CytD arrests PsV translocation from the ECM to the cell body.

(A) In the absence (Control) or presence of 10 µg/ml CytD (CytD), HaCaT cells were incubated with PsVs at 37 °C for 5 h. Then, cells were fixed, washed and stained with the cell membrane dye TMA-DPH (gray lookup table (LUT)). PsVs (magenta LUT) were visualized through coupling a dye (6-FAM Azide) to the encapsidated plasmid by click-chemistry. Indirect immunolabeling was employed for staining of HS (AlexaFluor 594; cyan LUT) and Itgα6 (STARRED; green LUT). Imaging was realized with epi-fluorescence microscopy. White lines delineate the main cell body; lines were created with reference to the TMA-DPH membrane staining. (B) Same pre-treatment of cells as in (A), with an additional condition where CytD was added 1 h after the PsVs (CytD after 1 h). Prior to fixation, membrane sheets were generated and F-actin was stained with phalloidin coupled to iFluor488 (green LUT). The capsid protein L1 of the PsVs and CD151 were stained by immunofluorescence using primary antibodies in combination with AlexaFluor 594-labelled (L1, magenta LUT) and STAR RED-labelled (CD151, not shown in this figure for clarity reasons) secondary antibodies. Images of phalloidin and L1 staining were acquired in the confocal and STED mode of a STED microscope, respectively. Arrows in the PsV-L1 images point towards accumulated PsVs that after CytD are more frequently observed than in the control (see text). (C) Analysis of images as shown in (B) using ROIs covering the whole image. PsV maxima were detected and their intensities were quantified in a circular 125 nm diameter region of interest (ROI), followed by background correction. Values are given as means ± SD (n = 3; one biological replicate includes per condition the average of 14 - 15 analyzed membranes (intensity values of one membrane sheet were averaged) with altogether at least 1000 maxima intensity values). (D) PsV maxima intensity distribution of the data in (C). The fraction of PsVs, expressed in percent, is plotted as histogram (10 a.u. bins) against the maxima intensity. Values are given as means ± SD (n = 3). (E) HaCaT cells were treated either for 5 h or 24 h with PsVs, together without (Control) or with 10 µg/ml CytD (CytD). In case of the 5 h incubation, after removal of the PsVs/CytD cells were incubated for another 19 h in medium (in total 24 h). After a total of 24 h incubation, the luciferase activity of lysed cells was measured, yielding the infection rate that was normalized to LDH, resulting in the normalized infection rate. The normalized infection rate was further related to the mean normalized infection rate of the 5 h control, set to 100%, yielding the relative infection rate. Values are given as means ± SD (n = 3 biological replicates; the value of one biological replicate is the average of three technical replicates). (C) and (E). Statistical differences between Control and CytD was analyzed by using the two-tailed, unpaired student’s t-test (n = 3, for details see methods). a.u., arbitrary units.

Transfer of PsVs from the ECM to the cell body requires PsV priming.

HaCaT cells were pre-incubated for 5 h at 37 °C with PsVs, in the presence of (A) 10 µg/ml CytD (CytD), (B) 10 µg/ml CytD and 100 µM leupeptin (CytD/leupeptin), or (C) 10 µg/ml CytD and 5 µM Furin inhibitor I (CytD/Furin inhibitor I). Afterwards, cells were washed and incubated without PsVs/inhibitors further for 0 min, 30 min or 60 min, before they were fixed and stained by indirect immunofluorescence for L1 (STAR GREEN, magenta LUT) and for F-actin by iFluor647-labelled phalloidin (green LUT). PsVs-L1 and F-actin staining were imaged in the confocal mode of a STED microscope. (D) For determination of the Pearson correlation coefficient (PCC) between PsV-L1 (magenta LUT) and Phalloidin (green LUT), we placed large ROIs onto the images that covered mainly the cell body but included parts of the cell periphery as well. The PCC was plotted over time. Values are given as means ± SD (n = 3 biological replicates). Statistical difference between CytD and CytD/inhibitors was analyzed by using the two-tailed, unpaired student’s t-test (n = 3, for details see methods).

Increased HS intensity after incubation with PsVs and CytD.

(A) HaCaT cells were incubated without (top) and with (bottom) PsVs at 37 °C for 5 h, in the absence (Control, upper panels) or presence of 10 µg/ml CytD (CytD, lower panels). Afterwards, cells were washed, fixed and stained. Immunofluorescence was used for L1 (STAR GREEN; magenta LUT) and for HS (AlexaFluor 594; cyan LUT). F-actin was stained by iFluor647-labelled phalloidin (green LUT). PsVs-L1, HS and F-actin staining were imaged in the confocal mode of a STED microscope. The open arrow marks a region where PsVs overlap with HS. The closed arrow marks a region devoid of PsVs showing strong HS staining. (B) For analysis of the mean HS intensity, we placed large ROIs onto the images covering mainly the cell body but including parts of the cell periphery as well. Values are given as means ± SD (n = 3 biological replicates). Statistical differences between Control and CytD were analyzed by using the two-tailed, unpaired student’s t-test (n = 3, for details see methods).

Fast diminishment of accumulated PsVs at the cell periphery after removal of CytD.

(A) HaCaT cells were incubated with PsVs at 37 °C for 5 h, in the absence (Control) or presence of 10 µg/ml CytD (CytD). Then, cells were washed and incubated for the indicated time periods without PsVs/CytD, before they were fixed and stained as in Figure 1 (t = 0 min is identical to Figure 1A; for clarity we show only the membrane (gray LUT; images are shown at different settings of brightness and contrast) and the PsV-DNA staining (magenta LUT; images are shown at the same settings of brightness and contrast). The white lines in the membrane images delineate the cell body from the periphery. They were created with reference to the membrane staining (for details see methods). Using the cell body delineation as starting point, an up to 30-pixel broad area was created (PsV channel, magenta LUT; see area enclosed by the smoother white lines and the cell body delineation lines). The area enclosed by the two white lines defines the cell periphery. (B) The PsV-DNA signal of the periphery was quantified as integrated density, background corrected, and plotted over time. Values are given as means ± SD (n = 3 biological replicates). The statistical difference between the same time points of Control and CytD were analyzed by using the two-tailed, unpaired student’s t-test (n = 3, for details see methods).

Association between PsVs and CD151 is an early event in the infection cascade.

(A) HaCaT cells were incubated with PsVs at 37 °C for 5 h, in the absence (Control, upper panels) or presence of 10 µg/ml CytD (CytD, lower panels). Afterwards, cells were washed and incubated without PsVs/CytD further for 0 min, 30 min, 60 min or 180 min, before they were fixed and stained by indirect immunofluorescence for L1 (STAR GREEN; magenta LUT) and for CD151 (AlexaFluor 594; green LUT), and for F-actin by iFluor647-labelled phalloidin (here not shown for clarity, please see Supplementary Figure 3 for F-actin illustrating the variability of filopodia after CytD treatment). The bottom rows show magnified views of the merged images from the regions marked by the white boxes. PsV-L1 and F-actin staining were imaged in the confocal and CD151 staining in the STED mode of a STED microscope, respectively. Therefore, compared to CD151, the PsVs are less resolved and also appear much larger than their real physical size (see magnified views). CytD/180 min, arrows mark presumably endocytic structures that formed in the central cell body region (for more examples see Supplementary Figure 5). For analysis, we placed large ROIs onto the images that covered mainly the cell body but included parts of the cell periphery as well (for an example ROI see Supplementary Figure 6A). (B) The mean CD151 intensity was measured and plotted over time. (C) The PCC between PsV-L1 (magenta LUT) and CD151 (green LUT) was calculated and plotted over time. (D) The fraction of PsVs (in percent) that have a distance to the next neighbored CD151 maximum ≤ 80 nm, which we define as closely associated, is plotted over time. Please note that the values in (D) were corrected for random background association (for details see Supplementary Figure 7). Two examples of PsVs (each marked by an asterisk) from the CytD/30 min (left) and CytD/60 min (right) conditions are shown. The value in the upper left states the shortest distance between the PsV and the next nearest CD151 maximum (marked by an arrow) in nm. Values are given as means ± SD (n = 3 biological replicates). Statistical differences between the same time points of Control and CytD were analyzed by using the two-tailed, unpaired student’s t-test (n = 3, for details see methods).

Association between PsVs and HS.

(A) HaCaT cells were incubated with PsVs at 37 °C for 5 h, in the absence (Control, upper panels) or presence of 10 µg/ml CytD (CytD, lower panels). Afterwards, cells were washed and incubated without PsVs/CytD further for up to 180 min, before they were fixed and stained. PsVs (magenta LUT) were visualized by click-chemistry (6-FAM Azide) and indirect immunolabeling was used for HS (AlexaFluor 594; cyan LUT) and for Itgα6 (STAR RED; green LUT). The bottom rows show magnified views of the white boxes in the merged images. PsV-DNA staining was imaged in the confocal and HS and Itgα6 staining in the STED mode of a STED microscope. For analysis, we placed large ROIs onto the images that covered mainly the cell body but included parts of the cell periphery as well (see example in Supplementary Figure 8A). For (D), smaller ROIs covering only the cell body region were used. (B) The mean HS intensity plotted over time. (C) The PCC between PsV-DNA (magenta LUT) and HS (cyan LUT) over time. (D) The PCC between PsV-DNA (magenta LUT) and HS (cyan LUT) in the region of the cell body over time. (E) The fraction of PsVs (in percent) closely associating with HS (distance ≤ 80 nm) plotted over time (for background correction see Supplementary Figure 9). Two examples of PsVs (each marked by an asterisk) from the CytD/0 min condition are shown. The value in the upper left corner states the shortest distance (in nm) between the marked PsV and its next nearest HS maximum (marked by an arrow). Values are given as means ± SD (n = 3 biological replicates). Using the two-tailed, unpaired student’s t-test (n = 3 biological replicates), we analyzed in (B), (C) and (E) the statistical differences between the same time points of Control and CytD, and in (D) the difference between CytD/30 min and CytD/0 min or CytD/180 min (for details see methods).

PsV-Itgα6 and PsV-HS distances over time.

(A) Definition of four PsV populations based on the PsV distances to Itgα6 and HS (the plot is taken from Control, 0 min and shown again in C). Dashed green rectangle, PsVs with a distance to HS < 250 nm and to Itgα6 > 250 nm. Dashed magenta rectangle, PsVs with a distance to HS > 250 nm and to Itgα6 < 250 nm. Dashed gray square, PsVs with a distance to HS > 250 nm and to Itgα6 > 250 nm. PsVs not included in the previous categories have a distance to HS < 250 nm and to Itgα6 < 250 nm (dashed black square). (B) From the PsVs analyzed in Figure 6, for the Control (left) and CytD (right) the PsV fraction size (in percent) of each population is illustrated. Shown are the means of three biological replicates. For means ± SD and statistical analysis see Supplementary Table 1. (C) For the Control (top) and CytD (bottom), for each PsV, we plotted the shortest distance to Itgα6 against the shortest distance to HS (pooling the three biological replicates; 3,043 – 4,080 PsVs per plot).

Association between PsVs and HS after blebbistatin treatment.

(A) HaCaT cells were incubated with PsVs at 37 °C for 5 h, in the absence (Control, upper panels) or presence of 30 µM blebbistatin (Blebbistatin, lower panels). Afterwards, cells were washed and incubated without PsVs/blebbistatin further for up to 180 min, before they were fixed and stained. Immunofluorescence was used for L1 (STAR GREEN; magenta LUT) and HS (AlexaFluor 594; cyan LUT) staining. F-actin was stained by iFluor647-labelled phalloidin (green LUT). The bottom rows show magnified views of the white boxes in the merged images. PsVs and F-actin staining were imaged in the confocal and HS staining in the STED mode of a STED microscope. For analysis, we placed large ROIs onto the images that covered mainly the cell body but included parts of the cell periphery as well. For (D), smaller ROIs covering only the cell body region were used. (B) Mean HS intensity over time. (C) PCC between PsV-L1 (magenta LUT) and HS (cyan LUT) over time. (D) PCC between PsV-L1 (magenta LUT) and HS (cyan LUT) in the region of the cell body over time. (E) The fraction of PsVs (in percent) closely associating with HS (distance ≤ 80 nm) plotted over time (for background correction see Supplementary Figure 11). Two examples of PsVs (each marked by an asterisk) from the blebbistatin/0 min condition are shown. The value in the upper left states the shortest distance between the PsV and its next nearest HS maximum (marked by an arrow) in nm. Values are given as means ± SD (n = 3 biological replicates). Statistical differences between the same time points of Control and Blebbistatin were analyzed by using the two-tailed, unpaired student’s t-test (n = 3), but the analysis yielded no p-values below 0.05.

Model of ECM events, actin transport, and receptor engagement in HPV16 entry.

(i) During 5 h of incubation with CytD, the PsVs bind to HS of the ECM, are primed, and become coated with HS cleavage products, enabling them for cell surface receptor engagement. (ii) After CytD removal, within 15 min, HS-decorated viruses move along filopodia to the cell body and associate with CD151 assemblies (completed within 30 min), likely located at the filopodia. (iii) Eventually, they lose their HS coat, and individual HPV16-CD151 assemblies agglomerate into larger structures (platforms), which are subsequently endocytosed. Dashed rectangles mark PsVs representing populations as defined in Figure 7. Dashed green rectangle, PsVs with a distance to HS < 250 nm and to Itgα6 > 250 nm. Dashed black rectangle, PsVs with a distance to HS < 250 nm and to Itgα6 < 250 nm. Dashed magenta rectangle, PsVs with a distance to HS > 250 nm and to Itgα6 < 250 nm.

PsV density at the adhered membrane of substrate-adhered grown and re-attached HaCaT cells.

(A) The shown HaCaT cells are from the 0 min time point (top, Control; bottom, CytD) of the experiment described in Figure 5. Only the PsV-L1 (STAR GREEN; magenta LUT) and the F-actin phalloidin (iFluor647; green LUT) staining are shown. (B) In order to count the PsVs at the basal membrane, we placed ROIs covering only the cell body. Within these ROIs the PsV maxima were counted and related to the size of the analyzed area. Values are given as means ± SD (n = 3 biological replicates). Statistical differences between Control and CytD were analyzed by using the two-tailed, unpaired student’s t-test (n = 3), but the analysis yielded no p-value below 0.05. (C) HaCaT cells were detached by a 15 min incubation with 10 mM EDTA (in PBS) and incubated with PsVs at 4 °C for 1 h under constant rotation. After washing the cells three times with PBS, they were seeded onto PLL-coated glass coverslips for 1 h. Then, cells were washed, fixed and stained for L1 (STAR GREEN; magenta LUT) by indirect immunofluorescence and for F-actin by iFluor647-labelled phalloidin (green LUT). PsV-L1 and F-actin staining were imaged as in (A) in the confocal mode of a STED microscope. (D) Analysis as in (B). Values are given as means ± SD (n = 3 biological replicates).

HS neo-epitope (Δ-HS) staining intensity after CytD treatment.

(A) HaCaT cells were incubated with PsVs at 37 °C for 5 h, in the absence (Control, upper panels) or presence of 10 µg/ml CytD (CytD, lower panels). Afterwards, cells were washed and incubated without PsVs/CytD further for up to 180 min, before they were fixed and stained. Immunofluorescence was used for the visualization of L1 (STAR GREEN; magenta LUT) and the HS neo-epitope, referred to as Δ-HS (AlexaFluor 594; cyan LUT). F-actin was stained by iFluor647-labelled phalloidin (green LUT). PsV-L1, Δ-HS and F-actin staining were imaged in the confocal mode of a STED microscope. For analysis, we placed large ROIs onto the images that covered mainly the cell body but included parts of the cell periphery as well. (B) The mean Δ-HS intensity over time. Values are given as means ± SD (n = 3 biological replicates). Statistical differences between the same time points of Control and CytD were analyzed by using the two-tailed, unpaired student’s t-test (n = 3), but the analysis yielded no p-values below 0.05.

Variability of filopodia/diminishment of PsVs from the cell border region after CytD removal.

(A) More images from the experiment described in Figure 5. We observed that many cells exhibit filopodia. Due to the large variability in number and shape we cannot show a representative image. Instead, we present for the CytD condition (0 min and 30 min) a variety of cells with CD151 positive filopodia. (B) Based on the CD151 image (green LUT), a cell border strip is defined and broadened by 20 pixels on each side, in the following referred to as the cell border region (for details see materials and methods). Please note that the cell border region is different from the cell periphery described in Figure 4 that delineates the cell body from the periphery. Instead, the cell border region covers both sides of the cell border (approximately two thirds inside and one third outside of the cell; for details see methods). (C) Diminishment of PsV maxima from the cell border region over time, expressed as percentage of all PsVs present in the image. Values are given as means ± SD (n = 3 biological replicates). Statistical differences between the same time points of Control and CytD were analyzed by using the two-tailed, unpaired student’s t-test (n = 3, for details see materials and methods).

Overview images.

(A) Upper row, micrographs from Figure 5, CytD/0min, showing as well F-actin staining (green LUT) and CD151 using the cyan LUT. Lower row, confocal overview. After taking the overview, the area in the white box was imaged as described in the legend of Figure 5, yielding the images shown in the upper row in A. (B) More overview images from the experiment described in Figure 5, condition CytD/0 min, showing examples of large and small PsV accumulations. (C) Upper row, micrographs from Figure 6, CytD/0 min. Lower row, confocal overview. The area in the white box was imaged as described in the legend of Figure 6, yielding the images of the upper row in C.

Examples of agglomerated CD151 maxima associated with PsVs that presumably represent endocytic structures.

(A) From the CytD/180 min time point described in Figure 5, we show more examples of agglomerated CD151 maxima (green LUT, see green patches) that associate with PsVs (magenta LUT). (B) Same experiment as in (A). Starting at the basal membrane, at confocal resolution we imaged CD151 (green LUT) and PsV-L1 (magenta LUT). Cells were further scanned by 400 nm steps in the axial direction. Some of the CD151 agglomerates noticed in the basal membranes appear to continue deeper into the cell, more than a micrometer in the second example from the left. We propose that the agglomerated CD151 maxima close to PsVs feature the characteristics of endocytic structures, as CD151 has been shown to co-internalize with PsVs (Scheffer et al., 2013), and as these structures invaginate into the cell, like PsV filled tubular organelles previously described by electron microscopy (Schelhaas et al., 2012). Images in (A) and (B) are shown at the same settings of brightness and contrast but at different settings compared to Figure 5.

PCC values of flipped images.

(A) For each image pair, we determined the PCC on respective non-overlapping images that have the same density of objects and intensities. To this end, one channel was flipped horizontally and vertically. Left, original images (from the CytD/0 min condition). The ROI for analysis is illustrated as white box. Right, in the ROI, the CD151 image (green LUT) was flipped horizontally and vertically. (B + C) Shown are the PCCs over time of Figure 5C obtained on the original images ((B) Control, (C) CytD) together with the PCCs of the flipped images. Values are given as means ± SD (n = 3 biological replicates). Statistical differences between the same time points of original and flipped images was analyzed by using the two-tailed, unpaired student’s t-test (n = 3, for details see methods).

Background correction of the fraction of PsVs closely associated with CD151.

(A) The fraction of closely associated PsVs (PsV-L1 maxima with a distance ≤ 80 nm to the next nearest CD151 maximum) in the Control of Figure 5 was analyzed on original and flipped images (for an example of a flipped image see Supplementary Figure 6A). On flipped images, we often find values more than half of the values of the original images, demonstrating that many PsVs have a distance ≤ 80 nm to CD151 merely by chance (background association). (B) Same as (A), for CytD. (C) Each time point in (A) and (B) obtained from flipped images is the average of three biological replicates. We use these altogether 24 data points, plotting the fraction of closely associated PsVs against the CD151 maxima density. The fraction increases with the maxima density, as the chance of random association increases with the maxima density. The fitted linear regression line describes the dependence of the background association from the maxima density. As a result, the background association (y) can be calculated for any maxima density (x) in original images with the equation y = 2.04x. Please note that the CytD/0 min may be overcorrected as we subtract background association with reference to the CD151 maxima density of the entire ROI (for an example ROI see Supplementary Figure 6A), although the local maxima density at distal PsVs is lower. On the other hand, PsVs at the cell border may have a larger local CD151 maxima density and consequently are undercorrected. (D) and (E), for each original and flipped image of (A) and (B), the background was calculated and subtracted from the uncorrected value. The original background corrected values in (D) and (E) are shown in Figure 5D. Values are given as means ± SD (n = 3 biological replicates). Statistical differences between the same time points of original and flipped images were analyzed by using the two-tailed, unpaired student’s t-test (n = 3, for details see methods).

PCC values of flipped images, HS-Itgα6 shortest distance and Itgα6 intensity.

(A) For each image pair, we determined the PCC on respective non-overlapping images that have the same density of objects and intensities. To this end, one channel was flipped horizontally and vertically. Left, original images (from the CytD/0 min condition). The ROI for analysis is illustrated as white box. Right, in the ROI, the HS image (cyan LUT) was flipped horizontally and vertically. (B + C) Shown are the PCCs over time of Figure 6C obtained with the original images ((B) Control, (C) CytD) together with the PCCs of the flipped images. (D) The shortest distance of HS maxima to the next nearest Itgα6 maxima over time. (E) The mean Itgα6 intensity over time. (F + G) Shown are the PCCs over time of Figure 6D obtained with the original images ((F) Control, (G) CytD) together with the PCCs of the flipped images. Values are given as means ± SD (n = 3 biological replicates). Statistical differences between the same time points of original and flipped images ((B), (C), (F) and (G)) or Control and CytD (E). (D) Difference between CytD/0 min and CytD/180 min. Statistical differences were analyzed by using the two-tailed, unpaired student’s t-test (n = 3, for details see methods).

Background correction of the fraction of PsVs closely associated with HS.

(A) The fraction of closely associated PsVs (PsV-DNA maxima with a distance ≤ 80 nm to the next nearest HS maximum) in the Control of Figure 6 was analyzed on original and flipped images (for an example of a flipped image see Supplementary Figure 8A). (B) Same as (A), for CytD. (C) From the altogether 24 analyzed flipped images, we obtained the equation y = 1.85x for calculating the background association (please see Supplementary Figure 7 for more details). (D) and (E), background corrected graphs of (A) and (B). For each original and flipped image the background was calculated and subtracted from the uncorrected value. The original background corrected values in (D) and (E) are shown in Figure 6E. Values are given as means ± SD (n = 3 biological replicates). Statistical differences between the same time points of original and flipped images were analyzed by using the two-tailed, unpaired student’s t-test (n = 3, for details see methods).

PCC values of flipped images.

(A) and (B) The PCC values between PsV and HS of Figure 8C ((A) Control, (B) Blebbistatin) plotted together with the respective PCC values of flipped images. (C) and (D) The cell body PCC values between PsV and HS of Figure 8D ((C) Control, (D) Blebbistatin) plotted together with the respective PCC values of flipped images. Values are given as means ± SD (n = 3 biological replicates). Statistical differences between the same time points of original and flipped images were analyzed using the two-tailed, unpaired student’s t-test (n = 3, for details see methods).

Background correction of the fraction of PsVs closely associated with HS upon blebbistatin treatment.

(A) The fraction of closely associated PsVs (PsV-L1 maxima with a distance ≤ 80 nm to the next nearest HS maximum) in the Control of Figure 8 was analyzed on original and flipped images. (B) Same as (A), for blebbistatin. (C) From the altogether 24 analyzed flipped images, we obtained the equation y = 2.15x for calculating the background association (please see Supplementary Figure 7 for more details). (D) and (E), background corrected graphs of (A) and (B). For each original and flipped image the background was calculated and subtracted from the uncorrected value. The original background corrected values in (D) and (E) are shown in Figure 8E. Values are given as means ± SD (n = 3 biological replicates). Statistical differences between the same time points of original and flipped images were analyzed by using the two-tailed, unpaired student’s t-test (n = 3, for details see methods).

Fraction of PsVs in percent of each of the four distance categories (see left column).

Values are means ± SD of the data shown in Figure 7B. For each time point and category, p-values between Control and CytD were calculated by using the two-tailed, unpaired student’s t-test (n = 3 biological replicates). P-values < 0.05 are illustrated in bold.