Characterization of long-term cultured 420Bla+ HCV stable cells.

<p>(A and B) Huh7 cells were respectively transfected with JFH1 and 420Bla RNAs and cultured for additional two days (A) or infected with the indicated viruses at an MOI of 0.01 for 12 hr and cultured for additional 3 days (B). Single clones from transfected or infected cells were isolated and grown in 10-cm culture dishes in the presence of 10 μg/ml of blasticidin. When reaching the confluence, the cells were harvested for analyses of the viral protein expression (left panel), extracellular viral infectivity (the numbers shown in the bottom of the left panel), and intracellular viral RNA amount (right panel). The “pool” represents the collection of all the remaining survival cells after single clone isolation whereas the “JFH1” indicates the JFH1 RNA-transfected (A) or JFH1 virus-infected (B) cells without blasticidin selection and cultured in parallel. The “No RNA” in (A) indicates the mock-transfected Huh7 cells, and the “Mock” in (B) indicates Huh7 cells without HCV infection. (C–E) Huh7 cells were transfected with JFH1- or 420Bla- RNAs and cultured for additional one day. Then one half each of transfected cells were cultured in the absence (−) or presence (+) of blasticidin. The extracellular viral infectivity (C), intracellular viral RNA amounts (D), and viral protein expressions (E) of samples harvested at the indicated times were determined. (F) JFH1, 420Bla-, and 420Bla+ cells collected at the indicated times after RNA transfection were fixed and analyzed by immunostaining of NS5A (green) and nuclei (blue). Data represents mean ± SEM (n = 3) (C).