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 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.


CONCLUSIONS
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.
Lemm
Julie A
JA
Department of Virology, Bristol-Myers Squibb Pharmaceutical Research Institute, Wallingford, Conn 06492, USA.
Liu
Mengping
M
Rose
Ronald E
RE
Fridell
Robert
R
O'Boyle Ii
Donald R
DR
Colonno
Richard
R
Gao
Min
M
D019943:Amino Acid Substitution; D005814:Genes, Viral; D016679:Genome, Viral; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics* / Q000502:physiology*; D009154:Mutation; D012093:Replicon; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000502:physiology; D014779:Virus Replication; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; D012324:RNA-Dependent RNA Polymerase
D017361:Viral Nonstructural Proteins; C084422:NS3 protein, hepatitis C virus; D012697:Serine Endopeptidases; D043583:Nucleoside-Triphosphatase; D053487:DEAD-box RNA Helicases; D000085242:Viral Proteases; C093542:NS4 protein, hepatitis C virus; C087036:NS-5 protein, hepatitis C virus; D012324:RNA-Dependent RNA Polymerase