Figure 3
(A) Effects of amantadine with or without interferon on the hepatitis A virus (HAV) internal ribosomal entry site (IRES) activities in Huh7 cells. Approximately 2 × 105 cells were seeded on a 6-well tissue culture plate (Iwaki Glass, Tokyo, Japan) 24 h prior to transfection. pSV40-HAV-IRES (0.3 μg) was transfected into Huh7 cells using the Effectene transfection reagent (Qiagen, Tokyo, Japan). 24 h after transfection, amantadine and/or IFN in various concentrations was added to cells. 48 h after transfection, cell extracts were prepared, and luciferase assays were performed using the Dual Luciferase assay system (Toyo Ink, Tokyo, Japan) according to the manufacturer's instructions [2]. For controlling the variations in transcription, IRES activity was assessed by measuring the ratio of Renilla and firefly luciferases. All samples were run in triplicate. Renilla and firefly luciferase activities were measured as relative light units using a luminescencer (JNRII-AB-2300; ATTO, Tokyo, Japan). (B, C) Effects of amantadine with or without interferon on the HAV subgenomic replicon replication in HuhT7 cells. (B) 48 h after transfection and (C) 72 h after transfection. Black columns, replication-competent HAV replicon; white columns, replication-incompetent HAV replicon (mut). Relative luciferase activities without any treatments were set at 1. Data are expressed as mean (columns) ± SD (vertical lines). *P < 0.05 and **P < 0.01, compared with untreated control by Student's t test. #P < 0.01 and ## P < 0.05, compared with amantadine alone or IFN-alpha alone by Student's t test.