Kinetics of HPV-16 endocytosis

First, we analyzed how rapidly infectious cell-bound HPV-16 particles were internalized by endocytosis in HeLa and HaCaT cells. To distinguish between external and internalized viruses, we used a high pH wash to inactivate the PsV located on the cell surface (see Figure S1 A, B, and material and methods for details). HPV-16 PsV were bound to cells in the cold, unbound virus was washed away, and the cells were warmed to 37°C. At different times post warming, cells were subjected briefly to a high pH wash and infection was scored after 48 h by the detection of GFP expression from the PsV reporter plasmid using flow cytometry.

This way the internalization exclusively of infectious particles was probed.

When using a low multiplicity of infection (MOI, 0.1 tdu/cell), so that a single infectious particle per cell caused infection, we found that about 2 h at 37°C were needed for the first viruses to be internalized. The half-time for the entire population was 11 h (Figure 1A, black). When a higher MOI (10 tdu/cell) was used, infectious internalization by the first (fastest) of several infectious particles caused infection providing information regarding the fastest possible internalization kinetics. The fastest particles provided a half time of internalization of 4 h post warming with no apparent lag time (Figure 1A, white). Similar internalization kinetics were observed in HaCaT cells (Figure 1B).

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Figure 1. HPV-16 internalization events are fast and occur asynchronously over several hours.

To test the average internalization kinetics of infectious HPV-16 PsV, HeLa (A) or HaCaT (B) cells were infected with HPV-16 PsV at MOIs of 0.1 vs. 10 transducing units/cell, i.e. about 10 vs. 1,000 particles/cell (black vs. white circles, respectively). Virus was bound to cells at 4°C, warmed to 37°C and at indicated times post warming, i.e. post infection (p.i.), cells were treated with pH 10.5 for 1 min. to inactivate extracellular virus. 48 h p.i. cells were analyzed for GFP expression from the PsV reporter plasmid by flow cytometry. The amount of GFP expressing cells relative to untreated control is given as relative internalization in %. (C) To test the internalization kinetics of single particles, we infected HeLa cells with HPV-16 PsV labeled with AF488 (5,000 particles/cell, HPV-16 AF488, green). Cells were transfected 16 h prior to infection with a construct expressing clathrin light chain-mRFP (red) to investigate any potential cointernalization. Depicted are TIRFM images of an uninfected (C, above) and infected (C, below) cell. Kymographs to the right of the images depict the formation and internalization of a CCP or virus (encircled in the images, also shown in Video S1), respectively. Internalization was defined as the loss of fluorescent signal. The time span required for CCP formation or virus internalization was determined from the initial detection of fluorescence to complete loss of fluorescence for CCPs, or from the moment of confinement to loss of fluorescence for HPV-16, respectively. The results were quantified manually and plotted (C, far below).

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When internalization of fluorescently labeled HPV-16 particles was analyzed by scoring cell-associated fluorescence after removing cell surface-bound viruses by protease digestion at different times post warming, similar kinetics as compared to the high MOI infectious internalization were observed (data not shown). This suggested that all particles, infectious and non-infectious, followed a similar itinerary.

With half times of internalization ranging from 4–12 h p.i. for the fastest and average particles, our data was consistent with previous observations, where bulk internalization of papillomaviruses and PsV was shown to be extremely slow [6], [23], [24]. To our knowledge, this describes the slowest internalization kinetics observed for a virus.

Endocytic internalization of individual viruses in live cells can be visualized using total internal reflection microscopy (TIRFM) by the loss of fluorescence as vesicles leave the evanescent field [25]. We used fluorophore labeled PsV that can be easily followed by microscopy and retain their infectivity after labelling [7]. As papillomavirus entry has been mostly attributed to CME, we analyzed internalization of AF488-labeled virions in cells expressing clathrin light chain tagged with mRFP (CLC-RFP) to follow any potential cointernalization.

After binding to cells, HPV-16 particles exhibit periods of lateral diffusion and directed transport, after which movement stops [7]. Such confinement is often associated with the interaction of plasma membrane receptors with the actin cytoskeleton or endocytic coat elements [26]. We analyzed how quickly the fluorescence of HPV-16 PsV particles was lost after confinement, an event consistent with internalization. The majority of particles stayed confined and were not internalized during data acquisition possibly due to HPV-16 anchoring to ECM factors and plasma membrane receptors [15], [27]. However, internalization of several particles (30 min p.i., n = 17; 2 h p.i., n = 31) could be observed, and when that happened it occurred on average within 120 s post confinement (Figure 1C, Video S1). The fastest event occurred 93 s after confinement. The formation and endocytosis of clathrin coated pits was readily observed by TIRFM in HeLa cells. Consistent with previous reports, the full cycle lasted about 60 s (Figure 1C) [28], [29]. Cointernalization of HPV-16 and clathrin was not observed. Thus we found that single virus internalization events were fast, but occurred only sporadically over a period of many hours. The absence of any clathrin signal during internalization suggested a clathrin-independent endocytosis event.

Analyzing HPV-16 entry by perturbations

This prompted us to reinvestigate the endocytic requirements of HPV-16 entry. We used small compound inhibitors, siRNA-mediated depletion, and overexpression of DN proteins to identify the cellular factors required or dispensable for infectious endocytosis.

To reduce potential cytotoxic effects of cellular inhibitors, the inhibitors were replaced after the entry phase was over by a less toxic inhibitor such as NH₄Cl that blocked acid-activation of viruses in endosomes (Figure S2A). Swapping for a second inhibitor allowed us to assess the effect of an inhibitor of interest on pre-acidification in the entry process such as binding, endocytosis and perhaps intracellular routing (compare Figure S2A, Table S1, and material and methods). The infection data was normalized to cells in which only the secondary inhibitor was used. In addition, we excluded any data that exhibited significant cytotoxic effects, i.e. a reduction of cell number by more than 50% for a given pertubation. Infection and cytotoxicity was analyzed by automated microscopy and computational analysis of cell number and infection (see Figure S2B and material and methods for details).

To minimize erroneous interpretation of data, and to exclude pleiotrophic effects beyond cell toxicity, we controlled for the specificity and efficiency of inhibition using prototype virus infection experiments using the same inhibitor. In addition, cell biological controls such as transferrin uptake for CME were more sensitive to pertubation than virus infection (data not shown). Here, we used SFV and VSV as controls entering by CME, and SV40 using caveolar/lipid-raft dependent endocytosis. Further controls included infection by VV for macropinocytosis, and IAV for penetration of late endosomal compartments. To stay within one experimental system for comparison of the different viruses, we used HeLa cells but verified key findings for HPV-16 in HaCaT cells. Since the dominant effect of mutant proteins often depends on the level of overexpression, we analyzed infection of transfected cells by flow cytometry and grouped the data into low, medium, and high mutant protein expressing cells (Figure S2C).

HPV-16 entry is clathrin-, caveolin-, and flotillin-independent

First, we analyzed whether infection of HPV-16 in HeLa cells was clathrin-dependent. To inhibit CME, we used siRNA to deplete the clathrin heavy chain or the μ subunit of the clathrin adaptor AP2. A depletion to 20% or less of the protein levels in control cells resulted in efficient decrease in SFV infection (Figure 2A, Figure S3A), a virus that enters exclusively by CME [30]–[32]. HPV-16 infection was not significantly affected (Figure 2A), indicating that HPV-16 entry was clathrin- and AP2-independent. This was consistent with the TIRFM results described above.

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Figure 2. HPV-16 entry is clathrin-, caveolin-, cholesterol- and flotillin-independent.

(A) HeLa cells were transfected with siRNA oligos directed against the AP2μ-subunit (AP2), clathrin heavy chain (CHC), or as control against luciferase. Cells were infected with HPV-16 PsV or SFV and infection was scored 24 or 6 h p.i. by flow cytometry, respectively. Depicted are the mean infection percentages relative to the luciferase control. (B) HeLa cells were pretreated for 30 min with chlorpromazine in the indicated concentrations. Cells were infected with HPV-16 PsV (black), SFV (grey), or SV40 (white) in the presence of inhibitor. Inhibitors were exchanged for HPV-16 and SV40 with NH₄Cl or DTT, respectively. Infection was scored by automated microscopy and image analysis. Depicted are results normalized to solvent treated control cells. See also Figure S2, and material and methods. (C) HeLa cells were pretreated overnight with nystatin/progesterone in the indicated concentrations. Cells were infected with HPV-16 PsV (black), SFV (grey), or SV40 (white) and analyzed as in (B). (D) HeLa cells were transfected with siRNA oligos directed against caveolin-1 or with the AllStar negative control (Qiagen). 48 h after siRNA transfection cells were infected with HPV-16 PsV (black) or VSV (grey). Infection was scored as in (B) and depicted relative to cells transfected with the AllStar negative control. (E) Mouse embryonic fibroblasts from caveolin-1 knockout mice or parental mice were infected with HPV-16 PsV at different MOI. Infection was scored by flow cytometry as described in material and methods. Depicted are infection percentages. (F) HeLa cells were transfected with siRNA oligos directed against flotillin-1 or −2 or with the AllStar negative control (Qiagen). 48 h after siRNA transfection cells were infected with HPV-16 PsV (black) or SV40 (white). Infection was scored as in (C) and depicted relative to cells transfected with the AllStar negative control. (G, H) Merge images of a single cLSM slice of caveolin-1 (J, red) or flotillin-1 (K, red) HPV-16 AF488 (green) fluorescence of HeLa cells infected for 8 h. Boxed regions were enlarged as insets. (J) HeLa cells were infected with HPV-16 AF488 for 2 or 8 h, fixed with paraformaldehyde and immunostained for caveolin-1 or flotillin-1. Samples were analyzed by confocal laser scanning microscopy (cLSM) and the colocalization indices were determined as described in material and methods. Asterisk indicates a significant Costes P-value.

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Chlorpromazine, generally considered an inhibitor of CME, abolished HPV-16 infection at 10 µg/ml in line with earlier studies [5], [8], [22]. At this concentration, chlorpromazine also partially affected infection of SV40, a virus that makes use of CME-independent endocytosis (Figure 2B). Since efficient clathrin and AP2 depletion did not affect HPV-16 infection, we suspected chlorpromazine to cause CME-independent inhibition of entry.

To test whether HPV-16 was using a caveolin/lipid raft-mediated pathway, we depleted cells of cholesterol with nystatin/progesterone, a combination of drugs often used to inhibit fomation of lipid rafts in the plasma membrane [33]. SV40 infection, which uses a combination of a caveolar and a caveolin-independent lipid raft-mediated mechanism [34]–[36], was efficiently inhibited (Figure 2C). SFV infection was also affected most likely due to its cholesterol-dependent membrane fusion activity [37]. However, HPV-16 entry was, if anything, increased (Figure 2D). Similarly, cholesterol extraction by methyl-ß-cyclodextrin had only little effect (Table S1). We found, moreover, that HPV-16 infected HeLa cells depleted of caveolin-1 and caveolin-1 knockout cells with the same efficiency as wild-type cells (Figure 2D, E, Figure S3B). Since SV40 can make use of a caveolin-independent lipid-raft dependent pathway [34], and since cholera toxin B, that can enter cells by caveolar endocytosis, can make use of multiple pathways [38], meaningful positive controls could unfortunately not be employed.

To test whether endocytosis of HPV-16 would involve flotillins, proposed to define a distinct endocytic mechanisms [39], we depleted cells of flotillin-1 or -2, or both by siRNA mediated knockdown. Since no viral cargo exists for flotillin endocytosis, and since cholera toxin B can use multiple pathways, again, no direct positive controls could be used. Independent or combined depletion of flotillin-1 and -2 to 20% protein levels or less of control cells had no effect on HPV-16 infection, whereas SV40 infection was enhanced after flotillin-2 but not -1 knockdown (Figure 2F, Figure S3C).

We observed no significant colocalization of HPV-16 with caveolin-1 or flotillin-1 at 2 h post warming by confocal microscopy (Figure 2G–J). At 8 h, the degree of colocalization between HPV-16 and caveolin or flotillin increased slightly (Figure 2H–J). The colocalizing objects were mostly located intracellularly and perinuclearly, which suggested that they belonged to a population of endosomal vacuoles (Fig. 2G, H; [35], [40]).

In summary, infectious HPV-16 entry was independent of clathrin, AP2, caveolin, lipid rafts and flotillin, and HPV-16 did not associate with caveolin or flotillin at the cell surface. This indicated that HPV-16 entry occurred by a distinct endocytic mechanism.

HPV-16 entry is dynamin-2 independent

Dynamin-2 facilitates membrane fission to generate endocytic vesicles in several endocytic pathways [41], [42]. To test its involvement in HPV-16 entry, we used overexpression of the DN K44A mutant, the inhibitor dynasore, and siRNA depletion of dynamin-2 as methods of pertubation. Overexpression of K44A dynamin-2 deficient in GTP hydrolysis reduced infection of SFV or SV40 (Figure 3B, C) in a dose dependent manner to 40% or 50% of control, respectively,whereas HPV-16 entry was only slightly perturbed (Figure 3A). Dynamin-2 depletion had no significant effect on HPV-16 infection (Table S1). Dynasore, a noncompetitive inhibitor of dynamin-2, reduced infection of SFV and SV40 to 30% and 40%, respectively (Figure 3D). In contrast, HPV-16 entry was enhanced by up to 50% relative to control (Figure 3D). Our data suggested that dynamin-2 was not required for HPV-16 endocytosis.

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Figure 3. HPV-16 entry is dynamin-2-independent.

HeLa cells were transfected with constructs expressing wildtype (WT, black) or the DN K44A (grey) dynamin-2-GFP. 24 h post transfection cells were infected with HPV-16 PsV dsRed (A), SFV (B), or SV40 (C), infection was scored by flow cytometry as described in Figure S2B in relation to the level of overexpressed dynamin-2 (low, medium high). Depicted are results normalized to GFP expressing control cells. (D) HeLa cells were pretreated for 30 min with dynasore in the indicated concentrations. Cells were infected with HPV-16 PsV (black), SFV (grey), or SV40 (white) and analyzed as in Figure 2C.

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HPV-16 entry is facilitated by actin polymerization dynamics independently of Rho GTPase signaling and actomyosin contractility

Actin polymerization plays a role in several endocytic pathways. During phagocytosis and macropinocytosis it causes outward protrusion of the plasma membrane. In addition, actin polymerization but not outward protrusion of the plasma membrane is required in caveolar/lipid raft endocytosis, the IL-2, the glycosphingolypid enriched endocytic compartment (GEEC), and the Arf6 pathways [41], [42].

To test the involvement of actin, we used the actin depolymerizing agent cytochalasin D and the stabilizing agent jasplakinolide. As expected, the drugs strongly inhibited SV40 infection, whereas SFV infection was unperturbed by jasplakinolide but strongly enhanced by cytochalasin D (Figure 4A, B). Since HPV-16 infection was efficiently inhibited by both drugs (Figure 4A, B, Table S1), we concluded that actin dynamics facilitated infection, in line with previous studies on HPV-16 uptake in BHPE cells or HPV-33 entry into COS-7 cells [23], [43].

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Figure 4. HPV-16 entry requires actin dynamics but not Rho-GTPase or Arf6 signaling.

HeLa cells were pretreated for 30 min (A, B, G) or overnight (C) with cytochalasin D (A), jasplakinolide (B), C3 toxin/toxin B (C), and EIPA (G) in the indicated concentrations. Cells were infected with HPV-16 PsV (black), SFV (grey), or SV40 (white) and analyzed as in Figure 2C. Alternatively, HeLa cells were transfected with Cdc42, Rac1, RhoA (D, E), or Arf6 (F) fused to mRFP. Either the wildtype (WT, black), the DN (grey), or the CA (white) mutant were used. 24 h post transfection cells were infected with HPV-16 PsV (D) or VV (E). Infection was scored by flow cytometry as in Figure 3A–C. Depicted are results normalized to mRFP expressing control cells.

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Since actin polymerization is most critical during macropinocytic/phagocytic protrusion of the plasma membrane, we checked if HPV-16 infection would be affected by inhibition of the Na⁺/H⁺ exchanger, considered to be a hallmark of macropinocytosis [44]. SFV and SV40 infection were only mildly affected upon EIPA treatment, whereas HPV-16 infection was effectively reduced (Figure 4G), so that we concluded that the Na⁺/H⁺ exchanger is involved.

During macropinocytosis, actin polymerization events are regulated by the Rho-like GTPases Cdc42 and Rac1, whereas phagocytosis and caveolar/lipid-raft endocytosis depend on RhoA [42], [44], [45]. We first inhibited RhoA by C3 toxin, and all Rho-like GTPases by toxin B. C3 toxin, as expected, partially inhibited SV40 infection, but did not affect HPV-16 entry (Figure 4C). Toxin B inhibited SFV infection and SV40 infection, but HPV-16 infection was unperturbed (Figure 4C).

Since Rho-like GTPases are key regulators of actin polymerization, we wanted to verify these results. We overexpressed Cdc42, Rac1, and RhoA, and their DN (T17/19N) or constitutively active (CA, Q61L, G14V) mutants. None of the mutants affected HPV-16 entry (Figure 4D). However, when we infected Cdc42 expressing cells with vaccinia virus (IHD-J/GFP), which enters cells by macropinocytosis [46], [47], the DN mutant blocked infection by about 50% (Figure 4E). Hence, we concluded that HPV-16 infection occurred by an endocytic mechanism dependent on actin polymerization but independent of signaling via the classical Rho-like GTPases.

Arf6, another small GTPase, has been implicated in macropinocytosis and in an elusive endocytic pathway that occurs in HeLa cells [48], [49]. Arf6 also has the potential to stimulate actin polymerization [50]. Hence, we tested whether Arf6 might be regulating actin dynamics during HPV-16 endocytosis. HPV-16 entry was unaffected by overexpression of the CA (Q67L) or DN (T27N) mutant (Figure 4F). In addition, siRNA mediated silencing of Arf6 did not affect HPV-16 entry (Table S1). We concluded that HPV-16 endocytosis did not involve an Arf6 mediated pathway, and that actin dynamics during HPV-16 entry were not regulated by Arf6.

Also, actomyosin contractility was not required for entry, as inhibitors of myosin II and myosin light chain kinase only mildly reduced infection consistent with their role in transport of HPV-16 along filopodia by actin retrograde flow (Table S1, [7]).

Signaling involved in HPV-16 infection

Specific cellular signals are required for activating different endocytic pathways [41], [42], [51]. Hence, we analyzed the role of major kinases and phosphatases involved in endocytic uptake and their effect on HPV-16 entry. Genistein-mediated inhibition of tyrosine kinases blocked HPV-16 entry (Figure 5A). As expected, SV40 infection was likewise affected (Figure 5A, [34], [36]), while SFV infection was unaffected (Fig. 5A). Similarly, inhibition of serine/threonine kinases with H-7 blocked HPV-16 and SV40 infection but not SFV infection (Figure 5B). When we inhibited phosphatases by the broad inhibitor orthovanadate, entry of HPV-16, SV40 and SFV were inhibited (Figure 5C). However, when the phosphatase classes PP1, PP2A, and PP2B were inhibited by okadaic acid, or PP1 by tautomycetin, SFV infection was largely unaffected whereas HPV-16 and SV40 infection was blocked (Figure 5C, Table S1).

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Figure 5. Signaling events required during HPV-16 entry.

(A–H) HeLa cells were pretreated for 30 min with genistein (A), H-7 (B), okadaic acid or orthovanadate (C), iressa (D), calphostin C (E), wortmannin (F), PI-103 (G), or IPA-3 (H) in the indicated concentrations. (A–C, E–G) Cells were infected with HPV-16 PsV (black), SFV (grey), or SV40 (white) and analyzed as in Figure 2C. (D) Cells were infected with HPV-16 PsV (black), SFV (grey), or VV (white). Infection was scored by automated microscopy and image analysis as in Figure 2C (HPV-16, VV) or by flow cytometry (SFV). (H) Cells were infected with HPV-16 PsV (black), SV40 (grey) or VV (white). Infection was scored by automated microscopy and image analysis as in Figure 2C (HPV-16, VV) or by flow cytometry (SV40). (I) HeLa cells were transfected with siRNA oligos directed against PAK-1 or with the AllStar Negative control (Qiagen). 48 h after siRNA transfection cells were infected with HPV-16 PsV (black), VSV (grey), and VV (white). Infection was scored as in Figure 2C and depicted relative to cells transfected with the AllStar negative control.

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To further analyze signaling in the context of HPV-16 endocytosis, we inhibited major kinases involved in macropinocytosis such as EGF receptor tyrosine kinase (EGFR), protein kinase C (PKC), PI3 kinase (PI3K), and p21-activated kinase (PAK-1) [44]. Inhibition of EGFR signaling by Iressa blocked HPV-16 and VV but not SFV entry (Figure 5D). Also, PKC signaling was required for HPV-16 infection as indicated by the inhibitory effects of calphostin C and rottlerin (Figure 5E, Table S1). Inhibition of PI3K by wortmannin or PI-103 reduced HPV-16 infection by 50–60%, which indicated some involvement (Figure 5F, G). Inhibition of PAK-1 by IPA-3 blocked HPV-16 and VV but not SV40 infection (Figure 5H). Also, HPV-16 and VV but not VSV entry were reduced in PAK-1 depleted cells (Figure 5I).

In summary, HPV-16 infection required activation of cellular signaling. The signaling factors included EGFR, phosphatases, PKC and PAK-1, possibly additional tyrosine and serine/threonine kinases, and potentially PI3K. This list of signaling components is similar to the signaling activating macropinocytosis.

To test whether the required cell factors were also critical in more physiologically relevant cells, we infected pharmacologically perturbed HaCaT cells, i.e. spontaneously immortalized keratinocytes. There was virtually no difference between HeLa and HaCaT cells suggesting that HPV-16 entry occurred by a similar endocytic mechanism (Figure S4).

Cell binding and endocytosis of HPV-16

To test whether any of the inhibitors affected HPV-16 binding to cells we measured the association of fluorescent viruses with perturbed HeLa cells. As a positive control, we blocked cell binding by preincubation of PsV with soluble heparin, as previously described [11], [13], [52]. Alternatively, chlorate treatment of cells was used, which results in undersulfation of GAG chains that the virus uses as attachment factors. In both cases, a dramatic reduction in binding was observed (Figure 6A). When we used a variety of inhibitors that did or did not perturb HPV-16 entry, no significant reduction in binding was observed (Figure 6B). Only cholesterol depletion of the plasma membrane by methyl-β-cyclodextrin showed a minor reduction in binding that correlated with a small reduction in infection, which suggested a general membrane-receptor perturbing effect of the drug as nystatin/progesterone had no effect.

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Figure 6. HPV-16 cell binding and internalization.

(A, B) HeLa cells were seeded in optical bottom 96-well plates to result in a confluent monolayer at the day of experimentation. Cells were pretreated with chlorate (A, overnight) or further small compound inhibitors (B, 30 min) in the indicated concentrations. Subsequently, HPV-16 AF488 (A, B) or HPV-16 AF488 preincubated with the indicated concentrations of heparin (A) was bound to cells for 1 h, after which cells were fixed with paraformaldehyde. Binding of HPV-16 to cells was analyzed by determining cell associated fluorescence in a sensitive plate reader. The cell associated fluorescence is depicted relative to solvent treated control cells. (C) HeLa cells were pretreated with small compound inhibitors (concentrations as in A, B). Subsequently, cells were infected with HPV-16 PsV in the presence of inhibitor. Cells were treated either treated with high pH buffer at 12 h p.i. to inactivate extracellular virus and infection was continued in the absence of inhbitor (infectious internalization) or the inhibitor was washed out and replaced by NH₄Cl. (infection). Note that orthovanadate, bafilomycin, and nocodazole inhibit infection but not internalization. The amount of GFP expressing cells relative to solvent treated control cells is given as relative internalization in %. (D) HeLa cells were pretreated with small compound inhibitors for 30 min (concentrations as in A, B). Subsequently, cells were infected with HPV-16 AF594 in the presence of inhibitor. At 6 h p.i., trypan blue (0.008%, w/v) was infused to quench the fluorescence of extracellular virus. Images depict the same cell prior and after trypan blue infusion.

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To analyze which factors contributed to endocytic internalization as opposed to intracellular events prior to acid-activation we employed the internalization assay for infective particles and compared it to the infection assay with inhibitor swap (compare Figure 1A, Figure S1, S2, material and methods). The relative amount of internalized infectious particles was analyzed at 12 h, when the drug was washed out, and a high pH wash inactivated extracellular virus, so that infection could only be caused by already internalized virus. Infection was continued the absence of an inhibitor for further 36 h. To control for the general reversibility of drug treatment and for cytotoxicity, we included samples where we did not subject cells to a high pH wash. Any samples where reversibility of the inhibitors of interest was significantly affected were excluded. As a positive control, we used chlorate treatment.

Infectious internalization and infection were found to be similarly reduced by cytochalasin D (actin polymerization), jasplakinolide (actin depolymerization), EIPA (Na⁺/H⁺ exchanger), genistein (tyrosine kinases), PI-103 (PI3K), IPA-3 (PAK-1), and H-7 (serine/threonine kinases) (Figure 6C). Although infection was reduced, internalization was only slightly perturbed by treatment of cells with orthovanadate (phosphatases), or unaffected by bafilomycin (endosomal pH), and nocodazole (microtubules) suggesting a role in intracellular routing of the virus. Nystatin/progesteron (cholesterol), blebbistatin (myosin II), and ML-7 (myosin light chain kinase) did not affect HPV-16 internalization or infection, as expected. We concluded that endocytic internalization of HPV-16 required actin polymerization dynamics, the function of the Na⁺/H⁺ exchanger, tyrosine and serine/threonine kinases, PAK-1, and PI3K signaling. Intracellular steps required phosphatases, microtubules, and acid-activation.

Then, we assessed internalization of HPV-16 particles (as opposed to infectious units) using a trypan blue based assay [35]. Addition of the membrane impermeable trypan blue to cells quenched the fluorescence of AF594 labeled particles bound to cells, whereas the fluorescence of internalized particles was unaffected (Figure S5). Microscopic analysis showed that e.g. cytochalasin D rendered almost all HPV-16 particles susceptible to trypan blue consistent with a block in internalization, whereas e.g. NH₄Cl and nocodazole did not (Figure 6D). Since this data correlated with our infectious internalization analysis (Figure 6C, D; data not shown), we concluded that HPV-16 particles were not internalized and not channeled into a non-infectious uptake pathway but stalled at the cell surface.

Morphology of HPV-16 internalization

Using thin section electron microscopy, we analyzed the morphology of HPV-16 internalization in HeLa and HaCaT cells. Bound viruses were readily detectable at the plasma membrane (Figure 7A). Occasionally, viruses appeared to cause a slight indentation in the plasma membrane (Figure 7B). From 1–24 h post warming, viruses were mainly observed in indentations 65–120 nm in diameter without a visible coat (Figure 7D, H, I) but not in clathrin coated pits (Figure 7F, data not shown). Occasionally, these indentations were wider or tubular and held several particles (Figure 7E, not shown). The particles had a distance of about 5–10 nm from the membrane. About 10–20% were associated with membrane protrusions resembling filopodia, which we have previously shown to be capable of binding and transporting HPV-16 (Figure 7H, [7]). Virus containing indentations were occasionally located at the base of such protrusions (Figure 7H). In close proximity to the plasma membrane, viruses were observed in small vesicles (70–140 nm in diameter) that held one or sometimes two particles (Figure 7C, D, J). Our data suggested that viruses were internalized via such small uncoated pits and vesicles that could accommodate one or possibly several particles.

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Figure 7. Ultrastructural morphology of HPV-16 internalization.

HeLa (A–G) or HaCaT (H–K) cells were infected with HPV-16 PsV (10–20,000 particles/cell) for different times prior to fixation by glutaraldehyde. Cells were either untreated (A–F; H–J) or treated with cytochalasin D (10 µg/ml) throughout infection (G, K). Cells were processed for thin section electron microscopy according to standard procedures. Depicted are representative micrographs for 2–4 h p.i. (A–F; H–J) or 6 h p.i. (G, K). Scale bars represent 100 nm.

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When virus internalization was blocked using cytochalasin D to inhibit actin polymerization, virus particles were found in deep, narrow, almost tubular invaginations of the plasma membrane that varied in length from a few 100 nm to microns where viruses occurred in tight rows (Figure 7G, K). Thus, the endocytic pits containing viruses seemed to grow into long tubules unable to separate from the plasma membrane by scission. In addition to implicating actin polymerization in scission, this suggested that viruses were internalized after sinking into plasma membrane rather than by protrusive engulfment, which would often be seen during macropinocytosis.

Intracellular trafficking of HPV-16

After endocytic internalization, most incoming animal viruses are delivered to endosomes and their penetration into the cytosol occurs either in early or late endosomes (EE, LE). If the viruses are endocytosed by macropinocytosis or related mechanisms, the agenda is less well characterized. Macropinosomes can recycle to the plasma membrane or fuse with endosomes or lysosomes at different levels of the pathway [44]. They acidify and it is possible that they undergo a maturation of their own before fusing with lysosomes.

Previous studies have shown that entry of a variety of papillomaviruses is blocked by agents that raise the pH in endosomes and lysosomes [5], [6], [8], [22], [53]. We confirmed that HPV-16 infection, both in HeLa and HaCaT cells, is reduced by the weak base NH₄Cl, which neutralizes endosomal pH, and by other inhibitors of endosomal acidification such as bafilomycin A1 and monensin (Table S1, not shown). Hence, endocytosis carries viruses to acidic compartments, where low pH-based activation for penetration or uncoating likely occurs.

To determine when the virus passed the acid-activated step during entry, we added NH₄Cl and bafilomycin A1 at various times post warming and scored for infection. If infection would no longer be perturbed at a certain time, the viruses must have passed the acid-activation step. Our results indicated that about half of the virus population had passed the low pH dependent step by 13–16 h in HeLa and HaCaT cells (Figure 8A). This means that the critical acid exposure occurred 2–3 h after endocytosis (compare Figure 1A, B). We confirmed previous findings that infection can be blocked by nocodazole-induced depolymerization of microtubules suggesting a need for intracellular transport of endocytic vacuoles or the virus itself (Figure 9A, [6], [23]). Since nocodazole add-on experiments followed a similar time course (not shown), we assume that microtubule-based intracellular sorting of endosomes is required for successful HPV-16 infection.

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Figure 8. Kinetics of HPV-16 acid-activation during entry.

(A) HeLa or HaCaT cells were infected with HPV-16 with different multiplicities of infection (MOI, in transducing units/cell). At different times p.i. NH₄Cl (straight lines) or Bafilomycin A1 (striated line) was added, and infection was continued in the presence of the drug. (B) HeLa were infected with HPV-16 at MOI 0.1 (10 particles/cell) in the presence of NH₄Cl (straight line). At different times p.i., the drug was washed out as indicated, and infection was continued in the absence of the drugs. (C) HeLa were infected with HPV-16 as in (B) in the presence of NH₄Cl. 8 h p.i., the drug was washed out, and infection was continued in the absence of the drug for indicated times, after which NH₄Cl was re-added and infection was continued in the presence of the drug, thus creating a time window of drug absence. (A–C) Infection was scored 48 h p.i by flow cytometry. Depicted are results normalized to untreated control cells.

https://doi.org/10.1371/journal.ppat.1002657.g008

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Figure 9. Intracellular vesicular transport of HPV-16 leads virus particles to the late endosomal/lysosomal compartments.

(A) HeLa cells were pretreated for 30 min with nocodazole in the indicated concentrations. Cells were infected with HPV-16 PsV (black), SFV (grey), or SV40 (white) and analyzed as in Figure 2C. (B, C) HeLa cells were transfected with Rab5 (B) or Rab7 (C) fused to a mRFP tag. Either the wildtype (WT, black), the DN (grey), or the CA (white) mutant were used. 24 h post transfection, cells were infected with HPV-16 PsV, SFV, VSV, or IAV as indicated. Infection was scored by flow cytometry as in Figure 3A–C. Depicted are results normalized to mRFP expressing control cells. (D) HeLa cells were infected with HPV-16 AF488 for 2 or 8 h, in the presence (white) or absence (black) of 20 mM NH₄Cl, fixed with paraformaldehyde and immunostained for EEA1, LAMP-1, or giantin. Samples were analyzed by confocal laser scanning microscopy (cLSM) and the relative amount of the colocalization index was determined as described in material and methods. Asterisk indicates a significant Costes P-value. (E–G) Merge images of a single cLSM slice of EEA1 (E), LAMP-1 (F), or giantin (G) (red) and HPV-16 AF488 (green) fluorescence of HeLa cells infected for 8 h.

https://doi.org/10.1371/journal.ppat.1002657.g009

Progression in the endocytic pathway involves the risk that viral particles are degraded. We infected cells at a low MOI in the presence of NH₄Cl and subsequently washed out NH₄Cl at various times. The virus remained infectious for prolonged times exhibiting 80% infectivity even after a washout at 24 h p.i. (Figure 8B). Thus, the viruses were not rapidly routed to compartments, where they were inactivated after acidification.

To analyze the average time that viruses require for acid-activation, we infected cells in the presence of NH₄Cl. After 12 h, we washed out NH₄Cl which results in a fast re-acidification of endosomes and lysosomes [54]. At various times after washout, we re-added NH₄Cl, and thus created a time window of virus exposure to low pH. When infectivity was quantified, we found that the halftime of activation was 6 h (Figure 8C), a surprisingly long time period suggesting that to be infectious the virus had to be exposed to low pH, or an enzyme requiring low pH for several hours. That the long time period was specific to HPV-16, is shown by the fact that the low pH based activation of VSV occurred almost completely within a time window of 15 min. (Figure S6).

Since most transport steps in the endocytic pathway are regulated by Rab GTPases, we analyzed which of several RabGTPases were required for HPV-16 infection. We overexpressed GFP- or RFP-tagged Rab GTPases either as wildtype, or in the form of DN or CA mutants.

The DN and CA mutants of Rab5 reduced HPV-16 infection when highly overexpressed (Figure 9B). To control for the efficiency of the dominant effect, we used SFV and VSV as controls, which both require Rab5-mediated transport to EEs for infection. Since the infectivity of both was similarly reduced (Figure 9B), we concluded that HPV-16 required Rab5. No requirement for Rab7 was detected (Figure 9C, left panel). As a positive control, we infected cells with IAV that requires Rab7 for transport to the LE compartment [55]. Since infection of IAV was reduced by the DN mutant, we concluded that HPV-16 was Rab7-independent (Figure 9C, right panel). Overexpression of Rab1, Rab4, Rab6, Rab11 and their mutants did not affect infection (Figure S7).

When the localization of HPV-16 with EE markers was analyzed, we found no significant colocalization of virus with the EEA1 from 30 min -2 h or at 8 h (Figure 9D, E, data not shown). However, we readily found AF488 labeled particles that co-migrated with Rab5 positive structures in live cells (Video S2). The time period spent in the Rab5-positive compartments may be fairly limited as exemplified in Video S2 by a virus entering a Rab5-positive, virus containing vacuole that loses the Rab5 signal about 90 s after this event.

Colocalization of HPV-16 with the LE and lysosome marker LAMP-1 was detectable at 8 h p.i. (Figure 9D, F). Since the localization of LAMP-1 in the crowded perinuclear space may introduce artifactual values for colocalization, we analyzed colocalization with giantin, a Golgi marker. No significant colocalization was observed (Figure 9D, G). We concluded that PsV were trafficked to perinuclear, LAMP-1 containing, acidic compartments. Virus was transported to these vacuoles in the presence of NH₄Cl (Figure 9D, G). Combined with the fact that after NH₄Cl washout the virus remained infectious (compare Figure 8B), this result suggested that the transport occurred to LEs or endolysosomes, and that this represented the infectious route.

EM showed the virus frequently in multivesicular bodies (Figure 10B, C) and lysosomes (Figure 10D), and occasionally in large endosomal vacuoles that lacked internal vesicles (Figure 10A). They were not observed in the Golgi complex, the endoplasmic reticulum (ER), or the cytosol. Interestingly, virus particles were detectable even late during infection in lysosomal compartments, which suggested that despite the fact that BrdU labeled DNA of PsV become accessible to antibody staining [56], they were not degraded and remained as recognizable particles.

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Figure 10. Ultrastructural morphology of HPV-16 intracellular trafficking.

HeLa cells were infected with HPV-16 PsV (10–20,000 particles/cell) for 12 h (A, B) or 18 h (C, D) prior to fixation by glutaraldehyde. Cells were processed for thin section electron microscopy according to standard procedures. Scale bars represent 250 nm.

https://doi.org/10.1371/journal.ppat.1002657.g010

In summary, our data were consistent with intracellular transport of HPV-16 to the LE/lysosomes. During transport the virus-containing vacuoles underwent a transient Rab5 association.

Cells, antibodies, small compound inhibitors, siRNAs, plasmids

HeLa and HaCaT cells, and mouse embryonic fibroblasts (caveolin-1 wildtype and knockout, [35]) were cultured in DMEM (Invitrogen) containing 10% fetal calf serum. For a full list of antibodies, inhibitors, plasmids, siRNAs, or reagents used, please refer to Table S2.

Viruses

HPV-16 PsV containing the pRwB or pCIneo-EGFP plasmid were produced with the p16sheLL plasmid by the propagation method described by Buck and colleagues [80]. The PsV were matured for 24 h in the presence of RNAse to maximize the purification of PsV containing the reporter plasmid, resulting in an improved particle to infectivity ratio [80]. All plasmids and production methods are fully described on the Schiller laboratory's website (http://ccr.cancer.gov/staff/staff.asp?profileid=5637). IAV (H1N1, A/PR 34/8), SFV, SV40, recombinant VSV (Indiana) expressing GFP (VSV-GFP), recombinant VV (IHD-J) expressing GFP were produced as described [32], [47], [57], [59], [81].

Fluorescent labeling of HPV-16 PsV

HPV-16 PsV were covalently labeled with fluorophores as described [7]. Briefly, purified HPV-16 PsV were incubated for 1 h at room temperature in PBS with a ten-fold molar excess of Alexa Fluor (AF) succinimidylesters (Molecular Probes) over the major capsid protein L1. PsV were separated from the labeling reagent by size exclusion chromatography using NAP5 columns (GE Healthcare) and stored at 4°C.

Transfection and transient expression

For transfection, cells were trypsinized, pelleted, washed with PBS and transfected with expression plasmids in Nucleofector solution R (Amaxa) utilizing program I13 of the Amaxa Nucleofector according to the manufacturer's instructions. Cells were seeded in 12-well plates or on 18 mm coverslips and used for infection or live cell imaging experiments, respectively, at 6–14 h post transfection.

TIRF microscopy

Microscopy was performed on a custom modified Olympus IX71 inverted microscope. Modifications included a heated incubation chamber that surrounded the microscope stage set to 37°C, an objective-type TIRFM setup from TILL Photonics (Grafeling, Germany), and a monochromator for epifluorescence excitation with a controller allowing hardware-controlled fast switching between total internal reflection fluorescence and epi-fluorescence excitation and acquisition (TILL Photonics). Images were acquired using a TILL Image QE chargecoupled device camera and TILLVISION software (both from TILL Photonics). The total internal reflection angle was manually adjusted for every experiment. Live HeLa cells on 18-mm coverslips were mounted in custom-made chambers. 5,000 HPV-16 PsV/cell labeled with AF dyes were added into the 0.5 ml of medium on the stage. Movies were recorded at a rate of 2–5 frames per s.

Live cell confocal microscopy

Live cell confocal microscopy was performed on a Zeiss 510 microscope with a confocal laser scanning setup. The confocal microscope was equipped with a heated incubation chamber set at 37°C. Cells were mounted on 18-mm coverslips in custom-made chambers, and cells were incubated with normal growth medium. HPV-16 PsV (500–1,000 particles/cell) were added into the 0.5 ml of medium on the stage. After 30 min – 4 h post addition of virus particles to cells image acquisition was performed. Movies were recorded at a rate of 2 frames per s.

Infection studies and analysis by automated microscopy

For siRNA mediated knockdown experiments, 3,000 cells were reverse transfected in optical bottom 96-well plates (Nunc) with the indicated amount of siRNA oligos using Lipofectamine RNAimax (Invitrogen), and infected 48 h post transfection of siRNAs. In case of oligos directed against clathrin heavy chain and the AP2 μ subunit, cells were transfected for a second time 24 h post initial transfection. For small compound inhibition studies, 5,000 cells were seeded 24 h prior to experimentation in 96-well optical bottom well plates. Inhibitors were added at indicated concentrations 30 min or as indicated 12–16 h prior to infection (Figure S2A). Cells were infected with HPV-16, SV40, SFV, or VV to result in about 20% infection. For inhibitor studies, the primary inhibitors were exchanged at 12 or 10 h p.i. against 10 mM NH₄Cl or 5 mM DTT in case of HPV-16 or SV40 infection, respectively, to reduce cytotoxicity (Figure S2A). After 4% paraformaldehyde fixation, cells were stained with Hoechst 33258 for cell nuclei and in case of SFV, SV40, and IAV with antibodies directed against SFV glycoproteins, T-antigen, and nucleoprotein, respectively, and secondary antibodies conjugated to Alexa Fluor 488 (Molecular Probes) directed against the primary antibodies. Nine images per well were acquired for each, nuclear stain (Hoechst 33258) and infection stain (GFP, or IF staining for SV40 T-antigen, SFV glycoproteins), on a Pathway 855 automated microscopy station (Becton Dickinson) using a 10× objective (Olympus) employing a laser-based autofocus every image. Cell numbers and raw infection indices for each well were determined using a MATLAB-based infection scoring procedure (The Mathworks) described previously ([35], Figure S2C). The raw infection indices were normalized to solvent treated control cells or AllStar negative siRNA transfected control cells to result in relative infection percentages.

Infection studies by flow cytometry

5×10⁴ cells seeded in 12-well plates were either transfected as described above or treated with small compound inhibitors 30 min prior to infection. Cells were infected with HPV-16 dsRed or GFP, VSV-GFP, VV-GFP, or IAV to result in 20% infection. Cells were trypsinized, fixed in 4% paraformaldehyde, and in case of IAV, immunofluorescence staining for the nucleoprotein was performed. Transfected cells were analyzed by flow cytometry for the level of transgene expression, grouped into low-, medium-, and high-expressing cells (Figure S2C), and the subpopulation was subsequently analyzed for the number of infected cells. The relative number of infected cells was normalized to cells expressing only the XFP-tag according to expression levels. Inhibitor treated cells were normalized to solvent treated control cells.

Immunofluorescence staining and analysis

5×10⁴ cells were seeded on 18 mm coverslips and infected 16–24 h post seeding with 500 AF488-labeled HPV-16 particles/cell for 2 or 8 h. Cells were fixed with 4% paraformaldehyde or in ice-cold methanol, stained against cellular structures with primary antibodies (Table S2) and AF594 labeled secondary antibodies (Molecular Probes). Cells were mounted in Citifluor AF1, and analyzed on a Zeiss 510 microscope with a confocal laser scanning setup. Per experiment, at least 10 fields of view were imaged in 5 confocal slices with 3–8 cells/field of view. The degree of colocalization between virus and cellular structures was assessed using Bioimage XD (www.bioimagexd.net). The thresholded pixel-per-pixel colocalization was analyzed in an automated fashion where significance of colocalization was attributed to a Costes P-value≥0.95 [82].

Binding of HPV-16

30,000 Hela cells were seeded into optical bottom 96-well plates 24 h prior to experimentation to reach confluency. Cells were pretreated with inhibitors as in the infection studies. AF488 labeled HPV-16 (5,000 particles/cell) was added to each well, and incubated for 1 h at 37°C, when cells were fixed in paraformaldehyde and stained with Hoechst 33258 for cell nuclei. Cellular and cell associated fluorescence was analyzed using a Safire² plate reader (Tecan), normalized to solvent controls, and depicted as relative cell associated fluorescence (relative binding).

Internalization of HPV-16 particles

5×10⁴ HeLa cells were seeded on glass coverslips 24 h prior to experimentation. Cells were infected with HPV-16 AF594 (500–1000 particles/cell) in the presence or absence of inhibitors. 6 h post addition, coverslips were mounted in custom made chambers, and the amount of fluorescent viruses/cell was analyzed in live cells on a Zeiss Observer Z1 microscope equipped with a Yokogawa spinning disc confocal unit and a heating chamber surrounding the microscope by acquiring Z stacks. After the initial acquisition, trypan blue (0.4% (w/v), Invitrogen) was added to live cells in a 1∶50 dilution. This immediately shifted the emission spectrum of viruses on the cell-surface and led to a loss of detectable fluorescence, whereas the fluorescence of intracellular viruses remained unaltered. Images were visually analyzed and representative examples were depicted as maximum intensity projections.

Infectious internalization of HPV-16

5×10⁴ HeLa or HaCaT cells were seeded 24 h prior to experimentation in 12 well plates. HPV-16 PsV were added to cells in the cold. After 4 h the plates were shifted to 37°C. At different time points post warming, cells were incubated with high pH buffer (PBS, pH 10.5) for 1 min, which was replaced by normal growth medium. The treatment did not cause any cell toxicity (not shown) but resulted in disassembly of surface bound virions rendering them non-infectious (Figure S1). Alternatively, HeLa cells were infected in the presence or absence of inhibitors with HPV-16 to result in about 20% infection of the unperturbed control (10–30 particles/cell) At 12 h p.i., cells were incubated for 2 min with the high pH buffer, which was replaced with normal growth medium without inhbitor causing infection by already internalized virus. 48 h p.i., cells were trypsinized, fixed in 4% paraformaldehyde, and analyzed for infection (GFP expression) by flow cytometry.

Electron microscopy

For thin-section EM, HeLa or HaCaT cells plated onto 12-mm coverslips were incubated with HPV-16 PsV (10–20,000 particles/cell) 37°C for 30 min, 2, 6, 12, and 24 h before fixation with 2.5% glutaraldehyde (in 0.05 M sodium cacodylate pH 7.2, 50 mM KCl, 1.25 mM MgCl₂, and 1.25 mM CaCl₂, 30 min at RT), followed by 1.5 h of incubation in 2% OsO₄ on ice. Dehydration, embedding, and thin sectioning were performed as previously described [58]. After transmission electron microscopy (Zeiss EM 91 microscope), images were exported as 8-bit TIFF files.

If not otherwise mentioned, all data is depicted ± standard deviation from at least three independent experiments.