FIG. 7.

RNA binding activity of NS5B is enhanced in CsA-resistant replicon cells. (A) An in vitro RNA-NS5B binding assay was conducted as described in Materials and Methods. Cell lysates of CsA-treated (8 μg/ml) or untreated GS5 or RS1-2 replicon cells were incubated with poly(U)-Sepharose or protein G-Sepharose beads in the presence or absence of 32 μg/ml of CsA. Precipitates were separated by SDS-PAGE and immunoblotted with anti-NS5B and anti-CypB antibodies. Twofold amounts of cell lysate were used for “input” in the RNA binding assay. “pU” represents poly(U), and “G” represents protein G-Sepharose beads. −, absence of; +, presence of. (B) The percentage of the NS5B bound to RNA was calculated by quantifying the band intensities. The ratio of “bound” versus “input” is plotted with the twofold difference in loading taken into consideration. (C) Direct comparison of the effect of CsA on the RNA binding activity of NS5B from GS5 and RS1-2 replicons. The percentage of bound RNA in the presence or absence of CsA is calculated and shown for both GS5 and RS1-2 cells. Error bars indicate standard deviations.