Figure 1.
HPV16 PsV infection decreases following SLPI treatment or anti-annexin A2 antibody inhibition of A2t.
HaCaT cells were infected with HPV16 pseudovirions containing a GFP plasmid. Infectivity was scored at 48 h post infection by enumerating GFP-positive cells by flow cytometry. (A) Cells were preincubated with increasing amounts of SLPI or BSA for one hour at 4° prior to PsV infection. The mean percentage of HPV16 PsV infected cells (GFP-positive) normalized to the PsV only group ± SD are presented. (**P<0.01 as determined by a two-tailed, unpaired t-test, as compared to the PsV only group). (B) Cells were incubated with increasing amounts of an anti-annexin A2 Ab or isotype control (mouse IgG1) for one hour prior to PsV infection The mean percentage of HPV16 PsV infected cells (GFP-positive) normalized to the PsV only group ± SD are presented (**P<0.01 as determined by a two-tailed, unpaired t-test, as compared to PsV only except where otherwise noted). Each graph is representative of at least two independent experiments.
Figure 2.
Surface expression of A2t on HaCaT and HeLa human epithelial cell lines. (
A) HaCaT and HeLa cells were incubated with an anti- S100A10 antibody, then incubated with fluorophore-conjugated secondary antibodies, and mounted with DAPI containing media. For control staining, cells were either stained with a mouse or rabbit IgG isotype control followed by secondary antibody staining. Images were acquired using an upright confocal fluorescent microscope. (B) HeLa and HaCaT cells were incubated with PBS supplemented with Ca2+ or PBS with increasing concentration of EDTA for 45 min. The supernatants were collected and the presence of ANXA2 and S100A10 were analyzed via Western blot.
Figure 3.
HPV16 binds to A2t on the cell surface of HeLa cells.
HeLa cells were incubated with HPV16 L1L2 VLP, HPV16 PsV or HPV16 L1 VLP for 1 hour at 37°C. The cells were washed and surface proteins cross-linked. HPV16 VLP and PsV were precipitated out of cell lysates with an anti-L1 antibody (H16.V5) conjugated to magnetic beads. Elutions were analyzed via Western blot for the presence of ANXA2 and S100A10. Band density was determined by Licor Odyssey imaging software. HPV L1 western blot shows equivalent amounts of HPV16 particles were precipitated in lanes 3–5. The table below the figure indicates which components were included in each treatment. Data are representative of at least three independent experiments.
Figure 4.
HPV16 L2108–126 is exposed on HPV16 L1L2 VLP and HPV16 PsV.
ELISA plate wells were coated with 500 ng of HPV16 L1 VLP, HPV16 L1L2 VLP or HPV16 PsV and subsequently incubated with anti-L1 H16.V5 or anti L2108–126 16L2.4B4 antibodies. Secondary HRP-conjugated secondary antibodies were added prior to the substrate. In control experiments, no VLP were used. The graph represents the mean absorbance measured at 490 nm ± SD of triplicate wells. Data was repeated in three independent experiments.
Figure 5.
HPV16 L2108–126 peptide binds to the S100A10 subunit of A2t protein with sequence specificity.
HPV16 L2 peptide was paramagnetic-labeled (*denotes the attachment of the paramagnetic-label) and analyzed for binding to purified A2t, S100A10 and ANXA2 protein in vitro by electron paramagnetic resonance. (A) The first derivative spectrum of L2108–126 peptide free from ligands in solution. (B) The first derivative spectrum of L2108–126 combined with A2t. (C) The first derivative spectrum of L2108–126 combined with A2t in the presence of the control peptide (5∶1). (D) The result of subtraction of spectrum A from spectrum B representing the bound spectrum of the labeled peptide. (E) The first derivative spectrum of L2108–126 combined with BSA. (F) The first derivative spectrum of L2108–126 combined with ANXA2. (G) The first derivative spectrum of L2108–126 combined with ANXA2 in the presence of the scrambled peptide (5∶1). (H) The first derivative spectrum of L2108–126 combined with S100A10. (I) The first derivative spectrum of L2108–126 combined with S100A10 in the presence of the scrambled peptide (5∶1). (J) The first derivative spectrum of L2108–126 combined with cysteine-blocked S100A10 (αCys-S100A10). (K) The first derivative spectrum of the paramagnetic-labeled scrambled peptide combined with cysteine-blocked S100A10. (L) Quantification of relative % bound of all treatments as measured through spectra subtraction and double integration expressed as the mean of three separate experiments ± SD.
Figure 6.
Mutations in HPV16 L2108–111 reduce PsV binding to A2t and PsV infectivity.
(A) ELISA plate wells were coated with 500 ng of A2t prior to overnight incubation with 400 ng HPV16 PsV or HPV16 L1–L2(GGDD) mutant PsV and subsequently incubated with mouse anti-L1 H16.V5 or goat anti-SLPI antibodies. Anti-mouse and anti-goat HRP-conjugated secondary antibodies were added prior to the substrate. In control experiments, no ligands were used. The graph represents the mean absorbance measured at 490 nm ± SD (***P<0.001 as determined by a two-tailed, unpaired t-test between WT and mutant PsV). (B) HaCaT cells were infected with wild type (WT) or mutant (L2108–111 LVEE to GGDD) HPV16 pseudovirions containing a GFP plasmid. Infectivity was scored at 48 h post infection by enumerating GFP-positive cells by flow cytometry. The mean percentage of HPV16 PsV infected cells (GFP-positive) normalized to the WT PsV group ± SD are presented of two combined independent experiments. Inset shows the L1 band of a coomassie blue stained SDS-PAGE gel loaded with an equivalent amount of WT and mutant PsV used in the infectivity assays. (***P<0.001 as determined by a two-tailed, unpaired t-test between WT and mutant PsV group).
Figure 7.
shRNA knockdown of A2t reduces internalization of HPV16 L1L2 VLP and infectivity of HPV16 PsV.
(A) HPV16 L1 VLP were fluorescently labeled with CFDA-SE, and the percent of infected cells was measured as the percent that were CFDA-SE positive after exposure to VLP for 1 hour as measured by FACS. To control for free label false positives, cells were treated with VLP pre-incubated with a neutralizing anti-L1 antibody (H16.V5). An identical experiment was performed using HPV16 L1L2 VLP. Both histograms are representative examples of two experiments done in triplicate. (B) HeLa cells were left untreated or transduced with a doxycycline inducible pTRIPZ Tet-On lentiviral vector containing an shRNA against ANXA2. Single cell clones treated with or without doxycycline were incubated with labeled HPV16 VLP for 3 hours at 37° and assessed by FACS. The mean percentage of uptake normalized to the wild type group ± SEM of three independent experiments is presented. (C) Protein was collected from cell populations used in the internalization assay for analysis of ANXA2 and S100A10 via Western blot. GAPDH served as a loading control. (D) mRNA was collected from cell populations in the uptake assay for quantitative RT-PCR analysis of ANXA2 and S100A10 expression. The mRNA expression levels were normalized to GAPDH and the graph is a representative example of an experiment performed in triplicate ± SD. (E) Wildtype HeLa cells or HeLa cells stably transduced with a doxycycline inducible lentiviral vector containing shRNA against ANXA2 or control non-target lentiviral vector were infected with GFP plasmid containing HPV16 pseudovirus. Infection was scored 48 h later by enumeration of GFP-positive cells by flow cytometry. The mean percentage of HPV16 PsV infected cells (GFP-positive) normalized to the no doxycycline treated groups ± SD are presented. (*P<0.05 and **P<0.01 as determined by a two-tailed, unpaired t-test, as compared to the no doxycycline-treated groups). Figure is representative of two independent experiments. (F) Protein was collected from cell populations used in the infection assay for analysis of ANXA2 and S100A10 via Western blot. GAPDH served as a loading control.