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/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.
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/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.
Authors: Rodney P Kincaid; Victor L Lam; Rachel P Chirayil; Glenn Randall; Christopher S Sullivan Journal: Proc Natl Acad Sci U S A Date: 2018-07-23 Impact factor: 11.205
Authors: Yueh-Ming Loo; David M Owen; Kui Li; Andrea K Erickson; Cynthia L Johnson; Penny M Fish; D Spencer Carney; Ting Wang; Hisashi Ishida; Mitsutoshi Yoneyama; Takashi Fujita; Takeshi Saito; William M Lee; Curt H Hagedorn; Daryl T-Y Lau; Steven A Weinman; Stanley M Lemon; Michael Gale Journal: Proc Natl Acad Sci U S A Date: 2006-04-03 Impact factor: 11.205
Authors: Thomas Pietschmann; Artur Kaul; George Koutsoudakis; Anna Shavinskaya; Stephanie Kallis; Eike Steinmann; Karim Abid; Francesco Negro; Marlene Dreux; Francois-Loic Cosset; Ralf Bartenschlager Journal: Proc Natl Acad Sci U S A Date: 2006-05-01 Impact factor: 11.205