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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

Structures of subgenomic replicons and mutants. Shown are diagrammatic representations of the replicons used in this study, with changes to the luciferase gene (L and l), deletion of the neomycin gene (N), and replacement of the GDD motif in NS5B with GND (or AAG in genotypes 3 and 4) to make it replication defective labeled.
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Figure 1: Structures of subgenomic replicons and mutants. Shown are diagrammatic representations of the replicons used in this study, with changes to the luciferase gene (L and l), deletion of the neomycin gene (N), and replacement of the GDD motif in NS5B with GND (or AAG in genotypes 3 and 4) to make it replication defective labeled.

Mentions: The Con1 subgenomic replicon was provided by R. Bartenschlager (11), SGR-JFH1 and SGR-JFH1/GND were provided by J. McLauchlan (20), and S52/SG-Feo(AII) and ED43/SG-Feo(VYG) were obtained from C. Rice (21). A synthetic DNA sequence used to replace the WT luciferase sequence was specified by using the program Sequence Mutate in the SSE package (22) and was cloned in the various replicons by using unique restriction sites at the 5′ end (AscI) and the 3′ end (PmeI) of the luciferase coding sequence (Fig. 1).


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)

Structures of subgenomic replicons and mutants. Shown are diagrammatic representations of the replicons used in this study, with changes to the luciferase gene (L and l), deletion of the neomycin gene (N), and replacement of the GDD motif in NS5B with GND (or AAG in genotypes 3 and 4) to make it replication defective labeled.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Structures of subgenomic replicons and mutants. Shown are diagrammatic representations of the replicons used in this study, with changes to the luciferase gene (L and l), deletion of the neomycin gene (N), and replacement of the GDD motif in NS5B with GND (or AAG in genotypes 3 and 4) to make it replication defective labeled.
Mentions: The Con1 subgenomic replicon was provided by R. Bartenschlager (11), SGR-JFH1 and SGR-JFH1/GND were provided by J. McLauchlan (20), and S52/SG-Feo(AII) and ED43/SG-Feo(VYG) were obtained from C. Rice (21). A synthetic DNA sequence used to replace the WT luciferase sequence was specified by using the program Sequence Mutate in the SSE package (22) and was cloned in the various replicons by using unique restriction sites at the 5′ end (AscI) and the 3′ end (PmeI) of the luciferase coding sequence (Fig. 1).

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