Effect of individual adaptive mutations on the production of infectious JFH1 HCV.

<p>(Panel A) Schematic of the wild-type (wt) and mutant JFH1 genomes. The location of HCV JFH1 adaptive mutations in the core, E2, NS3, NS5A and NS5B genes are indicated by * and are specified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044965#pone-0044965-g002" target="_blank">Figure 2</a>. (Panel B) Cell culture production of recombinant viruses with specific mutations. <i>In vitro</i> transcribed RNAs with specific mutations were electroporated into Huh-7.5 cells to generate recombinant mutant viruses. The transfected cells were passaged for six days. Infectivity titers of the culture supernatants were measured by titration assays. Viral titers are expressed as focus-forming units per milliliter (f.f.u./ml) (done in triplicate and performed twice). The data are presented as mean ± standard deviation (n = 6). (Panel C) HCV-RNA levels in cells six days after transfection. Intracellular-HCV-RNA levels were analyzed by quantitative RT-PCR. The mean ± standard deviations for two independent experiments are presented (qPCR assays, n = 6) (Methods).