HPV Pseudovirion Binding and Disassembly Assays

For the binding assay, HaCaT cells were preincubated for 1 h with the original or tannin-free (Section 2.6) extracts or DMSO and were then infected for 15 min with 500 HPV pseudovirions per cell. Cells were washed five times with PBS and collected in SDS sample buffer for Western blotting. Cell-bound HPV16 particles were stained with anti-L1 antibody 312F [62] (link). β-Actin (loading control) was stained using a murine antibody (Sigma-Aldrich) and relative band intensities were quantified densitometrically.
For the HPV capsid disassembly assay, HaCaT cells were grown on coverslips and treated with 20 µg/ml of the extracts for 1 h before HPV16 pseudovirion infection for 7 h at 37°C. Cells were fixed with methanol and stained with mouse anti-L1 antibody (33L1-7) as described previously [63] (link), [64] (link). L1-7 recognizes an epitope located inside of the pseudovirion capsid and is only accessible after uncoating [65] . Fluorescence was recorded using a Zeiss Axiovert 200 M microscope. For quantification, the relative amount of internalized particles was determined based on the L1-7-positive pixels relative to the cell nucleus signal (DNA/Hoechst 33342-positive pixels) out of 100 randomly selected cells from two independent experiments. A threshold value was set to exclude background.