig. 3A). All five cell lines selected constitutively expressed detectable but variable levels of SEC14L2 protein, while it was undetectable in the parental Huh7.5 cell line (labeled “P”).

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