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Proc Natl Acad Sci U S A. Dec 1, 2009; 106(48): 20458–20463.
Published online Nov 17, 2009. doi:  10.1073/pnas.0908502106
PMCID: PMC2787115
Microbiology

The initial steps leading to papillomavirus infection occur on the basement membrane prior to cell surface binding

Abstract

Using a murine challenge model, we previously determined that human papillomavirus (HPV) pseudovirions initially bind preferentially to the cervicovaginal basement membrane (BM) at sites of trauma. We now report that the capsids undergo a conformational change while bound to the BM that results in L2 cleavage by a proprotein convertase (PC), furin, and/or PC5/6, followed by the exposure of an N-terminal cross-neutralization L2 epitope and transfer of the capsids to the epithelial cell surface. Prevention of this exposure by PC inhibition results in detachment of the pseudovirions from the BM and their eventual loss from the tissue, thereby preventing infection. Pseudovirions whose L2 had been precleaved by furin can bypass the PC inhibition of binding and infectivity. Cleavage of heparan sulfate proteoglycans (HSPG) with heparinase III prevented infection and BM binding by the precleaved pseudovirions, but did not prevent them from binding robustly to cell surfaces. These results indicate that the infectious process has evolved so that the initial steps take place on the BM, in contrast to the typical viral infection that is initiated by binding to the cell surface. The data are consistent with a dynamic model of in vivo HPV infection in which a conformational change and PC cleavage on the BM allows transfer of virions from HSPG attachment factors to an L1-specific receptor on basal keratinocytes migrating into the site of trauma.

Keywords: furin, HSPG, mouse model, proprotein convertase

Papillomaviruses (PVs) infect mucosal and cutaneous stratified squamous epithelia, resulting in benign lesions, some of which can progress to invasive cancer. Human papillomavirus (HPV) 16 and several other PVs that preferentially infect the anogenital mucosa are responsible for virtually all cases of cervical cancer, as well as a number of other mucosal epithelial malignancies. It has been recognized for decades that efficient induction of experimental PV infection of animals requires local wounding in conjunction with virus exposure (14). However, there has been little molecular insight into the in vivo steps by which PVs initiate the infectious process.

We recently described a murine cervicovaginal challenge model of PV transmission that is amenable to examination of the early in vivo events of PV infection (5, 6). It employs high titer PV pseudoviruses, which are authentic PV capsids composed of the L1 major and L2 minor structural proteins that have encapsidated a non-PV plasmid encoding a quantifiable reporter gene to monitor successful infection (7). PV pseudoviruses abrogate the exquisite species specificity of the viral gene expression program of authentic PV virions, while retaining the early events of PV infection, including the preferential infection of keratinocytes in vivo. In the initial characterization of the mouse genital tract model, we confirmed that wounding by the transient disruption of cervicovaginal epithelial integrity, by physical or chemical means, is essential for efficient infection (5). It was shown that instillation of the detergent, nonoxynol-9, results in large areas of transient squamous epithelial ulceration resulting in the removal of the upper basal epithelial layer and exposure of the BM. In addition, we discovered that the pseudovirions did not bind intact stratified squamous or columnar epithelia of the genital tract. Rather, they preferentially bound the BM exposed at sites of trauma and became cell-associated only at later time points.

In this study we have used the murine cervicovaginal tract model to undertake a detailed analysis of the steps required for in vivo pseudovirus infection by a mucosal PV type, HPV16. Surprisingly, we have found that instead of being a passive reservoir for the accumulation of PV pseudovirions, the BM is the site of a series of conformational changes in the capsid that leads to protease digestion of L2 and exposure of its N terminus. These modifications apparently occur before transfer of the capsid to the surface of the epithelial cell. Thus, the PV life cycle utilizes an extracellular site for initial critical steps that lead to infection.

Results

Capsid Conformational Changes Occur on the BM.

In the mature virion, the amino terminus of L2 is not exposed. Studies in cultured cell lines (as distinguish from studies performed in the animal, which are exclusively designated “in vivo” herein) have revealed that after binding to heparan sulfate proteoglycans (HSPG) on the cell surface, the PV capsid of the mature virus particle undergoes a series of conformational changes before entry into the cell (8, 9). Specifically, surface binding during infection of cultured cells is associated with the N terminus of L2 becoming sensitive to cleavage by a proprotein convertase (PC), furin, and/or PC5/6. This cleavage leads to exposure of a cryptic L2 cross-neutralization epitope between amino acids 17 and 36, immediately downstream from the furin-PC5/6 cleavage site, which can be monitored by efficient antibody binding to this epitope. Infection of cultured cells requires L2 cleavage by furin-PC5/6, as a PC inhibitor (decanoyl-RVKR-cmk) that prevents cleavage also prevents infection (10, 11). Pseudovirions precleaved by furin in vitro can bypass the dependence on cellular furin, thereby allowing infection under conditions of PC inhibition or of furin-deficient cell lines. Furin precleaved pseudovirions are designated herein as “FPC” pseudovirions; mature pseudovirions that have not been exposed to furin are designated herein as “untreated” pseudovirions.

Here we studied exposure of the L2 epitope in vivo by examining staining of untreated pseudovirions with a polyclonal antiserum to amino acids 17–36, at 2, 4, or 18 h after instillation of pseudovirus into the murine vaginal tract. This antiserum does not bind mature virions in solution. We anticipated that antiserum binding to the L2 epitope would not occur until after transfer of the virus to the epithelial cells, as in cultured cells. As expected, we did not detect significant anti-L2 staining at the 2 h time point (Fig. 1D), despite strong pseudovirus binding to the BM as evidenced by anti-L1 colocalization with laminin 5 (Fig. 1 A and B). However, at the 4 h time point, there was strong anti-L2 staining that colocalized with L1. Surprisingly, this staining was most predominant on the BM (Fig. 1 E and F). By the 18 h time point, there was consistently strong anti-L2 staining on the cell layers that re-epithelialized over the BM (Fig. 1 G and H).

Fig. 1.
Binding of capsids to the BM and timecourse of L2 epitope exposure. To demonstrate the initial association of the PV capsids with the BM murine genital, tract sections of tissue harvested at 2 h following virus instillation were stained with a rat anti-L1 ...

Furin Inhibition Decreases In Vivo Infection Through Loss of Pseudovirions from BM.

The above results suggest that, in contrast to what happens with cultured cells, the L2 epitope exposure can occur on the BM before virion transfer to cells, implying that the furin-PC5/6 cleavage of L2 also occurred on the BM. To determine if furin or PC5/6 is expressed on the BM or basal epithelial cells, we assessed their presence in the intact vagina and at the 4 h time point (i.e., 8–9 h after the chemical disruption used for pseudovirus infection). The data indicated that in the intact genital tract, furin is present throughout the epithelium, and following wounding (by the nonoxynol-9 treatment used before infection), the furin signal is increased, especially in the basal cells (Fig. S1 A and B). Interestingly, PC5/6 was strongly associated with the BM of intact and wounded epithelium, in addition to being evenly distributed among the epithelial layers, with no apparent change in its expression following wounding (Fig. S1 C and D). Thus, PC5/6 was preferentially associated with the BM, while furin was highly expressed on basal cells, from which it might be shed or secreted to act on virions bound to the BM (12, 13).

To determine if furin and/or PC5/6 cleavage is essential for in vivo infection, we instilled the PC inhibitor decanoyl-RVKR-cmk intravaginally before and coincident with pseudovirus delivery. In the presence of the inhibitor, pseudovirus infection, as measured by delivery of a luciferase-encoding marker plasmid, was reduced by about 78% compared to that of the mock-treated controls (P < 0.0001) (Fig. 2). This incomplete inhibition may reflect a combination of the high levels of furin in the wounded epithelium and the topical application of the PC inhibitor.

Fig. 2.
In vivo PC inhibition. The effect of the PC inhibitor, decanoyl-RVKR-cmk on infection of wild-type, untreated pseudovirus was determined. The average luminescence values obtained 48 h following virus instillation is shown. There were five mice in each ...

We then studied at which point this treatment interfered with in vivo infection by examining pseudovirion localization at various times post-instillation. At 4 h (Fig. 3A), the pseudovirions were distributed on the BM similarly to that of untreated controls (Fig. 1E), although the amount of bound pseudovirions was reproducibly reduced compared to untreated tissue. However, at 18 h, few pseudovirions could be found located on either the BM or the epithelial cells (Fig. 3B), in contrast to the controls (Fig. 1G). By 30 h, no pseudovirions were evident (Fig. 3C). Untreated pseudovirions not subjected to the PC inhibitor in vivo were readily detected at 30 h (Fig. S2), and displayed a distribution similar to that found at 18 h. To rule out the possibility that the results with PC inhibitor were due to an alteration of capsid processing that might have interfered with antiserum binding, the experiment with the PC inhibitor was also carried out with Alexa-488-conjugated pseudovirions, and similar results were obtained (Fig. S3).

Fig. 3.
Capsid association with the vaginal epithelium. Pseudovirion capsids were evaluated for their ability to interact with the BM and epithelium following various treatments. Untreated pseudovirus in PC-inhibitor-treated tissue is shown in panels A–D ...

We also used the PC inhibition conditions to examine exposure of the L2 17–36 epitope, which under normal conditions is not well exposed until the 4 h time point. As expected from the low amounts of virions present and the requirement of PC cleavage for exposure of this epitope, no staining with the L2 antibody was detected at 18 h (Fig. 3D).

Furin Precleaved Virus (FPC) Overcomes the Infectivity Block Induced by PC Inhibition, But Is Inhibited by Heparinase III.

During infection of cultured epithelial cell lines, mature pseudovirus can only bind and infect cells that express cell surface HSPG, while FPC pseudovirus can bind and infect cells that lack surface HSPG (9). Thus, furin precleavage bypasses the requirement for furin-PC5/6 and permits binding to a non-HSPG receptor on the surface of cultured cells that leads to infection. This effect was exemplified by our examination of primary keratinocytes. Reasonable binding and infection was achieved with the FPC pseudovirus but not with the untreated pseudovirions. We attributed this to alterations in HSPG modifications induced by adaptation to ex vivo cell culture. We therefore examined the in vivo infectivity and binding of the FPC preparation under various conditions.

Consistent with the infectivity studies in cultured cells, cervicovaginal infection by FPC pseudovirus was not significantly affected by the presence of the PC inhibitor in vivo (P = 0.3730) (Fig. 4, left panel). Thus, the FPC pseudovirus is resistant to the block to infectivity induced by the PC inhibitor. This result also implies that the main effects of the PC inhibitor on infection can be attributed to its effects on the untreated pseudovirus, rather than its possible effects on the cervicovaginal tissue, which would have been expected to inhibit FPC virus as well.

Fig. 4.
Inhibition of FPC infection in vivo. Infection of FPC was evaluated in untreated mice or under conditions of PC inhibition by treatment with the PC inhibitor decanoyl-RVKR-cmk, indicated as RVKR. The luminescence values are shown in the left panel. The ...

We have previously found that genital tract instillation of heparinase III, which cleaves HSPGs, severely inhibited in vivo BM and cell surface binding and thereby infection by untreated virus (6). Given the ability of FPC virus to infect cultured cells that were HSPG-deficient, we expected that FPC virus would overcome the heparinase III-induced block to in vivo infectivity. However, heparinase III treatment drastically reduced the in vivo infectivity of FPC virus (Fig. 4, right panel) (P = 0.0024).

By microscopy, we found that, under normal conditions, the FPC pseudovirions bind robustly to the BM and somewhat to the cell surface at 4 h (Fig. 3E) and throughout the epithelium at 18 h (Fig. 3F), although cell surface binding is not seen with untreated pseudovirions until the 18 h time point. When the vaginal tract was treated with the PC inhibitor, the binding characteristics of the FPC pseudovirions were unchanged consistent with the lack of effect of the inhibitor on the infectivity of FPC virions (Fig. 3 I and J, 4 and 18 h, respectively). Thus, while the PC inhibitor reduced the infectivity of untreated pseudovirions and reduced their BM and cell surface binding, FPC pseudovirions can bind to the BM and the epithelial cells in the presence of the PC inhibitor.

When the vaginal tract was treated with heparinase, the FPC virus still bound strongly to the epithelial cell surface, but not to the BM (Fig. 3 G and H, 4 and 18 h, respectively). This suggests that FPC virus interacts with HSPG on the BM, but a non-HSPG receptor on epithelial cells. However, direct engagement of FPC virus with the epithelial cell receptor did not lead to infection in vivo. As demonstrated previously, untreated virus is not found associated with either the BM or cells at any time point following heparinase treatment (6).

L1 Virus-Like Particles (VLP) Bind the BM and Epithelial Cells.

L1 has the capacity to self-assemble into noninfectious VLPs whose binding characteristics can also be studied in vivo to evaluate the relative roles of L1 and L2 in cervicovaginal binding of the capsids. We found that L1 VLPs bind well to both the BM and epithelial cells at 4 h (Fig. 3K and Fig. S4A) and, as expected, that the PC inhibitor, whose effects are attributable to its cleavage of L2, had no effect on VLP binding (Fig. S4B). We also found that, similarly to FPC pseudovirus, the association with epithelial cells was not abrogated by heparinase treatment, while BM binding was prevented (4 h, Fig. 3L). The results indicate that L1 is the primary determinant of both BM and epithelial cell surface binding in vivo.

Discussion

While virion binding to the cell surface is usually described as the first obligate step during the life cycle of a virus, it is not the case for in vivo PV infection. Instead, the virus has apparently evolved to use the BM to mediate early changes that are essential for infection. Here we have confirmed that the BM is the first binding site for PVs, and our combined descriptive and mechanistic data strongly suggest that HSPG-dependent binding is the first of several essential steps that take place on the BM. These BM-associated steps include the conformational change in the virion that permits L2 cleavage by furin-PC5/6 and a subsequent conformational change that results in exposure of the L2 cross-neutralization 17–36 epitope and transfer of the virion to the cell surface. Overall, it appears that PVs have adapted their life cycle to the wound-healing process, binding initially to the BM during the “open wound” phase and subsequently transferring to basal epithelial cells during the “wound healing” phase.

We reported previously that heparinase treatment of the vaginal tract inhibits the binding of untreated virus to the BM and epithelial cells (6). Here we show that this treatment also prevents infection and BM-binding by FPC virus. However, it did not affect the ability of the FPC virus to bind to epithelial cells. The lack of infectivity under these conditions was surprising since the FPC capsids are HSPG-independent for infection of cultured cells, as shown in experiments using the pgsa-745 cells, which are genetically deficient for glycosyl amino glycans (9). This finding implies that productive infection by FPC virus, under normal conditions, results primarily from the BM-bound virus, as is true of untreated virus, rather than from the FPC virus bound directly to epithelial cells. There are at least three possible explanations for this lack of infectivity. First, the conformational changes in the capsid that result from HSPG binding may have a critical function(s) in addition to exposure of the L2 N-termini to PC cleavage. Second, the FPC virus may be binding a nonphysiologic cell surface receptor on the epithelial cells. Although this possibility is inconsistent with the results in cultured cells, it cannot be ruled out, as the identity and distribution of the cellular receptor is unknown. Third, the epithelial cells to which the FPC virions bind in vivo may be largely nonpermissive for infection. This possibility may be most easily reconciled with the HSPG-independent infection of FPC capsids in cultured cells. One possible reason for the lack of infection could be that the FPC virus binds throughout the layered epithelium, which is primarily composed of nondividing cells. It has been shown that the mitoses of murine epithelium are almost exclusively confined to the basal cell layer (14). Studies in cultured cells indicate that cell division is required for successful pseudovirus infection (15). By contrast, the BM-bound virus would preferentially transfer to the leading edge of basal keratinocytes that migrate to fill the open wound. Following closure of the wound these cells undergo cell division as the epithelial layering is re-established (16). If this speculation is correct, it implies that the prolonged association with the BM and the occurrence of critical conformational changes while at this locale serve to maximize the likelihood of infection of the basal keratinocytes that migrate into the site of trauma. The increased expression of furin in the basal cells following wounding may further promote viral infection. Similarly the localization of PC5/6 on the BM, likely associated with HSPG (17), may aid the infectious process.

The binding characteristics of L1 VLPs are also noteworthy. In most respects, these were similar to those of FPC pseudovirus, in that VLPs could bind to both the BM and the epithelial cells at early time points under normal conditions or in the presence of the PC inhibitor. The results reinforce the idea that BM and epithelial cell receptor binding are both L1 functions, and imply that before furin cleavage and exposure of the 17–36 epitope, the position of the N terminus of L2 in mature, untreated pseudovirus causes occlusion of the binding site for the epithelial surface receptor. Additionally, following heparinase treatment, L1 VLPs, like FPC pseudovirus, lost their ability to bind the BM, supporting the conclusion that HSPG binding in vivo is an L1-mediated activity.

While the analysis of pseudovirus infection of cultured cells has produced many useful observations, the studies with the mouse cervicovaginal model have revealed that the process of in vivo infection has some notable differences. The recognition of this may influence how results from other systems are interpreted, and underscores the need for in vivo analyses when possible. As noted earlier, the most salient is that furin-PC5/6 cleavage and exposure of the L2 17–36 epitope occur on the BM, while in cultured cells these changes occur following adsorption to the cell surface (11). During infection of cultured cells, mature virus can bind to the extracellular matrix (ECM) deposited by these cells, as well as to the cell surface (18, 19). However, the ECM is apparently not equivalent to the BM, in that, the virus does not undergo the same series of changes that occur in vivo after BM binding. One key difference between ECM and BM is the importance of HSPG for the BM binding of mature virus, while HSPG are not required for the majority of the ECM binding (20), although, it has been reported that HSPG on the ECM of HaCaT cells (an immortal human keratinocyte line) can influence subsequent infectivity (8).

These considerations have enabled us to develop the model for in vivo infection shown in Fig. 5. As discussed above, the virus first binds HSPG on the BM, where it undergoes a conformational change that permits cleavage of the L2 N terminus by furin-PC5/6, leading to the exposure of the L2 17–36 epitope. The virions then transfer to a receptor on the basal epithelial cell, which is permissive for infection because it is migrating to repair the epithelial wound and is therefore undergoing cell division. In the presence of a PC inhibitor, virus initially bound to the BM, but was lost at later time points, perhaps because the initial conformational change without L2 cleavage reduces virion affinity for the BM. This result supports a model in which the initial conformational change that exposes the furin-PC5/6 cleavage site on L2 also results in a decreased affinity of the virion for HSPG. The conformational change that follows cleavage is necessary for binding to the non-HSPG epithelial cell receptor. Therefore, a block at this step results in loss from the HSPG on the BM and a lack of association with the epithelial tissue.

Fig. 5.
Model for events leading to in vivo infection. Untreated, mature capsids initially bind to HSPG on the BM. This interaction leads to a conformational change that allows the accessibility of the L2 N terminus to PC cleavage (either furin shed from the ...

Several of the insights provided by this study may be relevant to the basic understanding of prophylactic HPV vaccines. First, if L1 VLPs have the binding determinants for both HSPG and the keratinocyte receptor exposed, whereas L1/L2 capsids only expose the binding determinants for HSPG, then vaccine developers may have fortuitously selected the superior immunogen in L1-only VLPs, since they, but not L1/L2 VLPs, have the potential of inducing neutralizing antibodies to both determinants. Second, L2 polypeptide-based vaccines are now being considered because of their potential to act as immunogens for a pan-HPV vaccine by capitalizing on the cryptic broad cross-neutralization epitopes present in L2 (21). These epitopes are not exposed on virions or L1/L2 VLPs in solution (22), and antibodies to them are apparently not induced following L1/L2 immunization (23, 24). Although other viruses also contain cryptic cross-neutralization epitopes, PVs are unusual in that preclinical PV models suggest that this vaccine approach may be highly effective at preventing infection. The results presented here indicate that the L2 17–36 cross-neutralization epitope (and, by extension, possibly other such epitopes on L2) is exposed for several hours on the BM and cell surface during the infectious process. Many other viruses, including HIV, expose critical functional domain transiently following cell surface attachment, thereby limiting the opportunity for induction of neutralizing antibodies directed against them (25). The long period of L2 epitope exposure may contribute to the exceptional effectiveness of L2-based vaccines.

Materials and Methods

Pseudovirions.

HPV 16-Luciferase pseudovirions and HPV 16 L1-only VLPs were produced as previously described (7). Furin precleaved pseudovirions were produced by incubation with recombinant human furin (Sigma) during the maturation process (9).

In Vivo Pseudovirus Delivery and Infection.

Six- to eight-week-old female BALB/cAnNCr mice were obtained from the National Institutes of Health and housed and handled in accordance with their guidelines. Experimental protocols were approved by the National Cancer Institute's Animal Care and Use Committee. The mice were treated as previously described (5, 6). On the day of pseudovirus infection, mice were pretreated with a 30-μL solution with a final concentration of 2% carboxymethylcellulose (CMC) containing 4% nonoxynol-9 and either buffer only, 6 μL PC inhibitor (200 μM) (furin inhibitor 1, decanoyl-RVKR-cmk, Calbiochem), or 5 μL heparinase III (1.7 U). Four hours later, a 30 μL solution with a final concentration of 2% CMC, 10 μg (L1 content) of HPV 16-Luciferase pseudovirus or 16 L1 VLPs and either buffer, PC inhibitor (6 μL of 200 μM stock), or heparinase III (3.3 U/mouse) was intravaginally delivered. Luminescence was measured daily for 1 week beginning 24 h after the initial pseudovirus infection. Intravaginal administration of 20 μL of D-Luciferin - K+ Salt (0.3 mg, Caliper Life Sciences) was followed by imaging with an IVIS 100 (Caliper Life Sciences), as previously described (6). The average radiance within the region of interest was determined. Data are representative of five mice per group, and experiments were performed in duplicate. Statistical analysis was performed with GraphPad Prism Software, in which a one-tailed, unpaired t-test was used to determine p values.

For binding analyses, mice were euthanized at 2, 4, 18, or 30 h post pseudovirus instillation. Genital tracts were excised, washed with PBS and snap frozen in tissue freezing medium (EMS). Six-micron tissue cryosections were obtained, transferred to glass slides, and fixed for 10 min in 100% ethanol at −20 °C. Tissues were stained within 48 h of sectioning. Images are representative of three to five mice per group. Experiments were performed in duplicate.

Antibodies and Immunofluorescent Staining.

The rabbit polyclonal antiserum against HPV16 L1 has been previously described (26). The rat anti-HPV16 L1 polyclonal serum was a gift from DelSite Biotechnologies, Inc. The rabbit antiserum, 17/36, that recognizes the 17–36 HPV16 peptide was obtained from Richard Roden (27). The rabbit anti-furin was purchased from Santa Cruz Biotechnolgy (H-220). The rabbit anti-PC5/6 was purchased from Alexis Biochemicals (210–336). Before staining, tissue sections were blocked with 10% donkey serum in PBS with 0.1% Brij58 for 30 min at room temperature. Antisera recognizing the HPV capsids were diluted 1:1,000. The 17/36 antiserum was diluted 1:500. Both the anti-furin and anti-PC5/6 sera were diluted 1:200. Bound antibody was detected with Alexa Fluor 488-conjugated donkey anti-rabbit serum or Alexa Fluor 594-conjugated donkey anti-rat serum (Invitrogen). Following staining, sections were mounted with Prolong Gold mounting solution (Invitrogen). All microscopy was performed on a Zeiss LSM 510 system.

Supplementary Material

Supporting Information:

Acknowledgments.

We gratefully acknowledge the gift of the 17/36 antiserum from Richard Roden, Johns Hopkins University and the rat anti-HPV16 L1 antiserum from Yawei Ni, DelSite Biotechnologies. This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research.

Footnotes

Conflict of interest statement: D.R.L. and J.T.S. are inventors on intellectual property owned by the United States government for the L2 vaccine.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0908502106/DCSupplemental.

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