Essential roles for soluble virion-associated heparan sulfonated proteoglycans and growth factors in human papillomavirus infections

Abstract

A subset of human papillomavirus (HPV) infections is causally related to the development of human epithelial tumors and cancers. Like a number of pathogens, HPV entry into target cells is initiated by first binding to heparan sulfonated proteoglycan (HSPG) cell surface attachment factors. The virus must then move to distinct secondary receptors, which are responsible for particle internalization. Despite intensive investigation, the mechanism of HPV movement to and the nature of the secondary receptors have been unclear. We report that HPV16 particles are not liberated from bound HSPG attachment factors by dissociation, but rather are released by a process previously unreported for pathogen-host cell interactions. Virus particles reside in infectious soluble high molecular weight complexes with HSPG, including syndecan-1 and bioactive compounds, like growth factors. Matrix mellatoproteinase inhibitors that block HSPG and virus release from cells interfere with virus infection. Employing a co-culture assay, we demonstrate HPV associated with soluble HSPG-growth factor complexes can infect cells lacking HSPG. Interaction of HPV-HSPG-growth factor complexes with growth factor receptors leads to rapid activation of signaling pathways important for infection, whereas a variety of growth factor receptor inhibitors impede virus-induced signaling and infection. Depletion of syndecan-1 or epidermal growth factor and removal of serum factors reduce infection, while replenishment of growth factors restores infection. Our findings support an infection model whereby HPV usurps normal host mechanisms for presenting growth factors to cells via soluble HSPG complexes as a novel method for interacting with entry receptors independent of direct virus-cell receptor interactions.

Figure 1. Normal HSPG biology and proposed model for extracellular interactions of HPVs in the context HS-GF complexes.

(A). Natural processes of HSPG shedding that occur in the absence of HPV. The lower edges of epithelial cell lipid bilayers are depicted interacting with the extracellular matrix (ECM). The ECM consisting of (e.g.) collagens, elastins, fibronectins, laminins is shown in pink. Laminin 332 (formerly laminin 5; orange) interacts with syndecan-1 (purple) and alpha-6 beta-4 integrin (dark blue) on the cell surface to provide cell anchorage to the ECM/basement membrane. Notably, these three molecules have been identified as HPV attachment factors (refs. in text). (i.) Sheddases including matrix metalloproteinase (MMP) and ADAM (a disintegrin and metalloproteinase) sheddases (green) normally catalyze the release or “shedding” (dotted arrows) of membrane-bound growth factors (GFs; light blue) and other bioactive molecules, the protein ectodomains of HSPGs like syndecan-1, and ECM residents like laminin 332 . (ii.) HSPGs in the plasma membrane and ECM act as local depots for soluble GFs and other bioactive molecules. The HS-GF and bioactive compounds can interact with their cognate receptors laterally, via soluble form after release (iii), or in the ECM when cells migrate over the HSPG-complexes. (iii.) Sheddases including MMPs and heparanases and proteolytic processing of laminin 332 liberate soluble complexes containing GFs and HS/syndecan-1. (iv.) Soluble HS-GF complexes bind to GFR/RTK (yellow) and activate intracellular signaling cascades. (B). The natural processes of HSPG decoration and release from the cells also occur in the presence of HPV particles (red). The virion image is based on the atomic structure from Modis et al. . By virtue of interaction with HS, HPV can join the complex at each stage where HSPG is involved (i–iv). HPV could associate with soluble HS-GF in a naïve infection site or during release from infected cells (v.). HPV association with syndecan-1 via HSPG and binding of syndecan-1 to laminin 332 and alpha-6 beta-4 integrin are consistent with the fact that HPV particles colocalize and interact with each of these extracellular molecules. The abundance of HSPG in the ECM can explain why HPVs bind at such high levels to the ECM (ii.). Cells can pick up HPV-HS-GF complexes in soluble form or by migrating over ECM-bound HPV-HS-GF complexes.

Figure 2. HPV16 and syndecan-1 release from the HaCaT cell plasma membrane is MMP dependent.

Immunoblot for syndecan-1 (snd-1) post starvation in SFM and after 6 h at 37°C in Tyrode's Buffer (A); immunoblot for HPV16 L1 released into CM post virus binding for indicated times (B) or 24 h (D). (C) Gelatin zymography showing protease activity present in HaCaT cell CM alone or with binding of HPV16. (D–F) Effect of MMP inhibitors batimastat (BM) or marimastat (MM) at the indicated concentrations on HPV16 release into media as in panel B and densitometric quantification with AlphaEaseFC software (E) and relative infection levels (F). Cells were untreated (U) or pre-treated with the indicated concentrations of BM, MM or TIMP3 for 1 h, then exposed to HPV16 PsV in the presence of inhibitors in CM. Panel D includes lanes spliced together from the same exposure of the same films. (F) Infection was assayed by quantifying luciferase levels at 24 h p.i. Data are represented as mean ± SEM of 3 experiments.

Figure 3. Sepharose 4B gel chromatography of media constituents from HPV-exposed HaCaT cells.

Sepharose 4B chromatography was performed on released components in the CM of HaCaT cells exposed to HPV16 PsV for 4 h; see Figure S2. (A) The void volume (HMW) fraction was divided into four parts that were untreated or incubated with 1 U heparinase III for 2 h at 37°C then solubilized in 6× sample buffer and incubated at 25°C or boiled for 7 min before SDS-PAGE and L1-immunoblot analysis. Separate lanes shown are from the same exposure of the same film. (B) Non-reducing SDS-PAGE of void volume Sepharose 4B fractions from released components in the CM of HaCaT cells mock-exposed or HPV16-exposed for 24 h. Immunoblot analysis was done to detect L1, amphiregulin (AREG), HB-EGF, EGF, HS and syndecan-1 (snd-1). (C) Non-reducing SDS-PAGE of released components following IP of HPV16 from CM of HaCaT cells mock- or HPV16-exposed. HPV16 exposed cells were allowed to bind virus at 4°C for 1 h, washed to remove unbound particles, then shifted to 37°C for 6 h or 24 h. CM were subjected to IP for HPV16 using affinity purified polyclonal anti-HPV16 VLP antibody covalently attached to magnetic beads. Immunoblot analysis was performed to detect HB-EGF, EGF, and syndecan-1.

Figure 4. Released HMW complexes including HSPG and HPV16 are required for infection.

(A–D) Schematic of the “donor” cell/“recipient” cell co-culture system indicating how cells were exposed to PsVs. PsVs were allowed to bind donor cells without internalization (A). Donor cells on coverslips were washed thoroughly to remove unbound PsVs and transferred to mesh inserts above the recipient cells (B) to co-culture with gentle rocking for 24 h and allow released HPV complexes from donors to access the recipient cells (C–D). All experiments employed CM. (E) Relative HPV16 infection levels of HaCaT donor cells and recipient HaCaT cells compared to mock infected cells as verification of the co-culture virus release model. (F) Non-reducing SDS-PAGE and immunoblot of syndecan-1 (polyclonal rabbit antisera) following IP of HPV16 (mouse monoclonal anti-L1). IP was performed by immobilizing anti-L1 in the lower chamber in place of cells (see panel B) to capture HPV16 released from mock exposed cells (M) or HPV16-exposed HaCaT donor cells at 2 or 20 h post virus exposure. Lower panel IgG detection is included as a loading control. (G) Relative infection levels in CHO-K1 and pgsd-677 cells used as donor cells bound to HPV16 PsV and co-cultured above the recipient cells. (H) Relative infection levels in CHO-K1 and pgsd-677 cells used as recipient cells co-cultured below the PsV-bound donor cells corresponding to the data in panel G. Infectivity data (E,G,H) were normalized to the mean value of the infected control set to 100% and represent the mean ± SEM of 4 replicate infections.

Figure 5. HPVs interact with growth factors and growth factor receptors on human keratinocytes.

Immunofluorescent confocal co-localization (arrowheads show examples of signal overlap) showing top view and side views of non-permeabilized HaCaT cells. (A) Co-localization of HPV16 (red) with EGF (green). Bars measure 10 µm. (B) Co-localization of HPV16 (green) with KGFR (red). Bars = 5 µm. (C) SDS-PAGE and immunoblot for EGFR or p-KGFR after IP of HPV16 from HaCaT cells exposed to PsV. Lane 1, HaCaT cell lysate (without HPV exposure) incubated with anti HPV antibody attached to magnetic beads (negative control); lane 2, IP of HPV16 (mouse monoclonal anti-L1) from virus-exposed HaCaT cells; lane 3, blank; lane 4, HaCaT cell lysate following EGF exposure as positive control.

Figure 6. HPV16 activates EGFR and KGFR signaling pathways.

(A–B) Immunoblot for p-EGFR, p-KGFR and downstream effector p-ERK1/2 following 10-min ligand exposure (lane 2; listed at the left of each blot: EGF, KGF, HPV16 PsV) in HaCaT cells serum-starved for 4 h. Mock exposed cells were negative controls (lane 1); ligand controls included 10 ng/ml EGF and 10 ng/ml KGF; HPV16 PsV dose was 20 vge/cell. Cells were pretreated with the indicated inhibitors in Tyrode's buffer (100 nM PD168393, 1 µM PD173074, 100 µM genistein, 100 µM daidzein, 600 nM cetuximab) and exposed to the ligands in the presence of inhibitors Tyrode's buffer. Actin is detected as a loading control. (C–D) Immunoblot of nuclear cell fractions and confocal microscopy localization of p-ERK1/2 following EGF and HPV16 exposure in serum-starved HaCaT cells at indicated times post-exposure. (C) Immunoblot for p-ERK1/2 in nuclear fractions from exposed cells. (D) Immunofluorescent confocal microscopy for localization of p-ERK1/2 (red) in PsV exposed cells. DAPI was used as a nuclear marker and was pseudocolored green to facilitate efficient co-localization of p-ERK1/2 in the nucleus. Parameters of lasers intensities were kept constant during the imaging.

Figure 7. GFR activation, EGFR expression levels, and serum components including GFs are important for HPV16 infections.

(A) Relative HPV16 infection of HaCaT cells in the presence of specific GFR and protein tyrosine kinase inhibitors. Subconfluent HaCaT cells were pre-treated 45 min with 1 µM AG1478, 100 nM PD168393, 100 µM genistein, 100 µM daidzein, 1 µM PD173074, or 100–600 nM cetuximab. Cells were exposed to HPV16 PsV at 100 vge/cell for 1 h at 4°C, then washed extensively and shifted to 37°C in the presence of the indicated inhibitor in CM for 24 h at which time they were analyzed for luciferase expression. Data are represented as mean ± SEM of 3 experiments. (B–C) EGFR knockdown in EGFR-siRNA transfected HaCaT cells was determined by immunoblot and compared by densitometry to EGFR levels in cells transfected with a negative control siRNA at 48 hours post transfection. Four separate transfections were analyzed (B) and HPV16 PsV infection levels were measured at 24 h post infection (48 h post transfection) (C). Error bars represent the average of triplicate luciferase readings from the four transfections. (D) HPV16 PsV infection levels (24 h post infection) in the presence of inhibitors following pre-treatment for 1 hr with 100 µM monastrol, pre-treatment with monastrol for 1 h plus 500 nM PD168393 for duration (monast.+PD), or pre-treatment with 500 nM PD168393 for 1 hr plus 100 µM monastrol for duration (PD+monast.). (E) Relative HPV16 infection is dependent upon medium constituents post primary HPV16 binding. HaCaT cells starved in SFM (4 h) were exposed to HPV16 in CM (positive control) or SFM. After washing away unbound virus, cells were incubated for 24 h in CM, SFM, syndecan-1-depleted CM (IP-snd), or EGF-depleted CM (IP-EGF). Infections were quantified by luciferase assay at 24 h post shift to 37°C. Data are represented as mean ± SEM of 3 experiments. (F) Relative HPV16 infection in SFM is enhanced by GFs. HaCaT cells starved in SFM were exposed to HPV16 in SFM for 1 h at 4°C. After washing away unbound virus, cells were incubated for 24 h in SFM, SFM containing GFs (concentrations indicated: ng/ml), or in CM. Infections were quantified by luciferase assay; bars represented the mean ± SEM of ≥3 individual experiments.