Fig 2: Integrin a3 and integrin a6 knockdown differentially affect the CD151 level of the cell membrane and PsV-binding. HaCaT cells were transfected with siRNA and two days later incubated without or with HPV16 PsVs for 5 h. Cells were fixed, permeabilized, immunostained for CD151 (green) and L1 (red), and imaged by STED microscopy. Images are displayed employing linear lookup tables. For A and D different scalings were applied, although all panels in A or D have the same arbitrary scaling. (A) Integrin a6 knockdown. For each condition, overlays from an overview and a magnified view are shown. (B) Average CD151 immunostaining intensity and (C) the PsVs particle density (100% correspond to 1.4 particles/µm2). (D–F) As (A–C), showing the integrin a3 knockdown (100% PsV density correspond to 1.2 particles/µm2). Values are shown as means ± SD (n = 60 cells collected from three biological replicates). The unpaired Student’s t-tests compare the conditions as indicated by the bars (B,E) or PsV to PsV and knockdown (C,F) (***p < 0.001; **p < 0.01; *p < 0.05).

Fig 3: Local crowding of clusters at virus attachment sites. To characterize the anatomy of a possibly forming viral platform area, we selected those PsVs from Fig. 4C,F with a distance smaller than 250 nm to both a bright CD151 and integrin maximum (for examples see centrally located PsVs in the left panels in A and B). In a PsV-centred 1 µm × 1 µm ROI the number of bright maxima (see methods) in the CD151 and integrin channels were counted. The same was performed after flipping the CD151/integrin channels, causing a randomized relationship between PsV-attachment sites and maxima (A and B, right panels). In the original images more closely associated maxima are present at sites of PsV-attachment. Images are displayed in cyan (PsV) green (CD151) and red (integrin) at arbitrary intensity scalings (linear lookup tables). (C,D), quantification of the bright maxima in the 1 µm × 1 µm ROI. Values are given as means ± SD (n = 678 (C) and 283 (D) 1 µm × 1 µm ROIs, respectively). Statistical analysis was performed employing the unpaired Student’s t-test comparing the original to the randomized condition (***p < 0.001). Please note, that the number of clusters per attachment site was highly variable. For a histogram illustrating the variability see Fig. S12.

Fig 4: Large CD151 patches coincide with intracellular actin accumulations. CD151-GFP transfected HaCaT cells were treated for 3 h without or with PsVs. Membrane sheets were generated, stained, and imaged by confocal microscopy. Green (CD151-GFP; GFP signal was enhanced by nanobodies), red (PsVs visualized by L1 antibody labeling) and cyan (filamentous actin; fluorescently labelled phalloidin). Images are displayed using a linear lookup table. For each channel, the same arbitrary scaling was applied. (A) For each condition a membrane sheet is shown. Magnified views from the white boxes are shown, illustrating the individual channels. (B) For the CD151 and the actin channels, within a freehand ROI excluding membrane edges, the pixels with an intensity higher than the average ROI intensity were selected. Then, the number of pixels positive in both channels were related to the number of all positive pixels in the CD151 channel. Values are given as means ± SD (n = 45 membrane sheets collected from three biological replicates). Unpaired Student’s t-test (***p < 0.001).

Fig 5: Characteristics of CD151 and integrin maxima. CD151-GFP was transfected into HaCaT cells. CD151-GFP and endogenous CD151 localize to the same domains (Fig. S7E,F) and CD151-GFP is as functional as non-tagged CD151 (Fig. S8). One day after transfection, cells were treated for 5 h without or with PsVs (Fig. S9), washed, membrane sheets were generated, stained and analyzed by three channel STED microscopy. Green lookup table, CD151-GFP visualized by nanobodies; red and cyan lookup tables, integrin a6 and integrin a3 stained by antibody labeling, respectively. Images are displayed at arbitrary intensity scalings (linear lookup tables). Staining with the integrin antibodies is highly specific (Fig. S1). (A) Large panel, membrane sheet (channel overlay). The white box marks an area from which magnified views of the individual channels are shown. From regions of interest (ROIs) we measured (B) maxima size and signal overlap (C). For (C), for all three channels within a ROI the pixels with an intensity higher than the average ROI intensity were selected. Then, the number of pixels positive in all three channels were related to the number of all positive pixels in one specific channel as indicated. (D) Shortest inter-maxima distances of CD151-GFP to CD151-GFP, integrin a3 or integrin a6. Values are given as means ± SD (n = 60 membrane sheets collected from three biological replicates).