Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon.
OBJECTIVE
Although the hepatitis C virus (HCV) subgenomic replicon system has been widely used in the study of HCV, this system is available only for a few related genotypes. To develop a new replicon system, the genotype 2a clone JFH-1 was isolated from a patient with fulminant hepatitis.


METHODS
A genotype 2a replicon was constructed by isolating the consensus sequence of JFH-1, transfecting G418-selectable subgenomic transcripts into Huh7 cells, and estimating the replication efficiency.


RESULTS
The colony formation efficiency of the JFH-1 replicon was 53,200 colonies/microg RNA, significantly higher than that of the genotype 1b cell-adapted replicon, at 909 colonies/microg RNA (P < 0.05). The JFH-1 replicon RNA was transmissible to naive Huh7 cells by transfection of cellular RNA from cells containing the replicon. Sequencing of cloned replicon RNAs revealed that all but 1 had at least 1 nonsynonymous mutation. One of these mutations was shown to enhance the colony formation efficiency of the JFH-1 replicon. Furthermore, the JFH-1 replicon RNA replicated efficiently without G418 selection in a transient replication assay.


CONCLUSIONS
The genotype 2a subgenomic replicon was established in Huh7 cells and replicated efficiently with or without G418 selection. This subgenomic replicon could replicate without common amino acid mutations; however, some of the mutations found in the clones might be important in conferring higher replication phenotypes. This system provides a powerful new tool for researching HCV.
Kato
Takanobu
T
Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Fuchu, Japan.
Date
Tomoko
T
Miyamoto
Michiko
M
Furusaka
Akihiro
A
Tokushige
Katsutoshi
K
Mizokami
Masashi
M
Wakita
Takaji
T
D002460:Cell Line; D016679:Genome, Viral; D005838:Genotype; D016174:Hepacivirus / Q000145:classification / Q000235:genetics*; D006801:Humans; D009154:Mutation; D012367:RNA, Viral / Q000096:biosynthesis; D012093:Replicon; D014162:Transfection; D014779:Virus Replication
D012367:RNA, Viral