A rapid transfer of virions coated with heparan sulfate from the ECM to CD151 defines an early step in the human papillomavirus infection cascade
- University of Bonn, Faculty of Mathematics and Natural Sciences, Membrane Biochemistry, Life & Medical Sciences (LIMES) Institute, Germany
- Institute for Virology, University Medical Center of the Johannes Gutenberg-University Mainz, Germany
- Research Center for Immunotherapy (FZI), University Medical Center of the Johannes Gutenberg-University Mainz, Germany
Figures
Figure 1
Cytochalasin D (CytD) arrests pseudovirion (PsV) recruitment from the extracellular matrix (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 hr. 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 heparan sulfate (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 hr after the PsVs (CytD after 1 hr). 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 regions of interest (ROIs) covering the whole image. PsV maxima were detected and their intensities were quantified in a circular 125 nm diameter 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 hr or 24 hr with PsVs, without (Control) or with 10 µg/ml CytD (CytD). In case of the 5 hr incubation, after removal of the PsVs/CytD cells were incubated for another 19 hr in medium (in total 24 hr). After a total of 24 hr 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 hr 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 were analyzed by using the two-tailed, unpaired Student’s t-test (n=3, for details see Materials and methods). a.u., arbitrary units.
Figure 2
Recruitment of pseudovirions (PsVs) from the extracellular matrix (ECM) to the cell body requires PsV priming.
HaCaT cells were pre-incubated for 5 hr 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 lookup table [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. Scale bar shown in (A) applies to (B) and (C) as well. (D) For determination of the Pearson correlation coefficient (PCC) between PsV-L1 (magenta LUT) and Phalloidin (green LUT), we placed large regions of interest (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 Materials and methods).
Figure 3 with 1 supplement
Increased heparan sulfate (HS) intensity after incubation with pseudovirions (PsVs) and cytochalasin D (CytD).
(A) HaCaT cells were incubated without (top) and with (bottom) PsVs at 37 °C for 5 hr, 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 lookup table [LUT]) and for HS (AlexaFluor 594; cyan LUT) staining. 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 regions of interest (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 Materials and methods).
Figure 3—figure supplement 1
Heparan sulfate (HS) neo-epitope (Δ-HS) staining after cytochalasin D (CytD) treatment.
(A) HaCaT cells were incubated with pseudovirions (PsVs) at 37 °C for 5 hr, 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 lookup table [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 regions of interest (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.
Figure 4
Fast diminishment of accumulated pseudovirions (PsVs) at the cell periphery after removal of cytochalasin D (CytD).
(A) HaCaT cells were incubated with PsVs at 37 °C for 5 hr, 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 (Figure 1A is from the same experiment; for clarity we show only the membrane, gray lookup table [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 Materials and methods). Using the cell body delineation as starting point, an up to 30-pixel broad area was created (PsV channel, magenta LUT; areas enclosed by the smoother white lines and the cell body delineation lines). The areas enclosed by the white lines define the cell peripheries. (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 Materials and methods).
Figure 5 with 6 supplements
Association between pseudovirions (PsVs) and CD151 occurs early in the infection cascade.
(A) HaCaT cells were incubated with PsVs at 37 °C for 5 hr, in the absence (Control, upper panels) or presence of 10 µg/ml cytochalasin D (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 lookup table [LUT]) and for CD151 (AlexaFluor 594; green LUT), and for F-actin by iFluor647-labelled phalloidin (here not shown for clarity, please see Figure 5—figure supplement 1A for F-actin staining 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 Figure 5—figure supplement 3). For analysis, we placed large regions of interest (ROIs) onto the images that covered mainly the cell body but included parts of the cell periphery as well (for an example ROI see Figure 5—figure supplement 4A). (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 background-corrected for random association (for details see Figure 5—figure supplement 6). 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 Materials and methods).
Figure 5—figure supplement 1
Variability of filopodia/diminishment of pseudovirions (PsVs) from the cell border region after cytochalasin D (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 lookup table [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 cell body and periphery (approximately two-thirds inside and one-third outside of the cell; for details see Materials and methods). (C) PsV maxima in the cell border region over time, expressed as a 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).
Figure 5—figure supplement 2
Overview images.
(A) Upper row, micrographs shown in Figure 5A, CytD/0 min, using for CD151 a cyan instead of a green lookup table (LUT), and showing as well F-actin staining (green LUT). Lower row, confocal overview images. After taking the overview images, the area in the white box was imaged as described in the legend of Figure 5, yielding the images shown in the upper row. (B) More merged overview images from the experiment described in Figure 5, condition CytD/0 min, showing examples of large and small pseudovirions (PsV) accumulations. (C) Upper row, same micrographs as shown in Figure 6A, CytD/0 min. Lower row, confocal overview images. 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.
Figure 5—figure supplement 3
Examples of agglomerated CD151 maxima associated with pseudovirions (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 lookup table [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, in the second example from the left more than a micrometer. We propose that the agglomerated CD151 maxima that overlap with PsVs are 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.
Figure 5—figure supplement 4
Pearson correlation coefficient (PCC) values of flipped images.
(A) For each image pair, we determined the PCC on non-overlapping images with 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 region of interest (ROI) for analysis is illustrated as a white box. Right, in the ROI, the CD151 image (green lookup table [LUT]) was flipped horizontally and vertically. (B, C) Shown are again the PCCs over time of Figure 5C of the original images ((B) Control, (C) CytD) together with the PCCs of the respective 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 by using the two-tailed, unpaired Student’s t-test (n=3, for details see Materials and methods).
Figure 5—figure supplement 5
Pseudovirion (PsV) density at the membrane of normally grown and re-attached HaCaT cells.
(A, B) In order to count the PsVs at the basal membrane of the experiments shown in (A) Figure 5 (PsVs visualized by antibody labeling) and (B) Figure 6 (PsVs visualized by click-chemistry), we placed regions of interest (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 cytochalasin D (CytD) at each time point were analyzed by using the two-tailed, unpaired Student’s t-test (n=3, for details see Materials and methods). (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 hr under constant rotation. After washing the cells three times with PBS, they were seeded onto PLL-coated glass coverslips for 1 hr. Then, cells were washed, fixed, and stained for L1 (STAR GREEN; magenta lookup table [LUT]) by indirect immunofluorescence and for F-actin by iFluor647-labelled phalloidin (green LUT). PsV-L1 and F-actin staining were imaged as in Figure 5 in the confocal mode of a STED microscope. (D) Analysis as in (A and B). Values are given as means ± SD (n=3 biological replicates).
Figure 5—figure supplement 6
Background correction of the fraction of pseudovirions (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 Figure 5—figure supplement 4A). As shown in (A), 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, in the following referred to as background association. (B) Same as (A), for cytochalasin D (CytD). (C) The value of each time point in (A) and (B) is the average of three biological replicates. We take the altogether 24 fraction values obtained on flipped images (12 values from Control and CytD each), and plot the fraction of closely associated PsVs against the average CD151 maxima density in the respective images. As can be seen in (C), the fraction increases with the maxima density, as the chance of a distance ≤80 nm increases with the maxima density. The fitted linear regression line describes how the background association depends on the maxima density. As a result, the background association (y) can be calculated for any maxima density (x) with the equation y=2.04 • x. The CytD/0 min condition may be overcorrected if it includes many images with CD151 flipped onto peripheral PsVs that actually are distal to CD151 (for an example ROI see Figure 5—figure supplement 4A). On the other hand, PsVs right at the cell border, where CD151 staining tends to be strong (Figure 5—figure supplement 4A), after flipping have less CD151 than before, contributing to undercorrection. (D) and (E), for each original and flipped replicate included in (A) and (B), the background was calculated using the above equation and subtracted from the uncorrected value. The average original background corrected values 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 Materials and methods).
Figure 6 with 2 supplements
Association between pseudovirions (PsVs) and heparan sulfate (HS) after cytochalasin D (CytD) treatment.
(A) HaCaT cells were incubated with PsVs at 37 °C for 5 hr, 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 lookup table [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 regions of interest (ROIs) onto the images that covered mainly the cell body but included parts of the cell periphery as well (see example in Figure 6—figure supplement 1A). For (D), smaller ROIs covering only the cell body region were used. (B) The mean HS intensity plotted over time. (C) The Pearson correlation coefficient (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 Figure 6—figure supplement 2). 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 Materials and methods).
Figure 6—figure supplement 1
Pearson correlation coefficient (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 with 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 region of interest (ROI) for analysis is illustrated as a white box. Right, in the ROI, the heparan sulfate (HS) image (cyan lookup table [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. (FG) 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), (E), (F), and (G)) or between CytD/0 min and CytD/180 min (D) were analyzed by using the two-tailed, unpaired Student’s t-test (n=3, for details see Materials and methods).
Figure 6—figure supplement 2
Background correction of the fraction of pseudovirions (PsVs) closely associated with heparan sulfate (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 Figure 6—figure supplement 1A). (B) Same as (A), for cytochalasin D (CytD). (C) From the altogether 24 fraction values obtained on flipped images (12 values from Control and CytD each), we obtain the equation y=1.85 • x for calculating the background association (please see legend of Figure 5—figure supplement 6 for more details). (D) and (E), for each original and flipped replicate included in (A) and (B), the background was calculated using the above equation and subtracted from the uncorrected value. The average original background corrected values 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 Materials and methods).
Figure 7
PsV-Itgα6 and PsV-HS distances over time.
(A) Definition of four pseudovirion (PsV) populations based on the PsV distances to Itgα6 and heparan sulfate (HS; please note that the data does not include distances of exactly 250 nm wherefore symbols as ≥and ≤ are omitted; 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 (n=3) and statistical analysis see 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; 3041–4080 PsVs per plot).
Figure 8 with 2 supplements
Association between pseudovirions (PsVs) and heparan sulfate (HS) after blebbistatin treatment.
(A) HaCaT cells were incubated with PsVs at 37 °C for 5 hr, 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 lookup table [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 regions of interest (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) Pearson correlation coefficient (PCC) between PsV-L1 (magenta LUT) and HS (cyan LUT) over time (for control with flipped images see Figure 8—figure supplement 1A and B). (D) PCC between PsV-L1 (magenta LUT) and HS (cyan LUT) in the region of the cell body over time (for control with flipped images see Figure 8—figure supplement 1C and D). (E) The fraction of PsVs (in percent) closely associating with HS (distance ≤80 nm) plotted over time (for background correction see Figure 8—figure supplement 2). 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.
Figure 8—figure supplement 1
Pearson correlation coefficient (PCC) values of flipped images.
(A) and (B) the PCC values between pseudovirion (PsV) and heparan sulfate (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 Materials and methods).
Figure 8—figure supplement 2
Background correction of the fraction of pseudovirions (PsVs) closely associated with heparan sulfate (HS) after 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 fraction values obtained on flipped images (12 values from Control and Blebbistatin each), we obtain the equation y=2.15 • x for calculating the background association (please see legend of Figure 5—figure supplement 6 for more details). (D) and (E), for each original and flipped replicate included in (A) and (B), the background was calculated using the above equation and subtracted from the uncorrected value. The average original background corrected values 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 Materials and methods).
Figure 9
Model of extracellular matrix (ECM) events, recruitment, and receptor engagement in HPV16 entry.
(i) During 5 hr of incubation with cytochalasin D (CytD), the pseudovirions (PsVs) bind to heparan sulfate (HS) of the ECM, are primed, and become coated with HS cleavage products, enabling them for cell surface receptor engagement. After CytD removal, within 15 min, HS-decorated viruses are actively recruited to the cell body and (ii) associate with CD151 assemblies (completed within 30 min). (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 belonging to 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.
Tables
Table 1
Fraction of pseudovirions (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.
| 0 min | 30 min | 60 min | 180 min | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Control [%] | CytD [%] | p-value | Control [%] | CytD [%] | p-value | Control [%] | CytD [%] | p-value | Control [%] | CytD [%] | p-value | |
| HS <250 nm and Itgα6 <250 nm | 62.31 ±5.05 | 47.83 ±3.26 | 0.0271 | 65.48 ±8.35 | 63.67 ±1.14 | 0.7764 | 59.06 ±10.64 | 67.16 ±9.84 | 0.4739 | 63.69 ±4.27 | 59.28 ±3.17 | 0.3065 |
| HS <250 nm and Itgα6 >250 nm | 12.18 ±5.51 | 46.04 ±3.67 | 0.0019 | 15.46 ±3.14 | 26.12 ±2.16 | 0.0167 | 13.25 ±1.94 | 19.87 ±3.12 | 0.0637 | 11.97 ±1.53 | 10.64 ±1.15 | 0.3808 |
| HS >250 nm and Itgα6 <250 nm | 21.59 ±4.07 | 4.34 ±0.37 | 0.0040 | 15.83 ±5.73 | 8.58 ±2.33 | 0.1723 | 23.49 ±6.55 | 9.60 ±4.81 | 0.0731 | 20.88 ±3.31 | 25.24 ±1.96 | 0.1842 |
| HS >250 nm and Itgα6 >250 nm | 3.93 ±0.30 | 1.79 ±0.60 | 0.0110 | 3.23 ±0.88 | 1.63 ±0.21 | 0.0675 | 4.19 ±2.43 | 3.38 ±2.20 | 0.7425 | 3.46 ±0.54 | 4.84 ±2.87 | 0.5393 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HaCaT cells | Cell Lines Services (CLS) | Cat. #: 300493; RRID:CVCL_0038 | Human immortalized keratinocytes used for experiments |
| Cell line (Homo sapiens) | HEK293TT | PMID:14694107 | RRID:CVCL_1D85 | PsV production cell line |
| Other | HPV16 PsVs | PMID:16350417 | Pseudovirions (PsVs) | |
| Antibody | Anti-CD151 (mouse, monoclonal) | Bio-Rad | Cat# MCA1856GA; RRID:AB_323247 | IF 1:200 |
| Antibody | Anti-HS F58-10E4 (mouse IgM, monoclonal) | Amsbio | Cat# 370255 S; RRID:AB_10891554 | IF 1:200 |
| Antibody | Anti-D-HS F69-3G10 (mouse, monoclonal) | Amsbio | Cat# 370260 S; RRID:AB_3096853 | IF 1:200 |
| Antibody | Anti-Itgα6 (rabbit, polyclonal) | Invitrogen | Cat# PA5-12334; RRID:AB_2128300 | IF 1:200 |
| Antibody | Anti-L1 K75 (rabbit, polyclonal) | PMID:15543569 | IF 1:1000 | |
| Antibody | Anti-rabbit STAR GREEN (goat, polyclonal) | Abberior | Cat# STGREEN-1002–500 UG; RRID:AB_2833016 | IF 1:200 |
| Antibody | Anti-rabbit AlexaFluor 594 (donkey, polyclonal) | Invitrogen | Cat# A21207; RRID:AB_141637 | IF 1:200 |
| Antibody | Anti-mouse AlexaFluor 594 (donkey, polyclonal) | Invitrogen | Cat# A21203; RRID:AB_2535789 | IF 1:200 |
| Antibody | Anti-mouse IgM AlexaFluor 594 (donkey, polyclonal) | Invitrogen | Cat# A21044; RRID:AB_2535713 | IF 1:200 |
| Antibody | Anti-rabbit STAR RED (goat, polyclonal) | Abberior | Cat# STRED-1002–500 UG; RRID:AB_2833015 | IF 1:200 |
| Antibody | Anti-mouse STAR RED (goat, polyclonal) | Abberior | Cat# STRED-1001–500 UG; RRID:AB_3068620 | IF 1:200 |
| Other | Phalloidin iFluor488 | Abcam | Cat# ab176753; RRID:SCR_012931 | IF 1:1000 |
| Other | Phalloidin iFluor647 | Abcam | Cat# ab176759; RRID:SCR_012931 | IF 1:1000 |
| Commercial assay, kit | Baseclick EdU 488 kit | Carl Roth | Cat# 1Y67.1 | |
| Commercial assay, kit | CytoTox-ONE Homogeneous Membrane Integrity Assay | Promega | Cat# G7891 | |
| Commercial assay, kit | Cell Culture Lysis 5 X Reagent | Promega | Cat# E153A | |
| Chemical compound, drug | (-)-Blebbistatin | Sigma-Aldrich | Cat# B0560-1MG | Stock solution 13.68 mM in dimethyl sulfoxide |
| Chemical compound, drug | Cytochalasin D | Life Technologies | Cat# PHZ1063 | Stock solution 10 mg/ml in dimethyl sulfoxide |
| Chemical compound, drug | Furin inhibitor I | Sigma-Aldrich | Cat# 344930–1 MG | Stock solution 5 mM in dimethyl sulfoxide |
| Chemical compound, drug | Leupeptin | Carl Roth | Cat# CN33.1 | Stock solution 100 mM in ddH2O |
| Software, algorithm | ImageJ | ImageJ (https://imagej.net/ij/) | RRID:SCR_003070 | Image analysis |
| Software, algorithm | GraphPad Prism 9.5.1 | GraphPad software | RRID:SCR_002798 | Visualization of data |
| Software, algorithm | Microsoft Excel | Microsoft | RRID:SCR_016137 | Statistics |
| Software, algorithm | CorelDRAW 2019 | CorelDRAW software | RRID:SCR_014235 | Visualization of data |
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/107139/elife-107139-mdarchecklist1-v1.docx
-
Source data 1
Numerical source data underlying the graphs of the manuscript.
- https://cdn.elifesciences.org/articles/107139/elife-107139-data1-v1.xlsx
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A rapid transfer of virions coated with heparan sulfate from the ECM to CD151 defines an early step in the human papillomavirus infection cascade
eLife 14:RP107139.
https://doi.org/10.7554/eLife.107139.4
