FIG. 4.

Antiviral effect of NHC on HCV RNA levels. (A) Dynamics of the antiviral effect of NHC over a 12-day treatment period are shown. ΔΔCt_{(HCV Ct−rRNA Ct−no-drug Ct)}; ♦, no treatment; ▪, NHC at 10 μM; ▴, NHC at 100 μM. The antiviral activities at day 4 of IFN-α-2a (100 IU/ml [left bar]) and ribavirin (100 μM [right bar]) are given as references. S.D., standard deviation. (B) Cells were treated for 4 days, followed by RNA extraction and Northern blot analysis. The upper gel shows the positive-strand (+) HCV RNA with a hybridization signal at approximately 8 kb. The lower gel shows the control hybridization of GAPDH mRNA at approximately 1.4 kb. Lane 1, parental Huh7 cell line; lane 2, HCV replicon cells, untreated; lane 3, 100 IU of IFN-α-2a/ml; lane 4, 65 μM NHC. (C) Cells were treated for 4 days, followed by protein extraction and Western blot analysis. The upper gel shows the α-NS5B reactivity of proteins transferred to the membrane; the lower gel is a Coomassie-stained gel illustrating equal amounts of protein in each lane (total cellular protein equivalent of 5 × 10⁴ cells per lane was loaded). Lane 1, recombinant HCV NS5B protein; lane 2, parental Huh7 cell line; lane 3, HCV replicon cells, untreated; lane 4, 100 IU of IFN-α-2a/ml; lane 5, 65 μM NHC. (D) HCV replicon cells were incubated with NHC and IFN-α-2a, either alone or in different combinations. After 94 h of incubation, total RNA was extracted and amplified in multiplex conditions. Analysis was performed by using Combostat software (Jasper, Ga.) (5).