Efficient replication of the genotype 2a hepatitis C virus subgenomic replicon

• Published in: Gastroenterology (New York, N.Y. 1943) Vol. 125; no. 6; pp. 1808 - 1817
• Main Authors: Kato, Takanobu, Date, Tomoko, Miyamoto, Michiko, Furusaka, Akihiro, Tokushige, Katsutoshi, Mizokami, Masashi, Wakita, Takaji
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-12-2003
• Subjects: Cell Line; Genome, Viral; Genotype; Hepacivirus - classification; Hepacivirus - genetics; Humans; Mutation; Replicon; RNA, Viral - biosynthesis; Transfection; Virus Replication; CFU; IRES; NS; neo r; 5′UTR; HCV; EMCV
• ISSN: 0016-5085; 1528-0012
• DOI: 10.1053/j.gastro.2003.09.023

Background & Aims:
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/μg RNA, significantly higher than that of the genotype 1b cell-adapted replicon, at 909 colonies/μg 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.