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

(A) In the absence (control) or presence of 10 µg/ml CytD, HaCaT cells are incubated with PsVs at 37 °C for 5 h. Then, cells are fixed and stained with the cell membrane dye TMA-DPH (grey). PsVs (magenta) are visualized through coupling a dye (6-FAM Azide) to the encapsidated plasmid by click-chemistry, and indirect immunolabeling is used for staining of HS (AlexaFluor™ 594; cyan) and Itgα6 (STARRED; green). Imaging was realized with epi-fluorescence microscopy. White lines mark the main cell body; they are generated with reference to the membrane staining. (B) Same pre-treatment of cells as in (A) with an additional condition where CytD is added 1 h after the PsVs (CytD after 1 h). Prior to fixation, membrane sheets are generated and F-actin is stained with phalloidin iFluor488 (green) and PsVs are stained by immunofluorescence using an antibody against L1 in combination with an AlexaFluor™ 594-labelled secondary antibody (magenta; not shown in this figure for clarity reasons is an additional staining of CD151 with STAR RED). Images of phalloidin and L1 are acquired in the confocal and STED mode of a STED microscope, respectively. Arrows in the magenta panels point towards accumulated PsVs. (C) Images as shown in (B) are analyzed. PsV maxima are detected and their intensities are quantified in a circular 125 nm diameter region of interest (ROI), followed by background correction. Values are given as means ± SD (n = 3 biological replicates; one biological replicate includes per condition 14 - 15 analyzed membranes with altogether at least 1000 maxima). (D) PsV maxima intensity distribution of the data in (C). The fraction of PsVs, expressed in percent, is plotted against the maxima intensity. Values are given as means ± SD (n = 3). (E) HaCaT cells are treated either for 5 h or 24 h with PsVs, with or without 10 µg/ml CytD. In case of the 5 h incubation, cells are washed and incubated for another 19 h in medium (in total 24 h). After a total of 24 h incubation, the luciferase activity of lysed cells is measured, yielding the infection rate that is normalized to LDH, resulting in the normalized infection rate. The normalized infection rate is 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). (D) and (E). Statistical differences between control and CytD is analyzed by using the two-tailed, unpaired student’s t test (n = 3). a.u., arbitrary units.

Fast diminishment of the accumulated PsV signal from the cell periphery after washing off of CytD.

(A) HaCaT cells are incubated with PsVs at 37 °C for 5 h, in the absence (control) or presence of CytD (10 µg/ml). Then, cells are washed and incubated for the indicated time periods without PsVs/CytD, before they are fixed and stained as in Figure 1 (t = 0 min is identical to Figure 1A; for clarity we show only the membrane (grey) and the PsV staining (magenta)). White lines in the membrane images are generated with reference to the membrane staining. One line marks the cell body. The cell body line was broadened by 30 pixels (see additional smoother white lines in the PsV channel, magenta). (B) Magenta images, the area enclosed by the two white lines mark the cell periphery. The PsV signal of the periphery is quantified as integrated density, background corrected, and plotted over time. Values are given as means ± SD. The statistical difference between the same time points of control and CytD is analyzed by using the two-tailed, unpaired student’s t test (n = 3 biological replicates).

Association between PsVs and CD151.

(A) HaCaT cells are 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 are washed and incubated without PsVs/CytD further for 0 min, 30 min, 60 min or 180 min, before they are fixed and stained by indirect immunofluorescence for L1 (magenta; STAR GREEN) and for CD151 (green; AlexaFluor™ 594), and for F-actin by fluorescent labelled phalloidin (iFluor647; here not shown for clarity, please see Figure 4 for the F-actin staining). The bottom rows show enlarged views of the merged images, from the regions marked by the white boxes. PsVs (L1 staining) and F-actin are imaged in the confocal and CD151 in the STED mode of a STED microscope, respectively (see enlarged views). 180 min/CytD, arrows mark presumably endocytic structures in the central cell body region (for more examples see also Supplementary Figure 1). For analysis, we place rectangular ROIs onto the images that cover mainly the cell body but include parts of the cell periphery as well (see example in Supplementary Figure 2A). (B) Within these ROIs, the average CD151 intensity is measured and plotted over time. In the same ROIs, (C) the Pearson correlation coefficient (PCC) between PsV-L1 (magenta) and CD151 (green) is 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 tightly associated, is plotted over time. The fraction of PsVs tightly associating with CD151 is corrected for random background association (for details see Supplementary Figure 3). (D) Two examples of PsVs (each marked by an asterisk) taken from the 30 min/CytD (left) and 60 min/CytD (right) conditions. Number in the upper left, the shortest distance between the PsV-L1 maximum and the next nearest CD151 maximum (marked by an arrow) is given in nm. Values are given as means ± SD (n = 3 biological replicates). Statistical difference between the same time points of control and CytD is analyzed by using the two-tailed, unpaired student’s t test (n = 3).

Gallery of images illustrating the variability of filopodia/the diminishment of the number of PsVs at the cell border region after CytD removal.

(A) Images are taken from the same experiment described in Figure 3. Many cells have filopodia. However, due to the large variability in number and shape it is impossible to show representative images. Therefore, the shown examples taken from the CytD condition are anecdotal images of cells with CD151 positive filopodia. (B) Based on the CD151 image, a cell border region is broadened to 40 pixels. Please note that this cell border region is different from the cell periphery described in Figure 2. In this analysis, the analyzed region covers both sides of the cell border (approximately 75% inside and 25% outside of the cell; for details see methods), and therefore is referred to as the cell border region. (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). Statistical difference between the same time points of control and CytD is analyzed by using the two-tailed, unpaired student’s t test (n = 3).

Association between PsVs and HS.

(A) HaCaT cells are 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 are washed and incubated without PsVs/CytD further for up to 180 min, before they are fixed and stained. PsVs (magenta) are visualized by click-chemistry (6-FAM Azide, see also above) and indirect immunolabeling is used for HS (cyan; AlexaFluor™ 594) and for Itgα6 (green; STAR RED). Shown in the bottom rows are enlarged views of the white boxes in the merged images. PsVs (DNA staining) are imaged in the confocal and HS and Itgα6 in the STED mode of a STED microscope, respectively (see enlarged views). For analysis, we place rectangular ROIs onto the images that cover mainly the cell body but include parts of the cell periphery as well (see example in Supplementary Figure 4A) (B) Average HS intensity over time. (C) PCC between PsV-DNA (magenta) and HS (cyan) over time. (D) PCC between PsV-DNA (magenta) and HS (cyan) in the region of the cell body over time. (E) The fraction of PsVs (in percent) tightly associating with HS (distance ≤ 80 nm) plotted over time (for background correction see Supplementary Figure 5). Two examples of PsVs (each marked by an asterisk) taken from the 0 min/CytD condition. Number in the upper left is the shortest distance in nm between the PsV-DNA maximum and the next nearest HS maximum (marked by an arrow). Values are given as means ± SD (n = 3 biological replicates). Statistical difference between the same time points of control and CytD is analyzed by using the two-tailed, unpaired student’s t test (n = 3).

Pattern of PsV-Itgα6 distances and PsV-HS distances over time.

(A) Definition of four PsV populations. 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. (B) Based on the experiment shown in Figure 5, the PsV fraction size (in percent) of the four populations defined in (A) is illustrated using the same colors as in (A), and using black for PsVs with a distance < 250 nm each to Itgα6 and HS. Shown is the mean of the three biological replicates. For means ± SD and statistical analysis see Supplementary Table 1. (C) The same data as in (B), plotting for each PsV the shortest distance to Itgα6 against the shortest distance to HS (pooling the three biological replicates; 3,043 – 4,080 PsVs per plot).

Model figure.

The structural activation of PsVs in the ECM is completed within 5 h (i). Within 15 min, the structurally- activated HPV16 translocate from the ECM to CD151 assemblies, likely located at the filopodia, and move toward the cell body (ii). During this process, the virions retain some heparan sulfate (HS) on their surface. (iii) Eventually, they lose their HS coat, and individual PsV-CD151 assemblies merge into larger complexes, which are subsequently endocytosed.

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

(A) From the data described in Figure 3 (180 min/CytD), we show more examples of agglomerated CD151 maxima (see green patches) that associate with PsVs (magenta) and likely are endocytic structures. (B) The same experiment as in (A), but cells are scanned with 400 nm steps in the axial direction at confocal resolution, starting at the basal membrane. The structures noticed in the basal membrane as agglomerated CD151 maxima continue deeper into the cell. In some cases, more than a micrometer (see second example from the left).

PCC controls.

(A) In order to determine the background colocalization, we analyze the PCC on flipped images as well. Left, original image taken from the 0 min/CytD condition. The ROI selected for analysis is shown as white box. Right, the green image within the ROI is flipped horizontally and vertically. Now, a pair of images with the same density of objects and intensity distributions can be analyzed. (B) PCC between PsV-L1 and CD151 of the control condition (Figure 3C) is shown again, together with the respective PCCs determined on flipped images. (C) The PCC between PsV-L1 and CD151 of the CytD condition (Figure 3C) is shown again, together with the respective PCCs determined on flipped images. Statistical difference between the same time points of original and flipped images is analyzed by using the two-tailed, unpaired student’s t test (n = 3). Values are given as mean ± SD.

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

(A) The fraction of tightly associated PsVs (PsV-L1 maxima with a distance ≤ 80 nm to the next nearest CD151 maximum) in the control of the experiment described in Figure 3, analyzed on original and flipped images (for an example of a flipped image see Supplementary Figure 2A). On flipped images, the fractions often are more than half of the original images. This demonstrates that many of the PsVs have a distance ≤ 80 nm to CD151 merely by chance (random background association). (B) Same as (A) for the CytD condition. (C) Each flipped data point in (A) and (B) is the average of three biological replicates. From the altogether 24 replicates of the flipped images, we plot the fraction of tightly associated PsVs against the CD151 maxima density. As expected, the fraction increases with the maxima density. The fitted linear regression line describes how the background association depends on the maxima density. With the equation of the regression line the random background association can be calculated, for any maxima density in an original images the random background association can be calculated. (D) For each replicate from the original and the flipped images in (A), the background fraction is calculated using the equation of the regression line in (C) and the respective CD151 maxima density, and subtracted from the fraction of tightly associated PsVs. (E) Same as (D) for the CytD condition. The corrected original values in (D) and (E) are shown in Figure 3D. Please note that the data point CytD/0 min likely is overcorrected because we flip a CD151 rich region onto the accumulated PsVs that in the original image are in a region that is low in CD151. Statistical difference between the same time points of original and flipped images is analyzed by using the two-tailed, unpaired student’s t test (n = 3). Values are given as mean ± SD.

PCC controls, HS-Itgα6 average shortest distance and Itgα6 intensity.

Additional analysis from the experiment shown in Figure 5. (A) Example of flipped HS image used as control (for explanation please see legend of Supplementary Figure 2). Left, original image taken from the 0 min/CytD condition. The ROI selected for analysis is shown as white box. Right, the cyan image within the ROI is flipped horizontally and vertically. (B) The PCC between PsV-DNA and HS of the control condition (Figure 5C) is shown again, together with the respective PCCs determined on flipped images. (C) The PCC between PsV-DNA and HS of the CytD condition (Figure 5C) is shown again, together with the respective PCCs determined on flipped images. (D) The average shortest distance of HS maxima to the next nearest Itgα6 maximum and (F) the average Itgα6 intensity. Statistical difference between the same time points of original and flipped images or control and CytD is analyzed by using the two-tailed, unpaired student’s t test (n = 3). Values are given as mean ± SD.

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

(A) The fraction of tightly associated PsVs (PsV-DNA maxima with a distance ≤ 80 nm to the next nearest HS maximum) in the control of the experiment described in Figure 5, analyzed on original and flipped images (for an example of a flipped image see Supplementary Figure 4A). (B) Same as (A) for the CytD condition. (C) From the altogether 24 replicates of the flipped images, the fraction of tightly associated PsVs is plotted against the HS maxima density, and a linear regression line is fitted to the data points. (D) For each replicate from the original and the flipped images in (A), the background fraction is calculated using the equation of the regression line in (C) and the respective HS maxima density, and subtracted from the fraction of tightly associated PsVs. (E) Same as (D) for the CytD condition. The background corrected original values in (D) and (E) are shown in Figure 5E. Statistical difference between the same time points of original and flipped images is analyzed using the two-tailed, unpaired student’s t test (n = 3). Values are given as mean ± SD.

Means ± SD of the data shown in Figure 6B.

P-values between control and CytD are calculated by using the two-tailed, unpaired student’s t test (n = 3). Significant p-values are illustrated in bold.