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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

In vitro translation assay of wild-type and low-CpG/UpA luciferase replicons. Shown are measurements of luciferase activity of translation products of WT and CpG/UpA-low HCV replicons of genotypes 1 to 4 after a 30-min incubation. Bars show the means of data from two replicates; error bars show standard deviations.
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Figure 5: In vitro translation assay of wild-type and low-CpG/UpA luciferase replicons. Shown are measurements of luciferase activity of translation products of WT and CpG/UpA-low HCV replicons of genotypes 1 to 4 after a 30-min incubation. Bars show the means of data from two replicates; error bars show standard deviations.

Mentions: The increased replication of replicons with alterations in dinucleotide composition may have originated from differences in the efficiencies of translation of the luciferase gene through associated alterations in codon usage or codon pair bias (25–27). Alternatively, as demonstrated for echovirus 7, changes in CpG and UpA frequencies may influence the cellular response to infection and induce greater restriction of replication (17). To investigate the effects of CpG and UpA dinucleotide frequency changes on translation, replicons from all four genotypes containing the original (L) or modified (l) luciferase genes were assayed for translation efficiency in an in vitro translation assay (Fig. 5). Despite the large differences in codon usage and codon pair bias between the insect-derived luciferase gene and the CpG/UpA-minimized mutant sequence (Table 1), expression levels of the original and mutant forms of the luciferase gene in all four replicons of genotypes 1 to 4 were similar. The two forms of the luciferase gene showed at most 2-fold differences in translation efficiency but with no evidence for any consistently higher expression levels of the CpG/UpA-low luciferase sequences than those of the wild type (Fig. 5). To ensure that the assay mixture was not saturated with RNA that narrowed differences in expression levels, the assay was repeated by using different RNA transcript amounts. Transfection of 4 times more and 4 times less RNA confirmed that the readouts for the assay concentrations used were in the linear range (see Fig. S2 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)

In vitro translation assay of wild-type and low-CpG/UpA luciferase replicons. Shown are measurements of luciferase activity of translation products of WT and CpG/UpA-low HCV replicons of genotypes 1 to 4 after a 30-min incubation. Bars show the means of data from two replicates; error bars show standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: In vitro translation assay of wild-type and low-CpG/UpA luciferase replicons. Shown are measurements of luciferase activity of translation products of WT and CpG/UpA-low HCV replicons of genotypes 1 to 4 after a 30-min incubation. Bars show the means of data from two replicates; error bars show standard deviations.
Mentions: The increased replication of replicons with alterations in dinucleotide composition may have originated from differences in the efficiencies of translation of the luciferase gene through associated alterations in codon usage or codon pair bias (25–27). Alternatively, as demonstrated for echovirus 7, changes in CpG and UpA frequencies may influence the cellular response to infection and induce greater restriction of replication (17). To investigate the effects of CpG and UpA dinucleotide frequency changes on translation, replicons from all four genotypes containing the original (L) or modified (l) luciferase genes were assayed for translation efficiency in an in vitro translation assay (Fig. 5). Despite the large differences in codon usage and codon pair bias between the insect-derived luciferase gene and the CpG/UpA-minimized mutant sequence (Table 1), expression levels of the original and mutant forms of the luciferase gene in all four replicons of genotypes 1 to 4 were similar. The two forms of the luciferase gene showed at most 2-fold differences in translation efficiency but with no evidence for any consistently higher expression levels of the CpG/UpA-low luciferase sequences than those of the wild type (Fig. 5). To ensure that the assay mixture was not saturated with RNA that narrowed differences in expression levels, the assay was repeated by using different RNA transcript amounts. Transfection of 4 times more and 4 times less RNA confirmed that the readouts for the assay concentrations used were in the linear range (see Fig. S2 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