Figure 3. Replication of JFH1 infectious clone carrying a Y325N mutation in the presence and absence of CsA.

(A) Infectivity of in vitro-transcribed RNA from JFH1-Y325N and JFH1-wt were determined by infectious center assay. Huh7.5 cells were electroporated with RNA transcripts and cleared supernatant was examined for number of infectious particles by serially diluting and passing on to fresh Huh7.5 cells. The infectious centers were monitored by immunofluorescence assay by using anti-NS5A monoclonal antibodies. Y axis shows number of infectious virus produced. The average of two independent experiments with error bar representing standard deviation is presented. Student's t test was used to calculate p values. JFH1-wt vs JFH1-wt-CsA, p = 0.012, indicated by star. (B) Northern blot analysis of total RNA from either non treated or pretreated Huh7.5 cells electroporated with RNA from JFH1-Y325N and JFH1-wt. Total RNA was isolated from these cells and analyzed by probing with ³²P-labeled random probe derived from C-terminal portion of NS5A. The signal was quantified with Storm and the reading was normalized with respect to actin control. The y axis represents relative transcript levels with JFH1-Y325N arbitrarily set to 100%.