Fig 5.

Clathrin heavy chain and Eps15 are required for LACV infection and replication. (A) Knockdown of clathrin heavy chain by siRNA. HeLa cells were serially transfected with siRNA (control [cont], GAPDH, or clathrin heavy chain [CHC]). Cell lysates were subjected to Western blotting for CHC, GAPDH, and ß-tubulin to confirm knockdown. (B) Knockdown of clathrin heavy chain decreases LACV infection. Twenty-four h after LACV infection (MOI, 0.5), HeLa cells were fixed and stained with anti-LACV Gc (green) and anti-clathrin (red) antibodies, followed by anti-mouse IgG and anti-rabbit IgG conjugated to FITC and R-phycoerythrin, respectively. Nuclei (blue) were stained with Hoecht's stain. (C) The number of LACV-infected cells was counted, and the percentage of cells infected with LACV was determined for each condition. The percentage of CHC siRNA-transfected cells was significantly decreased compared to levels of control or GAPDH siRNA-transfected cells (*, P < 0.01 by Student's t test). (D) Knockdown of CHC decreases LACV titers. HeLa cells were infected with LACV (MOI, 0.01) 24 h following the second siRNA transfection. Supernatants were harvested 24 h postinfection. Viral titers were determined by standard plaque assays. LACV titers were significantly reduced in CHC siRNA-transfected cells compared to control or GAPDH siRNA-transfected cells (*, P < 0.001 by Tukey test). (E) Overexpression of dominant-negative Eps15 (Eps15Δ95/295) inhibits LACV infection. Twenty-four h after transfection with control Eps15 (Eps15-3Δ2-GFP) or dominant-negative Eps15 (Eps15Δ95/295-GFP), HeLa cells were infected with LACV. Twenty-four h later, cells were fixed and stained with anti-LACV Gc (red) followed by anti-mouse IgG conjugated to R-phycoerythrin. The numbers of LACV-positive transfected and untransfected cells were counted, and the percentage of cells (transfected [GFP⁺] or untransfected [GFP⁻]) infected with LACV was determined for each condition (*, P < 0.01 by Student's t test). (F) Representative images of the immunofluorescence assay. The results are means ± SD of 3 independent experiments. The dashed line represents the limit of detection.