Annexin A2 and S100A10 regulate human papillomavirus type 16 entry and intracellular trafficking in human keratinocytes

Abstract

Human papillomaviruses (HPVs) cause benign and malignant tumors of the mucosal and cutaneous epithelium. The initial events regulating HPV infection impact the establishment of viral persistence, which is requisite for malignant progression of HPV-infected lesions. However, the precise mechanisms involved in HPV entry into host cells, including the cellular factors regulating virus uptake, are not clearly defined. We show that HPV16 exposure to human keratinocytes initiates epidermal growth factor receptor (EGFR)-dependent Src protein kinase activation that results in phosphorylation and extracellular translocation of annexin A2 (AnxA2). HPV16 particles interact with AnxA2 in association with S100A10 as a heterotetramer at the cell surface in a Ca(2+)-dependent manner, and the interaction appears to involve heparan-sulfonated proteoglycans. We show multiple lines of evidence that this interaction promotes virus uptake into host cells. An antibody to AnxA2 prevents HPV16 internalization, whereas an antibody to S100A10 blocks infection at a late endosomal/lysosomal site. These results suggest that AnxA2 and S100A10 have separate roles during HPV16 binding, entry, and trafficking. Our data additionally imply that AnxA2 and S100A10 may be involved in regulating the intracellular trafficking of virus particles prior to nuclear delivery of the viral genome.

Fig 1

HPV16 exposure induces Src kinase-dependent AnxA2 phosphorylation at Tyr23. HaCaT cells serum starved for 4 to 6 h were mock (M) or HPV16 (H16) exposed (100 vge/cell) for the indicated periods of time (A, E, and F) before harvest for immunoprecipitation (IP). (A) IP was performed with rabbit anti-Src Ab followed by immunoblotting with mouse pTyr Ab (upper blot). IgG was detected as a loading control (lower blot). (B) Cells were treated with 100 nm PD168393 prior to and during virus exposure (30 min) or with 5 ng/ml EGF (30 min postaddition) as a positive control. IP was performed with anti-Src Ab followed by immunoblotting for anti-pTyr Ab. IgG was detected as a loading control (lower blot). (C) Levels of pTyr Src normalized to IgG loading controls for various treatments based on data in panel B from three independent IP experiments; bars represent standard deviations (*, P = 0.0297 as determined by Student's t test). Ø, no treatment. (D) HaCaT cells were mock or HPV16 infected (100 vge/cell) in the presence of different concentrations of PP2. Infectivity was determined 24 h later, and data were graphed from three independent experiments; bars represent standard deviations (*, P = 0.0268 by Student's t test). (E) IP was performed with rabbit anti-AnxA2 Ab followed by immunoblotting with mouse anti-pTyr23 AnxA2 Ab (upper blot). Mouse anti-AnxA2 Ab was used to detect total protein as a sample loading control (lower blot). (F) Levels of pTyr 23 AnxA2 normalized to total AnxA2 protein for each time based on data from three independent IP experiments from panel E; bars represent standard deviations (*, P = 0.0038 by Student's t test). (G) Cells were treated with 1 μM PP2 inhibitor, 10 μg/ml N terminus anti-AnxA2 Ab, or 100 nM PD168393 (PD) prior to and during virus exposure for 30 min. IP was performed with rabbit anti-AnxA2 Ab followed by immunoblotting with mouse anti-pTyr23 AnxA2 Ab (upper blot). Mouse anti-AnxA2 Ab was used to detect total protein (lower blot). (H) The fractions of cells in G₁ (1n), S (intermediate), and G₂/M (2n) phases were expressed as percentages of the total cells counted. HaCaT cells were untreated (M) or treated with anti-AnxA2 Ab (15 μg/ml), anti-p11 Ab (15 μg/ml), control Ab (15 μg/ml), EGTA (0.1, 0.5, or 1.0 mM), or PP2 (0.1, 0.5, 1.0, or 5.0 mM) for 24 h.

Fig 2

AnxA2 translocates to the cell surface in HPV16-exposed cells. (A) Immunoblot of AnxA2 levels in the cytoplasmic and membrane fractions isolated from mock- and HPV16-exposed (100 vge/cell) HaCaT cells at 30 min p.i. α-Tubulin was detected as a cytoplasmic protein marker and Na/K ATPase α 1 as a membrane protein indicator. (B) The AnxA2 bands from immunoblots in panel A were analyzed with ImageJ, and the value of the raw integrated density (Raw IntDen) was plotted from two independent experiments (*, P = 0.0107 by Student's t test). (C) Confocal microscopy images of representative mock- and HPV16-exposed (100 vge/cell) cells at 30 min p.i. Nonpermeabilized cells were immunostained for AnxA2 (green) and HPV16 (red; anti-HPV16 VLP Ab). Arrows indicate yellow colocalization of red virus particles with green AnxA2. Bars = 10 μm. (D) Acquired confocal microscopy z-stack images were analyzed for FITC channel intensity representing AnxA2 staining at the plasma membrane. Cells were randomly selected for analysis based on the DAPI staining (n = 51 cells per group quantified). Error bars represent standard errors of the means (***, P = 0.0009 by Student's t test).

Fig 3

HPV16 associates with AnxA2 at the cellular membranes in the presence of calcium. (A, B) HPV16 colocalizes with AnxA2 and p11 at the plasma membrane of nonpermeabilized cells. HaCaT cells were either mock or HPV16 exposed (5,000 vge/cell) at 37°C for 30 min after the temperature shift. Images are two-dimensional (2D) views, with cell cross sections shown to the right of each frame. Bars = 10 μm. Yellow indicates overlap of green and red signals. (A) Mock- and HPV16-exposed cells were immunostained for AnxA2 (green) and HPV16 (red, anti-HPV16 VLP Ab). (B) HPV16-exposed cells were immunostained for S100A10/p11 (green) and HPV16 (red, anti-HPV16 VLP Ab). (C to E) HaCaT cells were mock or HPV16 exposed (100 vge/cell) at 37°C for 1 h after the temperature shift prior to cell lysis and IP. Immunoblotting was performed to detect HPV16 L1 and AnxA2. (C) Cell lysates were used for IP with anti-HPV16 VLP Ab followed by immunoblotting for AnxA2 in the presence of 5 mM CaCl₂. (D) Cells were either directly lysed in the absence or presence of calcium (−Ca2+ and +Ca2+) or cross-linked with DTSSP or formaldehyde (FA) in the absence of calcium prior to lysis. Cell lysates were used for IP with anti-AnxA2 Ab, followed by analysis of copurifying HPV16 L1. (E) Cell lysates were fractionated by differential centrifugation to separate membranes and cytosol. Each fraction was subjected to IP with anti-AnxA2 Ab. Blots were probed with anti-HPV 16 L1 Ab. Asterisks, nonspecific bands (D and E). Input lysates (prior to IP) and supernatants that remained unbound to the Ab–protein A-Sepharose beads (unb) are indicated. (F and G) HaCaT cells were pretreated with the indicated concentrations of EGTA for 1 h at 37°C, then were either mock or HPV16 exposed in the presence of EGTA for 1 h at 4°C to allow for cell surface binding. (F) Media were analyzed for unbound virus particles (top) and AnxA2 released from cell surface (bottom) upon EGTA treatment. (G) Infections were analyzed 24 h later; additionally, HPV16 PsVs were EGTA treated for 1 h at 37°C, repurified, and used to infect HaCaT cells (white bar). Data were graphed from three independent experiments. Error bars represent standard deviations (*, P = 0.0173 by Student's t test). (H) Morphology of the untreated (left) and EGTA-treated HPV16 (right) visualized by transmission electron microscopy. Bars = 100 nm.

Fig 4

AnxA2 regulates HPV16 infection. (A) HaCaT cells were pretreated with increasing concentrations of antibodies to the AnxA2 N terminus (amino acids 1 to 50), p11, or nonspecific control antibodies C_a (affinity-purified rabbit anti-mouse IgG) and C_b (mouse anti-PCNA antibody), followed by HPV16 PsV exposure (100 vge/cell). Virus was allowed to attach at 4°C in the presence of antibodies. Unbound viruses were removed, and cells were refed with fresh medium containing the corresponding antibody. Infections were continued at 37°C for 24 h. Alternatively HPV16 infection in the presence of 0.01% NaN₃ was monitored. Graphs were generated based on three independent experiments. Error bars represent standard deviations (***, P < 0.0001 as determined by Student's t test). (B) HaCaT cells were transfected with control or AnxA2-specific siRNAs for 6 days; AnxA2 protein knockdown was determined by immunoblotting, with tubulin detected as a control. The left graph represents quantified AnxA2 levels normalized to tubulin from immunoblots from three separate transfections. Error bars represent the average AnxA2 levels normalized to α-tubulin in control and AnxA2 siRNA-transfected cells from the three independent experiments (*, P = 0.029 by Student's t test). The middle graph shows data for siRNA-transfected cells infected with HPV16 PsVs (100 vge/cell) 6 days posttransfection. Error bars represent the averages of luciferase readings from the three independent infections (***, P = 0.0001 by Student's t test). The right graph represents cell cycle analyses of HaCaT cells transfected with control and AnxA2 siRNA (100 nM) for a period of 5 days. The fractions of cells in G₁ (1n), S (intermediate), and G₂/M (2n) phases were expressed as percentages of the total cells counted. (C to J) HaCaT cells were exposed to HPV16 PsVs (5,000 vge/cell) in the absence of Ab (C, I) or presence of control antibody (C_a) (D), rabbit anti-AnxA2 N terminus Ab (E), or anti-p11 Ab (F, G, H, J). All antibodies were used at a 10-μg/ml concentration. Confocal microscopy was performed 24 h p.i. by staining for HPV16 (red), plasma membrane (WGA, green), and nuclei (DAPI, blue) (C to F), for HPV16 (red) and EEA1 (green) (G), for LAMP1 (green) (H, I), or for AnxA2 (green) (J). Quantification of HPV16 and plasma membrane colocalization is depicted at each image (C to F). Numbers represent the percentages of the total HPV16 signal that colocalizes with plasma membrane signal as an average of 6 cells per group. Side views of images were used for colocalization analysis. Yellow indicates colocalization of green and red signal. Bars = 10 μm.

Fig 5

Exogenous expression of AnxA2 increases the susceptibility of the AnxA2-deficient C4-2 cell line to HPV16 infection. (A) C4-2 cells were transfected with a p11 expression plasmid, an AnxA2 expression plasmid, or an EGFP expression plasmid as a control (C) for 24 h prior to infection. Cells were mock or HPV16 infected (100 vge/cell) for 24 h, at which point they were analyzed for pseudovirus infection. Infection of C4-2 cells is expressed as % of infection relative to HPV16 infection of untreated HaCaT cells (set as 100%). Graphs were generated based on four independent experiments. Error bars represent standard deviations (*, P = 0.0442 by Student's t test). (B) Representative immunoblot with specific rabbit anti-AnxA2 Ab of untransfected C4-2 cells, control- and p11 vector-transfected C4-2 cells, and AnxA2 plasmid-transfected C4-2 cells. (C to J) 2D confocal microscopy images and side views of C4-2 cells. C4-2 cells untransfected or transfected with AnxA2 expression plasmid for 24 h were mock or HPV16 exposed for 6 h postbinding. (C) Untransfected C4-2 cells stained for AnxA2 in permeabilized cells. (D, E) AnxA2 plasmid-transfected mock-exposed C4-2 cells analyzed for intracellular AnxA2 expression (green) and cell surface AnxA2 presence in permeabilized cells (D) and AnxA2 cell surface expression in transfected, HPV16 exposed, nonpermeabilized cells (E). (F to H) HPV16 PsV localization (red) at 6 h p.i. in AnxA2-negative C4-2 cells (F) and transfected-AnxA2-expressing C4-2 cells (green; G and H). (I, J) C4-2 cells were WGA stained (green) prior to permeabilization. HPV16 PsV localization (red) in AnxA2-negative C4-2 cells (I) and AnxA2-transfected C4-2 cells (J). Cells in panels C, D, and F to J were permeabilized with 0.1% Triton X-100. Bars = 10 μm. Yellow indicates colocalization of green and red signal. Blue arrows, cell surface-bound HPV16; white arrows, intracellularly located HPV16.

Fig 6

AnxA2 associates with HPV16 and EGFR at the cell surface of HPV16-exposed HaCaT cells. (A, B) 2D confocal microscopy images and side views of nonpermeabilized cells. Bars = 10 μm. (A) Immunostaining of EGFR (green) and AnxA2 (red) at the cell surface 30 min after HPV16 PsV exposure. (B) Immunostaining of HPV16 (red, anti-HPV16 VLP Ab) with EGFR (green) at the cell surface 30 min after infection with HPV16 (5,000 vge/cell). (C) Co-IP of EGFR and HPV16 with AnxA2. Mouse anti-AnxA2 Ab was used to IP AnxA2 from cell lysates of mock- and HPV16-infected HaCaT cells at 30 min p.i. Immunoblots were probed with rabbit anti-AnxA2, rabbit anti-HPV16 VLP, and rabbit anti-EGFR antibody. EGFR was detected in untreated cells and cells cross-linked with DTSSP. (D) Co-IP of HSPG and AnxA2 at 30 min p.i. in mock- and HPV16-exposed HaCaT cells. (E, F) Co-IP of HPV16 with AnxA2. Rabbit anti-AnxA2 was used to IP AnxA2 from HPV16-infected cells at 30 min p.i. Immunoblots were probed with mouse anti-HPV16 L1. IgG was used as a loading control. (E) Cells were untreated or treated with 5 U/ml of heparinase I (Hep. I) 30 min prior to virus exposure. (F) Heparin (500 μM, low molecular weight) was added to culture medium 30 min prior to and during HPV16 binding (Pre) or after virus binding (Post).

Fig 7

Proposed model for AnxA2 function in HPV16 infection. (A) HSPG- and EGF (also heparin-binding EGF- or amphiregulin)-decorated HPV16 binds EGFR and activates MAPK and PI3K signaling cascades (17, 69). (B, C) Src protein kinase is subsequently activated, which in turn phosphorylates AnxA2 at Tyr23. (D) Association of AnxA2 with p11 and Tyr23 phosphorylation induces the translocation of AnxA2 to the extracellular leaflet of plasma membrane (31) (shown as a bent arrow as the translocation mechanism is not entirely clear). (E) Phosphorylated A2t adopts a conformation wherein the membrane interaction is independent of Ca²⁺. (F) HPV16 recognizes AnxA2 and binds to it in a Ca²⁺-dependent manner. This interaction may be directed, in part, through virus-associated HSPGs, as the common heparin-binding site is situated at the convex face of AnxA2's domain IV (74). AnxA2 associates with cholesterol-containing lipid rafts to regulate endocytic pit formation and initiate endocytosis of AnxA2-bound ligands in an EGFR-dependent or independent mode (94).