FIG 2.

NeoB suppressed HCV RNA replication. (A) Schematic representation for the HCV life cycle and the assay systems used to evaluate each step of the life cycle. HCV pseudoparticle (HCVpp) assay permits evaluation of effects on the early step of the life cycle including attachment and entry. HCV replicon reproduces the middle step, consisting of translation and RNA replication. An HCV replicon carrying a GND substitution within the polymerase active motif of NS5B (replicon-GND) reproduces translation in the absence of RNA replication. The HCVcc assay permits evaluation of effects on the whole HCV life cycle including the late steps, assembly and release. Double-membrane vesicles (DMVs) are proposed to be sites for the formation of viral replication complexes. (B) For an HCVpp assay, Huh7.5.1 cells were left untreated or pretreated with 20 μM NeoB or 2 nM bafilomycin A1 (a known inhibitor of viral entry) for 2 h and then infected with HCVpp for 4 h in the presence or absence of compounds (left). After free virus and compounds were washed out, cells were incubated for an additional 72 h and then lysed for measurement of luciferase activity driven by HCVpp infection. Replicon assays evaluating translation/replication (middle) and translation (right) were also performed. Huh-7 cells were transfected with HCV JFH-1 subgenomic replicon RNA or the RNA encoding a GND substitution in the NS5B polymerase active motif GDD, and then cells were cultured in the presence or absence of 20 μM NeoB or 5 μg/ml cyclosporine (a known inhibitor of replication) for 48 h or in the presence or absence of 5 μM acriflavine (a known inhibitor of translation) for 4 h. Luciferase activity was quantified, and the relative values are indicated. (C) Effect of NeoB on the IFN signaling pathway. Huh7.5.1 cells transfected with a reporter plasmid carrying IFN-sensitive responsive elements (ISRE) upstream of the luciferase gene were treated with or without 20 μM NeoB and/or 100 IU/ml IFN-α for 24 h, and the luciferase activity was measured (left). HCV-infected Huh7.5.1 cells were treated with or without 20 and 30 μM NeoB and/or 100 IU/ml IFN-α to detect IFN-α downstream genes, ISG56 and MxA, and actin as an internal control by immunoblotting (right). Band intensities are indicated as described in the legend of Fig. 1B. **, P < 0.01, N.S., not significant.