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 naive 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.
Liang
Chengyu
C
Department of Molecular Genetics and Microbiology, Stony Brook University, Stony Brook, NY 11790, USA.
Rieder
Elizabeth
E
Hahm
Bumsuk
B
Jang
Sung Key
SK
Paul
Aniko
A
Wimmer
Eckard
E
D000998:Antiviral Agents / Q000494:pharmacology; D045744:Cell Line, Tumor; D024882:Drug Resistance, Viral / Q000235:genetics*; D005838:Genotype; D016174:Hepacivirus / Q000187:drug effects* / Q000235:genetics*; D006801:Humans; D009154:Mutation; D011691:Puromycin / Q000494:pharmacology*; D012367:RNA, Viral; D012093:Replicon; D014779:Virus Replication
D000998:Antiviral Agents; D012367:RNA, Viral; D011691:Puromycin