A rapid transfer of virions coated with heparan sulfate from the ECM to CD151 defines an early step in the human papillomavirus infection cascade

Reviewer #1 (Public review):

Summary:

The author's goal was to arrest PsV capsids on the extracellular matrix using cytochalasin D. The cohort was then released and interaction with the cell surface, specifically with CD151 was assessed.

The model that fragmented HS associated with released virions mediates the dominant mechanism of infectious entry has only been suggested by research from a single laboratory and has not been verified in the 10+ years since publication. The authors are basing this study on the assumption that this model is correct, and these data are referred to repeatedly as the accepted model despite much evidence to the contrary. The discussion in lines 65-71 concerning virion and HSPG affinity changes is greatly simplified. The structural changes in the capsid induced by HS interaction and the role of this priming for KLK8 and furin cleavage has been well researched. Multiple laboratories have independently documented this. If this study aims to verify the shedding model, additional data needs to be provided.

Note on revisions:

The authors did an excellent job in their revision to include data from the effect of proteolytic priming on their observed virion transfer to the cell body. All other minor issues were addressed adequately.

The work could be especially critical to understanding the process of in vivo infection.

Reviewer #2 (Public review):

The study design involves infecting HaCaT cells (immortalised keratinocytes mimicking basal cells of a target tissue) and observing virus localization with and without actin polymerization inhibition by cytochalasin D (cytoD) to analyze virion transfer from the ECM to the cell via filopodial structures, using cellular proteins as markers.

In the context of the model system, the authors stress in the revised version the importance of using HaCaT cells as a relevant 'polarized' cell model for infection. The term 'polarized' is used in the cell biological literature for epithelial cells to describe a strict apical vs. basolateral demarcation of the plasma membrane with an established diffusion barrier of the tight junction. However, HaCat cells do not form tight junctions. In squamous epithelia, such barriers are only found in granular layers of the epithelium. The published work cited in support of their claims either does not refer to polarity or only in the context of other cells such as CaCo-2 cells.

Overall, the matter of polarity would be important, if indeed the virus could only access cell-associated HSPGs as primary binding receptor, or the elusive secondary receptor via the ECM in the used model system (HaCaT cells), if they would locate exclusively basolaterally. This is at least not the case for binding, as observed in several previous publications (just two examples: Becker et al, 2018, Smith et al., 2008). With only a rather weak attempt at experimental verification of their model system with regards to polarity of binding, the authors then go on to base their conclusions on this unverified assumption.

This is one example of several in the manuscript, where claims for foundational premises, observations, and/or conclusions remain undocumented or not supported by experimental data.

Another such example is the assumption of transfer of the virus from ECM to the tetraspanin CD151. Here, the conclusions are based on the poorly documented inability of the virus to bind to the cell body, which is in stark contrast to several previous publications, and raises questions. Thus, association with CD151 likely occurs both from ECM derived virus AND virus that binds to cells, so that any conclusions on the mode of association is possible only in live cell data (which is not provided). Overall, their proposed model thus remains largely unsubstantiated with regards to receptor switching.

There are a number of important additional issues with the manuscript:

First, none of the inhibitors have been tested in their system for efficacy and specificity, but rely on published work in other cell types. This considerably weakens the confidence on the conclusion drawn by the authors.

Second, the authors aim to study transfer from ECM to the cell body and effects thereof. However, there are still substantial amounts of viruses that bind to the cell body compared to ECM-bound viruses in close vicinity to the cells. This is in part obscured by the small subcellular regions of interest that are imaged by STED microscopy, or by the use of plasma membrane sheets. This remains an issue despite the added Supple. Fig. 1, where also only sub cellular regions are being displayed. As a consequence the obtained data from time point experiments is skewed, and remains for the most part unconvincing, largely because the origin of virions in time and space cannot be taken into account. This is particularly important when interpreting the association with HS, the tetraspanin CD151, and integral alpha 6, as the low degree of association could be originating from cell bound and ECM-transferred virions alike.

Third, the use of fixed images in a time course series also does not allow to understand the issue of a potential contribution of cell membrane retraction upon cytoD treatment due to destabilisation of cortical actin. Or, of cell spreading upon cytoD washout. The microscopic analysis uses an extension of a plasma membrane stain as marker for ECM bound virions, this may introduce a bias and skew the analysis.

Fourth, while the use of randomisation during image analysis is highly recommended to establish significance (flipping), it should be done using only ROIs that have a similar density of objects for which correlations are being established. For instance, if one flips an image with half of the image showing the cell body, and half of the image ECM, it is clear that association with cell membrane structures will only be significant in the original. But given the high density of objects on the plasma membrane, I am not convinced that doing the same by flipping only the plasma membrane will not also obtain similar numbers than the original.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public review):

The authors' goal was to arrest PsV capsids on the extracellular matrix using cytochalasin D. The cohort was then released, and interaction with the cell surface, specifically with CD151, was assessed.

The model that fragmented HS associated with released virions mediates the dominant mechanism of infectious entry has only been suggested by research from a single laboratory and has not been verified in the 10+ years since publication. The authors are basing this study on the assumption that this model is correct, and these data are referred to repeatedly as the accepted model despite much evidence to the contrary.

We stated in the introduction on line 65/66 ´Two release mechanisms are discussed, that mutually are not exclusive´. This implies that we do not consider the shedding model as ‘the accepted model’. Furthermore, we do not state in the discussion neither that the shedding model is the preferred one. However, we referred to the shedding model in the discussion, because we find HS associated with transferred PsVs, which is in line with this model.

The discussion in lines 65-71 concerning virion and HSPG affinity changes is greatly simplified. The structural changes in the capsid induced by HS interaction and the role of this priming for KLK8 and furin cleavage have been well researched. Multiple laboratories have independently documented this. If this study aims to verify the shedding model, additional data need to be provided.

Our findings are compatible with both models, and we do not aim to verify the shedding model neither want to disprove the priming model. However, as we understand, the referee wishes more visibility of the priming model. Therefore, using inhibitors previously used in the field, we tested whether inhibition of KLK8 or furin reduces PsV translocation to the cell body (after CytD wash off). Leupeptin blocks transport, while Furin inhibitor I still allows some initial translocation. We incorporated this new data as Figure 2 (line 265): “…we would expect that inhibition of L1 processing during the CytD incubation prevents the recovery of PsV translocation from the ECM to the cell body (Figure 2A and D). To test for this possibility, as employed in earlier studies, the protease inhibitor leupeptin was used to inhibit proteases including KLK8 which is required for L1 cleavage (Cerqueira et al. 2015). Employing this inhibitor, the PCC between PsV-L1 and F-actin staining remains negative after CytD removal, showing that for translocation indeed the action of proteases is required (Figure 2B and D). In contrast, inhibition of L2 cleavage by a furin specific inhibitor has no effect on the PCC (Figure 2C and D). However, it should be noted that we occasionally observe PsVs not completely translocating but accumulating at the border of the F-actin stained area (for example see Figure 2C (60 min)). This results in an increase of the PCC almost equal to complete translocation, explaining why the PCC remains unaffected despite a furin inhibitory effect. Hence, furin inhibition may have some effect on translocation that, however, is undetected in this type of analysis.’

Moreover, we have added a paragraph discussing how our data integrates into the established model of the HPV infection cascade (line 604): ‘HPV infection is the result of several steps, starting with the initial binding of virions via electrostatic and polar interactions (Dasgupta et al. 2011) to the primary attachment site HS (Richards et al. 2013), which induces capsid modification (Feng et al. 2024; Cerqueira et al. 2015) and HS cleavage (Surviladze et al. 2015), enabling the virion to be released from the ECM or the glycocalyx. Next, virions bind to the cell surface to a secondary receptor complex that forms over time, and become internalized via endocytosis, before they are trafficked to the nucleus (Ozbun and Campos 2021; Mikuličić et al. 2021). Regarding the transition from the primary attachment site to cell surface binding, as already outlined in the introduction, two models are discussed. In one model, proteases cleave the capsid proteins. After priming, the capsids are structurally modified and the virion can dissociate from its HS attachment site. It has been suggested that capsid priming is mediated by KLK8 (Cerqueira et al. 2015) and furin (Richards et al. 2006). In our system, KLK8 inhibition blocks PsV transport, while furin inhibition has some effect that, however, cannot be detected in this analysis (Figure 2) suggesting furin engagement at later steps in the infection cascade. This is in line with earlier in vitro studies on the role of cell surface furin (Surviladze et al. 2015; Day et al. 2008; Day and Schiller 2009). In any case, our results align with both models of ECM detachment: one involving HS cleavage (HS co-transfer) and another involving capsid modification (by e.g., KLK8).’

The model should be fitted into established entry events,…

Please see our reply above.

or at minimum, these conflicting data, a subset of which is noted below, need to be acknowledged.

(1) The Sapp lab (Richards et al., 2013) found that HSPG-mediated conformational changes in L1 and L2 allowed the release of the virus from primary binding and allowing secondary receptor engagements in the absence of HS shedding.

(2) Becker et al. found that furin-precleaved capsids could infect cells independently of HSPG interaction, but this infection was still inhibited with cytochalasin D.

(3) Other work from the Schelhaas lab showed that cytochalasin D inhibition of infection resulted in the accumulation of capsids in deep invaginations from the cell surface, not on the ECM

(4) Selinka et al., 2007, showed that preventing HSPG-induced conformational changes in the capsid surface resulted in noninfectious uptake that was not prevented with cytochalasin D.

(5) The well-described capsid processing events by KLK8 and furin need to be mechanistically linked to the proposed model. Does inhibition of either of these cleavages prevent engagement with CD151?

The authors need to consider an explanation for these discrepancies.

We do not see any discrepancies; our observations are compatible with aspects of both the shedding and the priming model. That PsVs carry HS-cleavage products doesn´t imply that HS cleavage is sufficient or required for infection, or that the priming model would be wrong. We do not view our data as being in conflict with the priming model. Most of the above-mentioned papers are now cited.

Altogether, we acknowledge that the study gains importance by directly testing the priming model within our experimental system. We are thankful for the above comments and addressed this issue.

Other issues:

(1) Line 110-111. The statement about PsVs in the ECM being too far away from the cell surface to make physical contact with the cell surface entry receptors is confusing. ECM binding has not been shown to be an obligatory step for in vitro infection.

Not obligatory, but strongly supportive (Bienkowska-Haba et al., Plos Path., 2018; Surviladze et al., J. Gen. Viro., 2015). As recently published by the Sapp lab (Bienkowska-Haba et al., Plos Path., 2018), ´Direct binding of HPV16 to primary keratinocytes yields very inefficient infection rates for unknown reasons.´ Moreover, the paper shows that HaCaT cell ECM binding of PsVs increases the infection of NHEK by 10-fold and of HFK by almost 50-fold.

This idea is referred to again on lines 158-159 and 199. The claim (line 158) that PsV does not interact with the cell within an hour needs to be demonstrated experimentally and seems at odds with multiple laboratories' data. PsV has been shown to directly interact with HSPG on the cell surface in addition to the ECM. Why are these PsVs not detected?

The reviewing editor speculated that HaCaT cells may be a model system in which the in vivo relevant binding to the ECM can be better studied as in non-polarized cell types. This is because binding to the ECM cannot be bypassed by direct cell surface binding. The observation that only few PsVs bind to the basal cell membrane indeed suggests restricted diffusional access of PsVs to binding receptors of the basal membrane. The reviewing editor asked for an experiment showing that more PsVs bind after cell detachment. We performed this experiment and indeed find more PsVs binding to the cell surface of detached cells. This point is very important for the understanding of the study and now we mention it in several sections of the manuscript, as outlined in the following.

Line 125: ‘Many PsVs that bind to the ECM may locate distal from the cell surface and are thus unable to establish direct contact with entry receptors. However, they are capable of migrating by an actindependent transport along cell protrusions towards the cell body (Smith et al. 2008; Schelhaas et al. 2008). We aimed for blocking this transport in HaCaT cells, a cell line that is widely used as a cell culture model for HPV infection. HaCaT cells closely resemble primary keratinocytes in key aspects: they are not virally transformed and produce large amounts of ECM that facilitates infection (Bienkowska-Haba et al. 2018; Gilson et al. 2020). In addition, HaCaT cells exhibit cellular polarity that enforces binding of virus particles to the ECM, as the virions cannot bind to receptors/entry components, such as CD151, Itgα6 and HSPGs that co-distribute on the basolateral membrane of polarized keratinocytes (Sterk et al. 2000; Cowin et al. 2006; Mertens et al. 1996), making them inaccessible by diffusion.’

Line 205: ‘During the CytD incubation, PsVs bind to HSPGs of the basolateral membrane for 5 h. Still, in the cell body area hardly any PsVs are present (0.14 PsV/µm², Supplementary Figure 1B). In the control, the PsV density is several-fold larger (Supplementary Figure 1B). This is expected, as the PsVs bind to the ECM and translocate to the cell body. We wondered whether there are more binding sites at the basal membrane that remain inaccessible to PsVs by diffusion because of the insufficient space between glass-coverslip and basolateral membrane. For clarification, we incubated EDTA detached HaCaT cells in suspension with PsVs for 1 h at 4 °C, followed by re-attachment for 1 h. Under these conditions, we find a PsV density 12.4-fold larger than after 5 h of CytD incubation of adhered cells (Supplementary Figure 1B and D). However, it should be noted that these values cannot be directly compared. Aside from the different treatments, another difference lies in the size of the basal membrane, as re-attachment of cells is not complete after only 1 h (compare size of adhered membranes in Supplementary Figure 1A and C). Therefore, the imaged membranes are likely strongly ruffled, which results in the underestimation of the size of the adhered membrane. As a result, we overestimate the PsVs per µm² (please note that we cannot re-attach cells for longer times as we would then lose PsVs due to endocytosis). On the other hand, we would underestimate the PsV density at the basal membrane if after re-attachment we image in part also some apical membrane. In any case, the experiment suggests that PsVs bind more efficiently if membrane surface receptors are accessible by diffusion. This is in support of the above notion that the basal membrane may provide more entry receptors than one would expect from the low density of PsVs bound after 5 h CytD (Supplementary Figure 1B). This suggests that under our assay conditions, PsVs cannot easily bypass the translocation from the ECM to the cell body by diffusing directly to the basal membrane. Hence, the large majority of PsVs that enter the cell were previously bound to the ECM. Therefore, HaCaT cells serve as an ideal model for studying the transfer of ECM bound HPV particles to the cell surface, which is similar to in vivo infection of basal keratinocytes after binding to the basement membrane (Day and Schelhaas 2014; Kines et al. 2009; Schiller et al. 2010; Bienkowska-Haba et al. 2018).’

Line 529: ‘Filopodia usage not only facilitates infection but also increases the likelihood of virions to reach their target cells during wound healing, namely the filopodia-rich basal dividing cells. In fact, several types of viruses exploit filopodia during virus entry (Chang et al. 2016), hinting at the possibility that for HPV and other types of viruses actin-driven virion transport may play a more important role than it is currently assumed. If this is the case, sub-confluent HaCaT cells, or even better single HaCaT cells, would be an ideal model system for the study of these very early infection steps that involve ECM attachment and subsequent filopodia-dependent transport. As shown in Supplementary Figure 1, HaCaT cells have many binding sites for the HPV16 PsVs. However, as they are polarized and the binding receptors are only at the basal membrane, they remain relatively inaccessible by diffusion. Therefore, the ECM binding that is also observed in vivo (Day and Schelhaas 2014) and subsequent transport via filopodia are used upon infection of HaCaT cells that locate at the periphery of cell patches. Here, PsVs bind to the ECM which strongly enhances infection of primary keratinocytes (Bienkowska-Haba et al. 2018). In contrast, HPV can readily bind to HSPGs on the cell surface of nonpolarized cells, and by this bypasses ECM mediated virus priming and the filopodia dependency. We propose that HaCaT cells are a valuable system for studying the very early events in HPV infection that allows for dissecting capsid interaction with ECM resident priming factors and cell surface receptors.’

Finally, please note that in the previous version of the manuscript, we did not question that in many cellular systems PsVs interact with heparan sulfate proteoglycans (HSPGs) present on the cell surface, or both on the cell surface and the ECM. We stated on line 59 ´While in cell culture virions bind to HS of the cell surface and the ECM, it has been suggested that in vivo they bind predominantly to HS of the extracellular basement membrane (Day and Schelhaas, 2014; Kines et al., 2009; Schiller et al., 2010).´

We hope that after adding the above explanations and the experiment requested by the reviewing editor it is now clear why only few PsVs bind directly (not via the ECM) to the cell surface. We appreciate the reviewer’s and the reviewing editor’s input that has significantly improved the manuscript.

(2) The experiments shown in Figure 5 need to be better controlled. Why is there no HS staining of the cell surface at the early timepoints? This antibody has been shown to recognize N-sulfated glucosamine residues on HS and, therefore, detects HSPG on the ECM and cell surface.

There is staining. However, as the staining at the periphery is stronger and images are shown at the same settings of brightness and contrast, the impression is given that the cell surface is not stained. We have added more images showing HS cell surface staining.

(i) Supplementary Figure 4C shows an enlarged view of the CytD/0 min cell shown in Figure 6A. In the area stained by Itgα6, that marks the cell body, HS staining is present, although less abundant in comparison to the ECM.

(ii) In Figure 8, CytD/30 min, a cell is shown with abundant HS in the cell body region (compare cyan and green LUT).

(iii) In newly added Figure 3A, lower panel, another cell with HS in the cell body region is shown.

Please note that the staining is highly variable. We indicate this by stating on Line 373: ‘The pattern of the HS staining (cyan LUT) and the overlap of HS with PsVs and Itgα6 are highly variable (Figure 6A).’

Therefore, the conclusion that this confirms HS coating of PsV during release from the ECM (line 430431) is unfounded. How do the authors distinguish between "HS-coated virions" and HSPG-associated virions?

The transient increase in the PCC at CytD/30 min can be interpreted as PsV/HS co-transport or as direct binding of PsVs to cell surface HSPGs. However, two arguments support co-transport.

First, we find that CytD/PsVs increases the HS intensity (see newly added Figure 3, confirming old Figure 5 that is now Figure 6). We state on line 290 ‘… that without actin-dependent PsV translocation HS cleavage products are retained in the ECM, consistent with the hypothesis that cleaved HS remains associated with PsVs (Ozbun and Campos 2021).

Second, the distance between HS and Itgα6 (the cell body marker) decreases over time after CytD removal, which suggests movement of HS to the cell body (Supplementary Figure 8D). We state on line 422: ‘The movement of HS towards the cell body after removal of CytD, which indirectly demonstrates that PsVs are coated with HS, is suggested by a shortening of the HS-Itgα6 distance over time (Supplementary Figure 8D).’

It is difficult to comprehend how the addition of 50 vge/cell of PsV could cause such a global change in HS levels.

Some areas are covered with confluent cells, to which hardly any PsVs are bound, because accessing their basolateral membrane is nearly impossible, and PsVs do not bind to the exposed apical membrane as well. We assume this is a major difference to cultures of unpolarized cells, where PsVs should distribute more or less equally over cells. This means that in our experiments the vge/cell is not a suitable parameter for relating the magnitude of an effect to a defined number of PsVs. In the ECM, the PsV density is very high, enabling one cell to collect, in theory, several hundred PsVs, much more than expected from the 50 vge/cell.

We state on line 135: ‘Frequently, we observe patches of confluent cells which are common to HaCaT cells. Cells at the center of these patches are dismissed during imaging, because there are no anterogradely migrating PsVs at these cells. A second reason for our dismissal of these cells is that hardly any PsVs are bound to them, possibly because their basal membranes are inaccessible by diffusion. Instead, we focus on isolated HaCaT cells or cells at the periphery of cell patches. In these cells, we find more PsVs per cell than one would expect from the employed 50 viral genome equivalents (vge) per cell, indicating that PsVs are unequally distributed between the cells.’

The claim that the HS levels are decreased in the non-cytochalasin-treated cells due to PsV-induced shedding needs to be demonstrated.

We did not claim that PsVs induce shedding, we rather believe they retain shedded HS. Without PsVs, the shedded HS is washed off from the ECM. We have reproduced the observation made in old Figure 5 (now Figure 6) in the newly added Figure 3 that also shows that PsVs alone have no effect on the HS intensity, only when present together with CytD. We state on line 277: ‘As outlined above, during the 5 h incubation with CytD, proteases in the ECM are expected to cleave HS chains. These cleavage products should be able to diffuse out of the ECM, unless they remain associated with nontranslocating PsVs. In the control, PsV associated HS cleavage products would leave the ECM through PsV translocation…. Using an antibody that reacts with an epitope in native heparan sulfate chains, only after CytD and if PsVs are present, the level of HS staining is significantly increased (Figure 3B). As shown in Figure 3A, stronger HS staining at PsVs (open arrows) and as well in PsV free areas (closed arrows) was observed… Collectively, our findings indicate that without actin-dependent PsV translocation HS cleavage products are retained in the ECM, consistent with the hypothesis that cleaved HS remains associated with PsVs (Ozbun and Campos 2021).’

If HS is actually shed, staining of the cell periphery could increase with the antibody 3G10, which detects the HS neoepitope created following heparinase cleavage.

We have tested the antibody by which we obtain only a very weak staining (Supplementary Figure 2), not allowing to differentiate between an increase in the cell periphery and the cell body area. We still include the experiment as it suggests that CytD has no effect on HS processing. We state on line 286: ‘As additional control and shown in Supplementary Figure 2, we use an antibody that reacts with a HS neo-epitope generated by heparitinase-treated heparan sulfate chains (Yokoyama et al. 1999; for details see methods). This neo-epitope staining is independent of the presence of CytD and the incubation time, suggesting that CytD does not directly affect HS processing.’

Reviewer #2 (Public review):

Summary:

Massenberg and colleagues aimed to understand how Human papillomavirus particles that bind to the extracellular matrix (ECM) transfer to the cell body for later uptake, entry, and infection. The binding to ECM is key for getting close to the virus's host cell (basal keratinocytes) after a wounding scenario for later infection in a mouse vaginal challenge model, indicating that this is an important question in the field.

Strengths:

The authors take on a conceptually interesting and potentially very important question to understand how initial infection occurs in vivo. The authors confirm previous work that actin-based processes contribute to virus transport to the cell body. The superresolution microscopy methods and data collection are state-of-the art and provide an interesting new way of analysing the interaction with host cell proteins on the cell surface in certain infection scenarios. The proposed hypothesis is interesting and, if substantiated, could significantly advance the field.

Weaknesses:

As a study design, the authors use infection of HaCaT keratinocytes, and follow virus localisation with and without inhibition of actin polymerisation by cytochalasin D (cytoD) to analyse transfer of virions from the ECM to the cell by filopodial structures using important cellular proteins for cell entry as markers.

First, the data is mostly descriptive besides the use of cytoD, and does not test the main claim of their model, in which virions that are still bound to heparan sulfate proteoglycans are transferred by binding to tetraspanins along filopodia to the cell body.

The study identifies a rapid translocation step from the ECM to CD151 assemblies. We have no data that demonstrates a physical interaction between PsVs and CD151. In the model figure, we draw CD151 as part of the secondary receptor complex. We are sorry for having raised the impression that PsVs would bind directly to CD151 and have modified the model Figure accordingly. In the new model figure (Figure 9), the first contact established is to a CD151 free receptor.

Second, using cytoD is a rather broad treatment that not only affects actin retrograde flow, but also virus endocytosis and further vesicular transport in cells, including exocytosis. Inhibition of myosin II, e.g., by blebbistatin, would have been a better choice as it, for instance, does not interfere with endocytosis of the virus.

As we focus on early events, we are not concerned about CytD blocking as well late steps in the infection cascade, like endocytosis. However, we agree that a comparison between CytD and blebbistatin would be very interesting. We added Figure 8, showing that blebbistatin only partially stops migration.

Line 429: ‘Actin retrograde transport, which underlies the here observed virion transport, is the integrative result of three components (Smith et al. 2008; Schelhaas et al. 2008)…. As CytD broadly interferes with F-actin dependent processes, we investigated the effects upon inhibition of only one of the three components, namely the myosin II mediated retrograde movement towards the cell body. Instead of CytD, we employed in the 5 h preincubation the myosin II inhibitor blebbistatin. For the control (0 min), we show in Figure 8A one example of a cell with comparatively many PsVs at the periphery (as mentioned above, the PsV pattern is highly variable) to better illustrate the difference to the PsV pattern occasionally seen with blebbistatin. After blebbistatin treatment (0 min), PsVs are still distal to the cell body but less dispersed than after CytD treatment, seemingly as if translocation started but stopped in the midst of the pathway (Figure 8A, blebbistatin). The PCC between PsVs and HS, like after CytD (Figure 6C), is elevated after blebbistatin, albeit the effect is not significant (Figure 8C). The cell body PCC, is not at 30 min (CytD) but already at 0 min elevated (compare Figure 6D to Figure 8D), which can be explained by partial translocation. This is further supported by the fact that only 8% of PsVs are closely associated with HS (Figure 8E; blebbistatin, 0 min) compared to 15% after CytD treatment (Figure 6E; 0 min). Furthermore, after 0 min PsV incubation with blebbistatin we observe no effect on the HS intensity (compare Figure 8B to Figure 3B and Figure 6B). Hence, in contrast to CytD, blebbistatin does not trap the PsVs in the ECM where they associate with HS, but ongoing actin polymerization pushes actin filaments along with PsVs towards the cell body.’

Third, the authors aim to study transfer from ECM to the cell body and the effects thereof. However, there are substantial, if not the majority of, viruses that bind to the cell body compared to ECM-bound viruses in close vicinity to the cells.

Please see our detailed reply to referee #1 that has raised the same issue. In brief, we agree that in multiple cell culture systems viruses bind preferentially to the cell surface directly. However, in HaCaT cells, the majority of PsVs does not bind directly to the basal membrane but gets there after initial binding to the ECM. Thus, we believe our system appropriately models the physiologically relevant scenario of ECM-to-cell transfer, as also speculated by the reviewing editor that has suggested an experiment showing that more PsVs bind to detached cells (please see above).

This is in part obscured by the small subcellular regions of interest that are imaged by STED microscopy, or by the use of plasma membrane sheets. As a consequence, the obtained data from time point experiments is skewed, and remains for the most part unconvincing due to the fact that the origin of virions in time and space cannot be taken into account. This is particularly important when interpreting association with HS, the tetraspanin CD151, and integral alpha 6, as the low degree of association could originate from cell-bound and ECM-transferred virions alike.

As already stated above, we observe massive binding of PsVs to the ECM, in contrast to very few PsVs that diffuse beneath the basolateral membrane of the polarized HaCaT cells and do bind directly to the cell surface. In other cellular systems, cells may hardly secrete ECM, are not polarized, and therefore virions can easily bypass ECM binding. Therefore, it is reasonable to assume that in HaCaT cells the large majority of PsVs found on the cell body originates from the ECM.

Fourth, the use of fixed images in a time course series also does not allow for understanding the issue of a potential contribution of cell membrane retraction upon cytoD treatment due to destabilisation of cortical actin. Or, of cell spreading upon cytoD washout.

The newly added blebbistatin experiment suggests that the initial translocation is exclusively dependent on retrograde actin flow. However, we agree that we are not able to unravel more details regarding the different possible contributions to the movement. Importantly, the lack of PCC increase after CytD/leupeptin removal (Figure 2D) suggest there is not much cell spreading into the area of accumulated PsVs. Please see our more detailed reply to the same issue raised by the same referee in the recommendations for the authors.

The microscopic analysis uses an extension of a plasma membrane stain as a marker for ECM-bound virions, which may introduce a bias and skew the analysis.

The dye TMA-DPH stains exclusively cellular membranes and not the ECM. The stain is actually used to delineate the cell body from the ECM area (please see Figure 1).

Fifth, while the use of randomisation during image analysis is highly recommended to establish significance (flipping), it should be done using only ROIs that have a similar density of objects for which correlations are being established.

We agree that the way of how randomization is done is very important. Regarding the association of PsVs with CD151 and HS, we corrected for random background association, which is now explained in more detail in in the Figure legend of Supplementary Figure 7: “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)… (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.’

For instance, if one flips an image with half of the image showing the cell body, and half of the image ECM, it is clear that association with cell membrane structures will only be significant in the original.

We are aware of this problem. For instance, it would produce ‘artificially’ low PCCs after flipping images of PsV/HS stainings (please see negative PCC value after flipping in Supplementary Figure 8). In this case, we do not use as argument that in flipped images the PCC is lower. Instead, we would argue that over time the PCC changes in the original images. We still provide the PCC values of flipped images, as additional information, showing that in most cases we obtain after flipping a PCC of zero, as expected

Hence, we fully agree that careful controls in image analysis is required, and used the above-described method for the correction of background association when the fraction of closely associated PsVs is analyzed. We do not use a lower PCC value in flipped images as argument if not appropriate.

I am rather convinced that using randomisation only on the plasma membrane ROIs will not establish any clear significance of the correlating signals.

Figure 6D and 8D show the PCC specifically of the cell body (only of plasma membrane ROIs). In flipped images (not shown in the previous version for clarity), we obtain significantly lower PCCs (Supplementary Figure 8F/G and Supplementary Figure 10C/D. We propose that in this case it would be appropriate to use a lower PCC of flipped images as argument for specific association. Still, also in this experiment we argue with a change in the PCC over time, and not with a PCC of zero after flipping. As above, we still provide the PCC values of flipped images as additional information.

Also, there should be a higher n for the measurements.

One replicate is based on the average of 14-15 cells for each condition (more for figure 4). Hence, in a typical experiment (Control and CytD with 4 time points) about 120 cells are analyzed, which is a broad basis for the averages of one replicate.

We realize that with three biological replicates we find significant effects only if we have strong effects or moderate effects with very low variance.

Recommendations for the authors:

Reviewing Editor:

The focus on the events of HPV infection between ECM binding and keratinocyte-specific receptor binding is unique and interesting. However, I agree with the reviewers that some of the conclusions could use more experimental support, as detailed in their comments. The failure to detect direct binding of the PsV to HSPGs on the cell surface in in vitro assays contradicts much of the published literature. For example, others have found that HPV capsids bind cultured cell lines in suspension, i.e, in the absence of ECM. Do EDTA-suspended HaCaT cells bind PsV? Is the binding HSPG dependent? If the authors think that failure to detect direct cell binding of HaCaTs is an unusual feature of these cell lines or culture condition,s then it would be helpful to provide an explanation. However, it is worth noting that an in vitro system where the cells do not directly bind capsids through HSPG interactions would be a much better model for studying the stages of HPV infection that are the focus of this study, since there is no direct binding of keratinoctyes in vivo.

We are thankful for this comment that had a strong influence on the revision. The suggested experiment has been incorporated as new Supplementary Figure 1. It shows that many more PsVs bind to the cell surface of cells in suspension than to adhered cells. As suggested by the reviewing editor, we explain now that HaCaT cells are a suitable model system for studying the in vivo transport from the ECM to the cell body that in these cells, due to their polarization, cannot be bypassed (for more details please see our replies above addressing these issues).

Because conclusions drawn regarding HS interactions are largely based on experiments using a single HS mAb, it is important that the specificity of this mAb is described in more detail, either based on the literature or further experimentation.

We provide now detailed information about the HS antibodies used in the study. We state on line 282 ‘Using an antibody that reacts with an epitope in native heparan sulfate chains…’ and on line 286 ‘we use an antibody that reacts with a HS neo-epitope generated by heparitinase-treated heparan sulfate chains…’ and in the methods section ‘For Heparan sulfate (HS) a mouse IgM monoclonal antibody (1:200) (amsbio, cat# 370255-S) was used that reacts with an epitope in native heparan sulfate chains and not with hyaluronate, chondroitin or DNA, and poorly with heparin (mAb 10E4 (David et al., 1992)). For HS neo-epitope (Yokoyama et al., 1999) detection, a mouse monoclonal antibody (1:200) (amsbio, cat#370260-S) was used that reacts only with heparitinase-treated heparan sulfate chains, proteoglycans, or tissue sections, and not with heparinase treated HSPGs. The antibody recognizes desaturated uronic acid residues (mAb 3G10 (David et al., 1992)).’

Reviewer #1 (Recommendations for the authors):

(1) The phrase "tight association" or similar is repeatedly used and is not acceptable for microscopic studies; use "close association", which has no affinity connotations.

Has been changed as suggested by the referee.

(2) Why are lysine-coated coverslips used for microscopy? HaCaT cells adhere tightly to untreated glass, and this coating could affect the distribution of ECM and extracellular PsV.

We believe a tight association of the basal cell membrane to its substrate, as in vivo, where the basal membrane is tightly adhered to other cells, is important in these experiments. In weakly adherent cells more PsVs may bind to the cell surface, bypassing the transport step. Hence, although HaCaT cells may not require the coat and would be able to adhere to glass, the association may not be tight enough to mimic in vivo conditions.

(3) What is the reason to use detection of the pseudogenome for some of the experiments instead of L1 detection throughout? The process of EdU detection is sufficiently denaturing to affect some protein epitopes. The introduction of this potential artifact doesn't seem warranted for capsid detection experiments.

The L1 and the Itgα6 antibody are from the same species, wherefore we have used in Figures 4 and 6 click-labeling of the reporter plasmid. We do not disagree with the notion of the referee, that EdU detection may denature the epitope of some proteins. For instance, we have observed a different staining pattern for CD151; for Itgα6 and HS we saw no obvious difference in the staining patterns. In double staining experiments using L1 antibody and click-labeling, both staining patterns overlapped very well, indicating that click-labeling is suitable to visualize PsVs.

(4) What concentration of TMA-DPH was used?

TMA-DPH is a poorly water-soluble dye that becomes strongly fluorescent upon insertion into a membrane. Because of its poor water solubility, a precise concentration cannot be given. We added 50 µl of a saturated TMA-DPH solution in PBS to 1 ml of PBS in the imaging chamber. We state this now in the methods section.

(5) Line 419: This statement is misleading. Although PsV interaction with HSPG on the ECM is crucial for infectious transfer to cells, the majority of the PsV binding on the ECM has been attributed to interaction with laminin 332. Treatment of PsV with heparin causes sequestration to the ECM.

We are sorry for the confusion and have removed the misleading statement.

(6) Some reference choices are poor:

Line 54: Ozbun and Campos, this is not the correct reference

In the review we cited, in the introduction it is stated that PsVs establish infection via a break in the epithelial barrier? However, we have replaced this reference by a review that focuses more on epithelial wounding: ‘Ozbun, Michelle A. (2019): Extracellular events impacting human papillomavirus infections: Epithelial wounding to cell signaling involved in virus entry. In Papillomavirus research (Amsterdam, Netherlands) 7, pp. 188–192. DOI: 10.1016/j.pvr.2019.04.009.’

Line 2012: Doorbar et al., this is not the correct reference.

Thank you for pointing this out (..we assume the referee refers to line 104 and not line 2012). We have noticed this error during revision. As it is difficult to get a specialized review on this topic, we now cite Ozbun and Campus, 2021 that states PsVs are ‘structurally and immunologically indistinguishable from lesion- and tissue-derived HPVs.’

Minor issues:

(1) It is difficult to appreciate the ECM and cell surface binding pattern from the provided images, which do not even contain an entire cell. We need to see a few representative field views with the ECM delineated with laminin 332 staining, as HS antibodies stain both the ECM and cell surface.

We now provide overview images in Supplementary Figure 4. The only experiment requiring a clear delineation between ECM and cell surface is the experiment of Figure 4. Here, we do not use the HS as a reference staining because it stains both the ECM and the cell surface.

(2) For Figure 1E, the cells were only infected for 24 hours. The half-time for infectious internalization of HaCaT cells was shown to be 8 hours for cell-associated PsV and closer to 20 hours for PsV that was associated with the ECM prior to cell association (Becker et al., 2018). Why was such a short infection time chosen?

During assay establishment it has been observed that after 24 h the luciferase activity is optimal.

(3) Figure 5, the staining of uninfected cells +/- cyto treatment needs to be included.

Now visible in new Figure 3.

I am confused by lines 54-57. It seems as if the authors are claiming that HSPGs are not present on the ECM. This sentence, as written, is misleading.

We agree, and state now on line 58 ‘Here, virions bind to the linear polysaccharide heparan sulfate (HS) that is present in the extracellular matrix (ECM) but as well on the plasma membrane surface. HS is attached to proteins forming so called heparan sulfate proteoglycans (HSPGs).’

Reviewer #2 (Recommendations for the authors):

There are further issues that are not pertaining to the study design that I find important.

(1) It remains speculative whether the virions that are transferred from the ECM are actually structurally modified.

The newly added Figure 2, showing that leupeptin blocks infection in our assay, suggests that virions indeed are primed.

(2) The origin of HS correlated with virions on the cell body after transfer is also not clear: does the virus associate with cell surface HS, or does it bring HS from the ECM? Simply staining HS against Nsulfated moieties does not allow such conclusions.

This issue has been already raised in the public review to which we replied above. In brief, we agree that the transient increase of the PCC between PsVs and HS in the cell body region can be also explained by PsVs coming from the ECM without HS and binding to cell surface HS, or from PsVs binding directly (not via the ECM) to cell surface HSPGs. However, there are two more arguments indicating that PsVs are coated with HS. Please see our detailed reply above.

(3) Figure 1: There are few, if any, filopodia in untreated cells. It would be good to quantify their abundance to substantiate that resting HaCat cells are indeed a good model for filopodial transport bs. membrane retraction / spreading. In HaCat ECM, the virus also binds to laminin-332 for a good part. Would this not also confound the analysis?

At first glance, the number of filopodia appears to be too low to account for such an efficient transport. However, please note that the formation of filopodia is very dynamic, and that they can form and disappear within minutes (see below). We also often observe many PsVs aligned at one filopodium. Moreover, not every cell periphery exhibits large accumulations of PsVs. Therefore, we believe it is in principle possible that filopodia are largely responsible for the transport. We cannot exclude that we overestimate the transport rate due to partial cell spreading after CytD removal, which, however, we consider as rather unlikely as in Figure 2 we observe no increase in the PCC when leupeptin was present during the CytD incubation. Under these conditions, PsVs do not translocate but cells could spread, and this would increase he PCC between PsVs and F-actin if cells would spread into the area of accumulated PsVs.

We now state on line 304: ‘This suggests that the half-time of PsV translocation from the periphery to the cell body is about 15 min. In fact, the half-time maybe longer, as we cannot exclude that cell spreading after CytD removal contributes to less PsVs measured in the cell periphery.’ and on line 477 ‘As mentioned above, the half-time could be longer if cell spreading is in part responsible for the translocation of PsVs onto the cell body. However, we assume that this is rather unlikely, as cell spreading would increase the PCC between PsVs and F-actin under a condition where filopodia mediated transport is blocked but not cell spreading, which is not the case (Figure 2B and D, CytD/leupeptin).’

(4) Figure 2: This would benefit from live cell analysis. There are considerable amounts of virions on the cell body, which partially contradicts statements from Figure 1.

Does the referee refer to the images shown in Figure 4 (old Figure 2)? Please note that at CytD/0 min there are hardly any PsVs in the cell body region, the fluorescence (magenta LUT) is autofluorescence (this is explained in the results section). Only at later time points PsVs are in the cell body region.

The fast transfer to the cell body after cyto D washout is based on the assumption that filopodia formation and transport along them (and not membrane extension) occur quickly. Is this reasonable?

We are no experts on filopodia, but one finds references suggesting that they grow at rates of several µm per minutes and have lifetimes between a few seconds and several minutes. Hence, within the 15 min we determine for the transport, cells may need a few minutes to recover from CytD, a few minutes to form filopodia that reach out into the ECM, and a few minutes for the transport itself. However, we agree that we cannot exclude membrane extension contributing to our observed transport, although we consider this as rather unlikely (see above).

(5) Figure 3: The rationale of claiming the existence of 'endocytic structures' needs to be better explained and quantified in the according supplementary figure.

We now state in the legend ‘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).’ For a proper quantification of these highly variable structures a much larger sample would be required.

The formation of virus-filled tubules upon cytoD treatment has been previously reported. Are these viruses that come from the cell body or from the ECM?

With the new data and explanations that have been added to the manuscript, it should be clear that it is reasonable to assume that they come largely from the ECM.

(6) Figure 4: How are the subcellular ROIs chosen? Is there not a bias by not studying a full cell?

We now explain better how we chose cells for analysis. We state on line 138 ‘Instead, we focus on isolated HaCaT cells or cells at the periphery of cell patches. In these cells, we find more PsVs per cell than one would expect from the employed 50 viral genome equivalents (vge) per cell, as PsVs are unequally distributed between the cells. Moreover, these PsVs usually are not homogenously distributed around the cell but concentrate at one region. We investigate the translocation of PsVs from these regions, defining ROIs for analysis that cover PsVs at the periphery and the cell body (see Supplementary Figures 6A and 8A).’

(7) Figure 5/6: The data needs a better analysis on correlation by using randomisation as explained above.

Please see our reply to the same point of the public review raised by the same referee.

(8) Figure 7: This model involves CD151 being a mediator in transfer, but this has not been functionally shown. There are HaCaT CD151 KO cells available (from the Sonnenberg lab), it would be good to use those to test the model and whether transfer indeed involves CD151.

As already stated above, we are sorry for having raised the impression that PsVs bind directly to CD151. The model Figure has been modified. Please see our reply above.

(9) The manuscript would benefit from a number of experiments addressing the most crucial issues:

(a) As mentioned before, the use of blebbistatin, which blocks myosin II function and arrests actin retrograde flow within seconds of addition, would be a good inhibitor to control for transfer in at least some of the most crucial experiments.

In Figure 8 we have tested blebbistatin. Please see our reply above.

(b) Live cell analysis would allow for monitoring of whether membrane retraction upon cytoD treatment would have to be taken into account for the analysis of the data. The same is true for the cytoD washouts, upon which most cells exhibit pronounced membrane spreading. The latter is important to support filopodial transport rather than membrane ruffling and spreading, leading to the clearance of extracellular virions from the ECM.

We agree that this would be desirable. As replied above, we now discuss the issue of possible membrane spreading and reason why we consider it as rather unlikely.

(c) To rid oneself of the issue of plasma membrane-bound virions as a confounding factor, one could use cells treated by sodium chlorate, which leads to undersulfation of HS on the cell surface, and seed them onto ECM with functional HSPGs. This would then indeed establish that the HS and virus are transferred together.

We agree that this would be a smart experiment. As the main focus of our study is not clarifying whether PsVs are coated with HS or not, we gave other experiments priority.

(10) The manuscript is, while carefully and thoughtfully worded on the issue of microscopy analysis, for a good part, extrapolating too strongly from the authors' data and unsubstantiated assumptions to conclude on their model. It would be good if the authors would support their claims with previous or their own experimental work. Just two examples of several: the assumption that cell-bound virions are negligible should be substantiated, as the literature would indicate otherwise.

We determined the PsV density in adhered, CytD treated cells, and find around 0.14 per µm² (Supplementary figure 1B), which is 4 to 5-fold less when compared to the PsV density quantified in an area covering the cell body and the periphery (Figure 1B, see line 174 for PsVs/µm² values). Quantifying the PsV density only in the periphery would yield a severalfold larger difference. However, due to the limited resolution of the microscope we would strongly underestimate the PsV density in the accumulations. We prefer not to discuss this in detail, as exact numbers are difficult to obtain.

Line 129: Cyto D should not inhibit the enzymes modifying HS or proteins (including virions). This is true, but cytoD may limit their secretion and abundance.

We show in Figure 3 that CytD does not reduce HS staining (e.g., by limiting HS secretion, as suggested by the referee), suggesting that it rather does not limit secretion.

We thank the referee´s and the reviewing editor for their helpful comments!