Fig. 5.

EbGP mediated viral infection is inhibited by siRNA-mediated knockdown of clathrin heavy chain.

(A) Co-transfection of HOS cells with eGFP and siRNA against clathrin heavy chain followed by incubation with 14 μg/ml of TR-transferrin for 30 min. Cells were fixed and stained for clathrin (shown in blue) using mouse monoclonal antibody against clathrin heavy chain and Cy5-conjugated anti-mouse secondary antibody. Arrows indicate transferrin endocytosis in the neighboring untransfected cells. Scale bar represents 15 μm. The side panels show individual channels in a single siRNA transfected cell (co-transfected with eGFP) and demonstrate that there is very little clathrin and no internalized transferrin in that GFP expressing cell. (B) HOS cells in 12 well plates were transfected twice with siRNA against clathrin followed by western blot detection of clathrin using mouse monoclonal antibody against clathrin heavy chain. GAPDH was measured as a loading control. (C) HOS cells on coverslips were transfected twice with siRNA against clathrin heavy chain and mCherry plasmid as a transfection marker. Control cells were transfected with mCherry plasmid alone. 48 h following the second transfection, the cells were incubated with virus for 4 h. 48 h post-infection, the cells were fixed and the DNA was stained with Hoechst. Several panels of images were collected from each sample and the number of transfected cells and transfected and infected cells were counted in each panel. Graph represents the fold decrease in viral infectivity in siRNA-transfected cells when compared to control cells. Error bars represent SEM for three independent experiments. * p value for fold change in EbGP mediated infectivity compared to either VSVg or HIV = 0.0006 (extremely significant). Typically, there was 20 % transfected and infected cells (double positive cells) in control cells infected with EbGP virus and 25 % transfected and infected cells in siRNA-treated as well as control cells for VSVg and HIV infection.