Replication-Competent Chimeric Hepatitis C Virus Subgenomic Replicons

Objective: To utilize chimeric hepatitis C virus (HCV) replicons to select adaptive mutation(s) that allow replication of a genotype 1a replicon. Methods: We used a genetic approach to gradually apply selective pressure by generating chimeric replicons through sequential replacement of nonstructural...

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Veröffentlicht in:Intervirology 2005-01, Vol.48 (2-3), p.183-191
Hauptverfasser: Lemm, Julie A., Liu, Mengping, Rose, Ronald E., Fridell, Robert, O’Boyle II, Donald R., Colonno, Richard, Gao, Min
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container_end_page 191
container_issue 2-3
container_start_page 183
container_title Intervirology
container_volume 48
creator Lemm, Julie A.
Liu, Mengping
Rose, Ronald E.
Fridell, Robert
O’Boyle II, Donald R.
Colonno, Richard
Gao, Min
description Objective: To utilize chimeric hepatitis C virus (HCV) replicons to select adaptive mutation(s) that allow replication of a genotype 1a replicon. Methods: We used a genetic approach to gradually apply selective pressure by generating chimeric replicons through sequential replacement of nonstructural genes of a 1b replicon with genotype 1a sequences. Results: A chimeric replicon containing a genotype 1a NS5A protein did not replicate in a transient assay, but could be used to establish stable cell lines using G418 selection. The cell lines contained a K1846T mutation in NS4B which functioned as an adaptive mutation that now allowed the chimera to replicate at levels similar to wild-type replicons. Similarly, replication of a 1a NS5A5B chimera was only observed after establishment of stable cell lines, even in the presence of the K1846T mutation. Sequence analysis of this cell line revealed an additional adaptive mutation of M1496L in NS3. Lastly, by including the K1846T mutation in a replicon that was entirely genotype 1a sequence, stable 1a cell lines could be established. Conclusion: These studies identify an NS4B adaptive mutation, K1846T, which allows establishment of a replication-competent 1a replicon and demonstrate the utility of this chimeric approach for establishing replicons for various HCV genotypes.
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source Karger Journals; MEDLINE
subjects Amino Acid Substitution
Genes, Viral
Genome, Viral
Genotype
Hepacivirus - genetics
Hepacivirus - physiology
Hepatitis C
Hepatitis C virus
Mutation
Original Paper
Replicon
Viral Nonstructural Proteins - genetics
Viral Nonstructural Proteins - physiology
Virus Replication
title Replication-Competent Chimeric Hepatitis C Virus Subgenomic Replicons
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