Replication of a novel subgenomic HCV genotype 1a replicon expressing a puromycin resistance gene in Huh-7 cells

Genotype 1a is a most prevalent genotype of hepatitis C virus in North America yet HCV replication has been studied predominantly with genotype 1b subgenomic replicons under neomycin selection in Huh-7 cells. Development of 1a-related dicistronic replicons under neo selection proved difficult and required either “conditioned” Huh-7 cells and/or chimeric genomes harboring pre-engineered adaptive mutations. We report the construction of a novel dicistronic genotype 1a(H77C) replicon expressing the puromycin N-acetyltransferase (PAC) gene as a selectable marker that, without prior introduction of adaptive mutations, allows establishment of puromycin-resistant Huh-7 colonies after transfection of naïve Huh-7 cells. The large majority of HCV1a/PAC replicons did not reveal any adaptive mutations on short-term passage of Huh-7 cells. Continued passage led to mutations in the non-structural genes although these mutations did not significantly enhance replication of the original replicon. Transfection with total cellular RNA isolated from HCV1a/PAC replicon-containing cells led to a significant increase in colony-forming ability. The data identify PAC as an efficient selectable marker for studies of HCV replication, which may be useful with different genotypes in different host cell systems.