Abstract
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.
Highlights
Infection that follows [3,4,5]
We show that JFH-1 can replicate in two other hepatocyte-derived cell lines, HepG2 and IMY-N9, an Hepatitis C virus (HCV)-replicable cell line formed by fusing human primary cultured hepatocytes and HepG2 cells [13]
We previously demonstrated that the genotype 2a replicon could efficiently replicate in Huh7, and here we showed that it can replicate in other hepatocyte-derived cells, HepG2 and IMY-N9 cells
Summary
Hepatitis C virus (HCV), one of the plus-strand RNA viruses, is a principal agent in post-transfusion and sporadic acute hepatitis [1, 2]. Total RNA and genomic DNA were simultaneously isolated from amplified clones using the ISOGENTM reagent (Nippon Gene, Tokyo, Japan). Another portion of the cell pellet was dissolved in radioimmune precipitation assay buffer containing 0.1% SDS for protein analysis. To confirm the integrity of the genomic DNAs isolated from replicon cells, the -globin gene was amplified using primers GH-20 (5Ј-GAAGAGCCAAGGACAGGTAC-3Ј) and GH-21 (5Ј-GGAAAATAGACCAATAGGCAG-3Ј) as described previously [16]. RT-PCR and Sequencing Analysis—The cDNAs of the HCV RNA replicon were synthesized from total RNA isolated from replicon RNAtransfected cells These cDNAs were subsequently amplified with DNA polymerase (TaKaRa LATM Taq, Takara Bio Inc., Otsu, Japan). Statistics—Statistical analysis was conducted with the Mann-Whitney U test, and p values of Ͻ0.05 were considered significant
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