Author response:
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.
Please note that we state in the introduction on line 65/66 ´Two release mechanisms are discussed, that mutually are not exclusive´. This is implying that we do not consider the shedding model as the one accepted model. HS may associate with PsVs despite of a decreased affinity and only after priming (see below the ‘priming model’) may translocate to the cell body.
Furthermore, we do not state in the discussion either that the shedding model is the preferred one; although it is correct that we refer to the shedding model more extensively, simply because we find HS associated with transferred PsVs, which is in line with this model and requires its citation.
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.
As outlined above, our finding is compatible with both models, and we do not aim to verify the shedding model or disprove the priming model.
It appears that the referee wishes more visibility of the priming model. Inhibition of KLK8 and furin should reduce the translocation to the cell body, no matter whether PsVs carry HS on their surface or not. For revision, we plan an experiment as in Figure 3 (CytD), testing whether either KLK8 or furin inhibition blocks the transfer to the cell body. Then, our data can be discussed also in the context of the priming model and by this increase its visibility.
The model should be fitted into established entry events, 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.
That PsVs carry HS-cleavage products doesn´t imply that HS cleavage is sufficient or required for infection. Therefore, we do not view our data as being in conflict with the priming model. In fact, our observations are compatible with aspects of both the shedding and the priming model.
Yet, we acknowledge that the study would gain importance by directly testing the priming model within our experimental system. As requested by the referee, we will discuss the above papers, and further plan to test KLK8 and furin inhibitors.
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?
We do 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 in the manuscript 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).´
Moreover, we ourselves detect these PsVs, for example, in Figure 5A (CytD, 0 min time point), a handful of PsVs localize to the cell body area. However, the large majority overlaps with the strong HS staining at the cell periphery, likely the ECM. An accurate quantification of the fractions of PsVs bound to the ECM/cell body is for the following reasons very difficult. First, the ECM PsVs are very dense and therefore not microscopically resolved into single PsVs, at least not completely (see Figure 1C; the high intensity spots are non-resolved PsVs, please see our discussion on line 148 - 152). For this reason, by just counting spots we strongly underestimate the ECM PsVs versus the cell body PsVs. Second, with the available immunostainings we cannot exactly delineate the ECM from the cell body. In particular, at the cell border region (for example see Figure 4B) we often observe PsV accumulations. Assigning these ´cell border region PsVs´ entirely to the cell body fraction, a preliminary analysis (correcting for the limitation of non-resolved ECM PsVs) suggests that about a quarter of the PsVs bind to the cell body. On the other hand, assigning them to the ECM, the cell body fraction would be much below 10%. Third, we observe that in regions devoid of ECM and cells PsVs apparently adhere unspecifically to the glass-coverslip. This suggests that some of the cell body PsVs are just unspecific background. Subtraction of a background PsV density from the ECM and cell body PsV density will reduce relatively more the cell body PsVs, and consequently decreases the fraction of cell body PsVs even more.
Moreover, in the course of the project we wondered whether at the basolateral membrane there are not many binding sites anyway. To address this question, in an unpublished experiment, we detached HaCaT cells with trypsin, incubated them with PsVs, and then allowed reattachment to assess the binding in suspension. We detected minimal to no binding, which, however, could also result from apical membrane adherence to the coverslip or trypsin-mediated cleavage of HSPGs. As suggested by the reviewing editor, we agree that repeating this experiment using EDTA for detachment—thus preserving HSPGs—would offer more definitive insight into binding efficiency in the absence of accessibility constraints. In summary, the reason why in our cellular system most PsVs do not bind to the cell surface could be a combination of several factors:
(1) The primary binding partners are more abundant in the ECM and the polarized HaCaT cells secrete more ECM when compared to other cultured cells used to study HPV infection. This promotes ECM binding.
(2) In the polarized HaCaT cells, the apical membrane is largely devoid of syndecan-1, CD151 and Itga6, wherefore PsVs infect the cell via the basolateral membrane. However, the accessibility to the basolateral membrane is restricted, PsVs must diffuse through a narrow slit between the glass coverslip and the attached cell to reach HS on the cell surface. This limits cell surface binding.
(3) If HaCaT cells secrete large amounts of ECM, the may become depleted from cell surface HS. As outlined above, we will try to find out how many PsVs bind to the basolateral membrane in the absence of restricted accessibility. If it turns out that HaCaT cells have not many binding sites anyway, this would additionally promote binding to the ECM.
The outcome of the above issues, and how we will mention them in the revised version of the manuscript, is open. In any case, we would like to point out that PsVs bound to the cell body do not weaken our main conclusion. Still, we recognize that this point merits attention and plan several modifications of the manuscript. We did already, but now we will mention more explicitly that PsVs have been shown to directly interact with HSPG on the cell surface, in addition to the ECM, but that it also has been shown that the ECM strongly supports infection in NHEK and HFK (Bienkowska-Haba et al., Plos Path., 2018). The following is a draft version of a paragraph we plan to incorporate, explaining the above issue and why we used in our experiments HaCaT cells:
´In vitro, PsVs bind to both the cell surface and the ECM, as has been widely documented. In vivo, however, it has been proposed that initial binding occurs predominantly to the basement membrane ECM, rather than directly to the cell surface (Day and Schelhaas, 2014; Kines et al., 2009; Schiller et al., 2010). This distinction reinforces the physiological relevance of ECM-bound particles in the early steps of HPV infection. Support for a functional role of ECM-mediated entry comes from a study showing that PsV binding to ECM derived from HaCaT cells significantly enhances infection of primary keratinocytes (Bienkowska-Haba et al., 2018). For these reasons, we specifically chose polarized HaCaT cells as a model system. These cells secrete abundant ECM from which the cells readily collect bound PsVs. On the other hand, the polarization limits the access of PsVs to basolateral receptors such as CD151 and Itgα6, and also cell body resident Syndecan-1, the most abundant HSPG in keratinocytes (Rapraeger et al., 1986; Hayashi et al., 1987; Kim et al., 1994). Hence, as polarization limits direct cell surface accessibility it biases binding toward the ECM, that in this culture system is abundant. Hence, in the HaCaT cell culture system, like probably in vivo, PsVs cannot circumvent binding to the ECM what they can do in unpolarized cell cultures that may not even secrete significant amounts of ECM. Altogether, this experimental situation closely mimics the in vivo situation where PsVs bind preferentially to the ECM (Day and Schelhaas, 2014; Kines et al., 2009; Schiller et al., 2010).´
We appreciate the reviewer’s input and believe these additions will strengthen the manuscript with regard to the relevance of the used cellular model system.
(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.
We have shown all images at the same adjustments of brightness and contrast. As the staining at the periphery is stronger, the impression is given that the cell surface is not stained, although there is some staining. Specific staining is documented in Figure 5D, showing the PCC between PsVs and HS only of the cell body. If there was no HS staining, the PCC would be zero, which is not the case. Yet, it is lower when compared to the PCC measured at the cell border region, with more strongly stained HS.
We will provide images at different contrast and brightness adjustments enabling the reader to see the staining on the cell surface. We will provide also more overview images to illustrate the strong variability of the HS staining between cells.
Therefore, the conclusion that this confirms HS coating of PsV during release from the ECM (line 430-431) is unfounded. How do the authors distinguish between "HS-coated virions" and HSPG-associated virions?
The HS intensity transiently increases on the cell body (Fig. 5D) only after releasing a cohort of PsVs, which can be only explained by PsVs that carry HS from the ECM to the cell body. However, the effect is not significant. Using the antibody 3G10 detecting the HS neoepitope (see the referees’ suggestion below) we will reanalyze this point. This should help clarifying the issue.
It is difficult to comprehend how the addition of 50 vge/cell of PsV could cause such a global change in HS levels.
The distribution of bound PsVs largely varies between cells. Some areas are covered with essentially confluent cells, to which hardly any PsVs are bound, because accessing the basolateral membrane of confluent cells is nearly impossible, and PsVs do not bind to the exposed apical membrane. This is different in cultures of unpolarized cells where we expect that PsVs distribute more 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 several hundred PsVs, much more than expected from the 50 vge/cell. We will point this out in the revised version.
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 just take shedded HS with them. Without PsVs, the shedded HS likely remains in the ECM or is washed out very slowly.
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.
As outlined above, we plan to test the suggested antibody 3G10. We also plan to repeat the 0 min time point (with and without PsVs, with and without CytD) to find out whether in the PsV absence the HS intensity (at 0 min) is unchanged between control and CytD.
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 the cell body. 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 will rephrase the respective section.
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.
We agree, and plan to test whether blebbistatin is equally efficient in blocking the transfer.
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.
We agree that in multiple cell culture systems viruses bind preferentially to the cell directly. But we respectfully disagree with the assertion that the majority of PsVs bind to the cell body of HaCaT keratinocytes. As noted above (e.g., Figure 5A, CytD, 0 min), only a small fraction of PsVs localize to the cell body, whereas the vast majority overlap with intense HS staining at the cell periphery, consistent with ECM association, as the accessibility to the basolateral expressed HSPG is limited (see above). Based on quantitative estimation from multiple images, ECM-bound PsVs largely outnumber cell-bound particles (see above). These features make HaCaT cells a suitable in vitro model for mimicking in vivo conditions, where HPV has been proposed to bind predominantly to the basement membrane ECM rather than the cell surface (Day and Schelhaas, 2014; Kines et al., 2009; Schiller et al., 2010) which also strongly enhances infection of primary keratinocytes in vitro (Bienkowska-Haba et al., 2018).
Thus, we believe our system appropriately models the physiologically relevant scenario of ECM-to-cell transfer, and the observed predominance of ECM binding supports the validity of our experimental focus.
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 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 (or maybe they are simply trapped between glass and basolateral membrane). PsVs are not expected to bind to the apical membrane that is depleted from CD151 and Itga6. In other cellular systems, cells may hardly secrete ECM, are not polarized, and do not adhere so tightly to the substrate. In other cultures, where virions can easily circumvent ECM binding, the large majority of PsVs will likely bind directly to the cell surface.
As outlined above, in order to quantify PsVs that can bind without restricted accessibility, we plan to detach HaCaT cells by EDTA from the substrate, incubate them with PsVs, and let them adhere again (please see above).
No matter what is the outcome, the fraction of PsVs that binds directly to the cell surface does not weaken our conclusion that we have identified a very fast and efficient transfer step from the ECM to the cell body.
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.
If blebbistatin works as expected, we can safely conclude that we observe the very same process as described in Scheelhas et al., PLoS Pathogens, 2008, showing that the PsVs migrate by retrograde transport to the cell surface and not that the cell spreads out and by this reaches the PsVs.
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.
Our plasma membrane stain does not stain the ECM. Please see Figure 1. The stain is actually used to distinguish the cell body from the ECM area.
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, based on flipped images, we generated a calibration curve used for the correction of random background. For details, please see Supplementary Figures 3 and 5.
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. I am rather convinced that using randomisation only on the plasma membrane ROIs will not establish any clear significance of the correlating signals.
Figure 5D shows the PCC specifically of the cell body. In flipped images (not shown in the manuscript for clarity, but can be added) we obtain a PCC of around zero. For CytD, the flipped images always have a significantly lower PCC compared to the original images. In the control, the PCC of the flipped images are significantly lower only for the 30 min and 60 min time point. The non-significance of the 0 min and 180 min time point is due to low PCCs also in the original images.
Also, there should be a higher n for the measurements.
One n is the average of 15 cells. We realize that with n = 3 we find significant effects only if the effect is very strong or moderate with very low variance.
