Masitinib Inhibits Hepatitis A Virus Replication

Figures

• 

  Figure 1. Construction of the HuhT7-HAV/Luc cells. (A) Insertion construct of HAV/Luc. (B) HuhT7 cells [14] and HuhT7-HAV/Luc cells were seeded at a density of 1 × 105 cells/well in 12-well plates (AGC Techno Glass, Haibaragun, Shizuoka, Japan). HuhT7-HAV/Luc was treated with interferon-α-2a (IFN-α-2a; Sigma-Aldrich, Saint Louis, MO, USA) at 0 or 0.1 μg/mL. After 48 h of incubation, firefly luciferase (Fluc) activity was measured [15,17]. Data are expressed as the means and standard deviations of duplicate determinations from two independent experiments. Statistical significance was determined using a two-tailed Student’s t test. * p < 0.05, ** p < 0.01 (versus untreated control). T7, T7 promoter; P2, HAV P2; P3, HAV P3; 5′UTR, 5′ untranslated region; 3′UTR, 3′ untranslated region; polyA, polyA tail.

• 

  Figure 2. Masitinib is a candidate drug for inhibition of the hepatitis A virus (HAV) HM175-18f genotype IB subgenomic replicon. Analyses of 1134 drugs screened by luciferase assays and dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium (MTS) assays [15,17]. The results of the drug screening are shown. All drugs were plotted on a scattergram in which the Y-axis and X-axis indicate the % cell viability of HuhT7-HAV/Luc cells and the relative luciferase activity (fold) of HAV/Luc, respectively. Masitinib, cetylpyridinium chloride, nebivolol, cyclosporine and thonzonium bromide are indicated as red circles. The red horizontal line indicates 90% cell viability. The orange vertical line indicates 0.33-fold relative luciferase activity.

• 

  Figure 3. Masitinib inhibits hepatitis A virus (HAV) HA11-1299 genotype IIIA infection. Huh7 cells infected with the HAV HA-11-1299 genotype IIIA strain were treated with 10 μM masitinib; 5 µM cetylpyridinium chloride, nebivolol and cyclosporine; and 1 µM thonzonium bromide for 72 h. HAV RNA levels were examined using real-time RT-PCR [15,17,20]. Actin mRNA was used as an internal control. HAV RNA levels were significantly inhibited in masitinib-treated Huh7 cells. Data are expressed as the means and standard deviations of triplicate determinations from three independent experiments. Statistical significance was determined using a two-tailed Student’s t test. ** p < 0.01.

• 

  Figure 4. Immunofluorescence analysis of hepatitis A virus (HAV) HM175-18f genotype IB-infected Huh7 cells. Antibodies against HAV VP1 are shown in green, and nuclei stained with Hoechst are shown in blue [17,21]. Positive immunofluorescence staining observed for HAV VP1 in HAV-infected cells but not in the uninfected cells (Mock) [21]. HAV HM175-18f genotype IB-infected Huh7 cells treated with 10 µM masitinib reduced HAV VP1 staining. HAV HM175-18f genotype IB-infected Huh7 cells treated with 0.1 μg/mL interferon-α-2a (IFN) stained with HAV VP1 as the control. Scale bar: 25 μm.

• 

  Figure 5. Masitinib inhibits hepatitis A virus (HAV) internal ribosomal entry site (IRES)-mediated translation. (A) COS7-HAV-IRES cells were seeded at a density of 1 × 105 cells/well in 12-well plates (AGC Techno Glass). COS7-HAV-IRES was treated with masitinib at 0, 1 and 5 μM. After 48 h of incubation, a luciferase assay was performed. (B) Huh7 cells were seeded 24 h prior to transfection at a density of 1 × 105 cells/well in 12-well plates. Cells were transiently transfected with 0.2 μg of pSV40-HAV-IRES using Effectene Transfection Reagent (Qiagen, Chuo-ku, Tokyo, Japan ). After 24 h of transfection, the cells were treated with masitinib at 0, 1, 5 and 10 μM. After 72 h of transfection, luciferase activities were determined. The cytotoxicity of masitinib on COS7-HAV-IRES and Huh7 cells was determined (C,D). COS7-HAV-IRES and Huh7 cells were treated with masitinib at 0, 5 and 10 μM for 48 h. Cell viability was measured via dimethylthiazol carboxymethoxyphenyl sulfophenyl tetrazolium (MTS) assays. Data are expressed as the means and standard deviations of triplicate determinations from three independent experiments. We compared statistical significance using the Student’s t test in two…