Genotype 2a hepatitis C virus subgenomic replicon can replicate in HepG2 and IMY-N9 cells.
A hepatitis C virus genotype 2a subgenomic replicon, JFH-1 replicon, was previously established using the consensus sequence of clone JFH-1 from a patient with fulminant hepatitis and, in a previous report, was indicated to replicate efficiently in Huh7. Here the replication of JFH-1 replicon was tested in HepG2, a human hepatocyte-derived cell line, and in IMY-N9, a cell line developed by fusing human hepatocytes and HepG2 cells. Following transfection with in vitro transcribed replicon RNA and selection by cultivation with G418, colonies formed in both cell lines although at efficiencies substantially lower than those of Huh7. The H2476L mutation identified in the Huh7 replicon in our previous study increased the colony formation efficiencies of the JFH-1 replicon in HepG2 and IMY-N9 cells. Higher amounts of replicon RNA were detected in IMY-N9 clones than in HepG2 clones by real time detection reverse transcription-PCR, and replicon RNA replication and viral protein expression were confirmed by Northern and Western blotting in isolated clones. Sequencing of replicon RNAs revealed that mutations found in hepatitis C virus-derived regions were not identical and that two of nine HepG2 clones and three of nine IMY-N9 clones had no or one synonymous mutation. This system with the JFH-1 replicon and three cell lines is useful not only for estimating the cellular factors affecting viral activity but also for clarifying the common gene response of the host.
Department of Microbiology, Tokyo Metropolitan Institute for Neuroscience, Tokyo 183-8526, Japan.
Tomoko
T
Date
Takanobu
T
Kato
Michiko
M
Miyamoto
Zijiang
Z
Zhao
Kotaro
K
Yasui
Masashi
M
Mizokami
Takaji
T
Wakita
D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004279:DNA, Viral; D019084:Fluorescent Antibody Technique, Indirect; D016679:Genome, Viral; D005838:Genotype; D016174:Hepacivirus / Q000235:genetics*; D022781:Hepatocytes / Q000378:metabolism / Q000821:virology; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry; D012367:RNA, Viral; D012093:Replicon / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D014779:Virus Replication
D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral; D012313:RNA