Figure 1.

Integrin α3 or integrin α6 knockdown inhibits viral capsid processing and infection. Integrin α3 or integrin α6 were knocked down in HaCaT cells by siRNA transfection (for knockdown efficiency see Fig. S1). (A) Two days after transfection, cells were incubated for 24 h with HPV16 PsVs, washed, lysed and analyzed by Western blot for the viral protein L1 and its ~25 kDa cleavage product. For clarity, lanes were cropped from original blots shown in full in Fig. S14 (L1) and S15 (actin). Values were related to the control which was set to 100% and are given as means ± SD (n = 3 independent experiments). (B,C) Two days after transfection, cells were incubated for 5 h with HPV16 PsVs, washed fixed, stained with an antibody that recognizes L1 after capsid disassembly, and imaged by confocal microscopy taking an optical section from the cell body. (B) Actin (cyan) and L1–7 (inverted greyscale) each are displayed at the same arbitrary scaling (linear lookup tables). From the optical section (B), an image analysis algorithm counted the number of detected vesicles per cell (C) and quantified the vesicle staining intensity (Fig. S2). Values are given as means ± SD (n = 60 analysed cells collected from three biological replicates). (D) HaCaT cells were transfected and incubated with PsVs as in (A) with the difference that on the encapsidated plasmid luciferase expression is under the control of the HPV16 promoter instead of the CMV promoter. One day after adding PsVs, cells were lysed and the infection rate was assessed by analysing the luciferase activity. For normalization to cell number, luciferase activity was related to the dehydrogenase activity. Values are expressed as percent of control (average of control was set to 100%). Values are given as means ± SD (n = 20–21 technical replicates collected from five biological replicates). Unpaired Student’s t-test, comparing control to knockdown conditions (***p < 0.001; **p < 0.01).