Usage of heparan sulfate, integrins, and FAK in HPV16 infection

Abstract

Human papillomavirus type 16 (HPV16) is the major causative agent of cervical cancer. Studies regarding the early binding and signaling molecules that play a significant role in infection are still lacking. The current study analyzes the role of heparan sulfate, integrins, and the signaling molecule FAK in HPV16 infection of human adult keratinocytes cell line (HaCaTs). Our data demonstrate that infection requires the binding of viral particles to heparan sulfate followed by activation of focal adhesion kinase through an integrin. Infections were reduced in the presence of the FAK inhibitor, TAE226. TAE226 was observed to inhibit viral entry to the early endosome a known infectious route. These findings suggest that FAK can serve as a novel target for antiviral therapy.

Fig 1. Heparan sulfate proteoglycans role in HPV16 binding and infection

(A) HaCaT cells were incubated for 2 hours only with purified ³[H] thymidine labeled HPV16 reporter virions or with labeled virions previously incubated with 50ug/ml soluble heparin. Bound radioactivity was precipitated with TCA and counted by scintillation counter. First bar shows background cellular cpm in the absence of virus, second bar represents virions cpm in the absence of soluble heparin, and third bar represents bound virions in the presence of soluble heparin. (B) Binding of ³[H] thymidine labeled HPV16 to HaCaT cells pre-treated with heparinase I. Radioactivity was precipitated with TCA and counted on a scintillation counter. Counts: Bar 1 buffer background; bar 2 control untreated HaCaTs; bar 3 total bound virions; bars 4-7 bound virions cpm in heparinase I treated cells with 1U, 2U, 4U and 10U respectively. Each sample was plated with 1×10⁵ cells. Experiments were done in triplicate, and error bars represent sample means +/- SD. Significance level was set at *p<0.05. (C) Cells were infected with non-radioactive HPV16 GFP reporter PsVs for 48 hours. Samples: Bar 1 uninfected HaCaTs; bar 2, virus infection; bar 3 HaCaT infected with HPV16 GFP reporter PsVs pre-incubated with 50ug/ml soluble heparin; bars 4-6 represent infection of HaCaT cells pre-treated with increasing units of heparinase I prior to addition of HPV16 GFP reporter PsVs; bar 7 represents soluble heparin treated cells prior to addition of HPV16 GFP reporter PsVs. Each condition was performed in triplicate and error bars represent sample means +/- SD. Significance level was set at *p < 0.05.

Fig 2. Integrin Profile of HaCaTs, KH-SV α6 +/+ and BOUA-SV α6 -/- cells

Integrin profiling of HaCaTs (A1), KH-SV α6 +/+ (B1) and BOUA-SV α6 -/- cells (C1) was performed using the Alpha/Beta Integrin mediated cell adhesion array combo kit. Baseline controls (cells alone) were used for standardizing and measuring the increase in relative fluorescence units for each cell type. Flow cytometry analysis of α6 expression on HaCaTs (A2), KH-SV α6 +/+ (B2) and BOUA-SV α6 -/- cells (C2).

Fig 3. HPV16 infection in human keratinocyte lines

HPV16 GFP reporter PsVs were added to HaCaTs, KH-SV α6 +/+, and BOUA-SV α6 -/- cells. Percent infection was determined via flow cytometry analysis. Infection of HaCaT cells bar 1, KH-SV α6 +/+ cells bar 2, BOUA-SV α6 -/- cells bar 3. Error bars show the standard deviation of three experiments in which 10,000 cells were analyzed for GFP expression to obtain the percent of infected cells. Significance level was set at *p < 0.05.

Fig 4. HPV16 induces FAK-Tyr³⁹⁷ phosphorylation in HaCaT cells

(A) Cells were incubated with MOI 0.0015 HPV16 PsV for 5, 10, 15 and 30 minutes (Lanes 4-7 respectively) and MOI 0.15 HPV16 PsV for 10 and 30 minutes (Lanes 2 and 3 respectively); uninfected cells are shown in lane 1. Control LPA (20 ng) induced FAK-Tyr³⁹⁷ is shown in lane 8. Equal amounts of NP-40 lysates resolved by 8% SDS- PAGE, were blotted for phosphorylated FAK-Tyr³⁹⁷ (pFAK blot), total FAK (tFAK blot), and actin. (B) Graphical representation of temporal pFAK-Tyr³⁹⁷ induction evaluated by Odyssey densitometry of western blot shown in C. Uninfected cells assigned a value of 1 for comparison lane 1. (C) HaCaT cells were infected with HPV16 (MOI 0.15) at the indicated times post-infection. Cell lysates were resolved on an SDS-PAGE and incubated with pFAK-Tyr³⁹⁷, total FAK (tFAK) and Actin antibodies. Protein levels were measured using the Odyssey densitometer. (D) Lysates from uninfected HaCaT cells (lane 1), mock-infected HaCaTs with 8fwb mock viral fraction (lane 2), or HPV16 PsVs (lane 3) were resolved on an SDS-PAGE and incubated with phosphorylated FAK-Tyr³⁹⁷ (pFAK), total FAK (tFAK) and Actin. Uninfected cells are assigned a value of 1 for comparison with values of pFAK fold increase. Protein levels were measured using the Odyssey densitometer. Significance level was set at *p< 0.05.

Fig 5. Phosphorylation of FAK is blocked in HaCaT using TAE226

(A) Cytotoxicity of TAE226 was not observed on HaCaTs up to 2 uM concentration. Increasing concentrations of 0um, 0.1uM, 1.0uM, 2.0uM, 5.0uM and 10uM were tested. Cell viability was assessed after 48 hours as a function of the level of ATP. Doses of 5.0uM and 10.0uM were cytotoxic to the cells (Significance level was set at *p<0.05). (B) HaCaT cells were infected with HPV16 virus (MOI 0.15) for: 5’, 10’ 15’ and 30 minutes in the absence of TAE226 (lanes 2-5), in the presence of TAE226 at 0.1, 1.0 and 2.0 uM of (lanes 6-8); or incubated with 20% FBS for 30 minutes (lane 9). Equal amounts cell lysates were run on an 8% SDS-PAGE. Levels of FAK phosphorylation (pFAK-Tyr³⁹⁷), total FAK (tFAK), and actin blots (actin) were determined using corresponding antibodies. Values were normalized based on tFAK values which were normalized to actin control. Uninfected cells are assigned a value of 1 for comparison with values of pFAK fold increase. The percent inhibition of FAK phosphorylation (Bottom of lanes 6-8) was determined by dividing the decrease in FAK phosphorylation in inhibitor treated cells post 30 minutes infection to FAK induction post 30 minutes infection in the absence of inhibition. (C-N) Visualization of FAK phosphorylation by immunofluorescence confocal microscopy. In panels E, G, I, K and M cells were infected with HPV16 MOI 0.15 for 0, 10, 30, 60 minutes and 4 hours respectively. In panels F, H, J, L, and N cells pre-incubated with 2.0uM of TAE226 were infected for 0, 10, 30, 60 minutes and 4 hours respectively. Immunofluorescence was performed with anti-pFAK-Tyr³⁹⁷; nuclei are visualized with Topro-3 (blue). Confocal images were obtained using a 60X oil objective. Control uninfected cells stained for pFAK and nuclei are shown (C, D).

Fig 6. FAK phosphorylation influences HPV16 infection

(A) TAE226 inhibits HPV16 PsV infection in a dose dependent matter. HaCaT cells were infected with HPV16 PsV carrying the GFP cDNA in the presence of 0uM, 0.1uM, 0.5uM, 1.0uM and 2.0uM TAE226. Infection was measured by FACS analysis for GFP positive cells. Error bar represents the standard deviations of the mean from three separate experiment, *p<0.02, **p<0.015 (Student's t test). (B) HPV16 PsV infection level was decreased with TAE226, but not by the Src Family kinase inhibitor PP2. The inactive PP2 analogue PP3 was used as control. Cells were infected with HPV16 GFP reporter PsVs, and percent infection was analyzed by flow cytometry. Error bar represents the standard deviations of the mean from three separate experiments (in triplicate). Significance was determined by Student's t test and p-value was set at *p< 0.05. (C) FAK expressing (DU17) and FAK knockout DU3 MEFs cells infected in the presence or absence of the FAK kinase inhibitor TAE226 (2uM). Cells were infected with HPV16 GFP reporter PsVs and percent infection was measured by flow cytometry. Error bar represents the standard deviations of the mean from three separate experiments (in triplicate). Significance was determined by Student's t test and p-value was set at *p<0.05. (D) FAK expressing MEFs (DU17) (uninfected control in lane 1) were infected with HPV16 PsV (lane 2), incubated with 20% FBS (lane 3), or infected with HPV16 in the presence of an anti-α6 funtional blocking integrin antibody (lane 4). No FAK expression was detected in FAK knockout cells (lanes 5-8). Lanes 1-8, NP-40 lysates were run on an 8% SDS-PAGE and levels of phosphorylated FAK-Tyr³⁹⁷ (Top bands), total FAK (middle band) and actin blots (lower band). Loading values were normalized based on total FAK expression, which was normalized to actin values. Uninfected cells FAK phosphorylation levels are assigned a value of 1 (lane 1) for comparison.

Fig 6. FAK phosphorylation influences HPV16 infection

(A) TAE226 inhibits HPV16 PsV infection in a dose dependent matter. HaCaT cells were infected with HPV16 PsV carrying the GFP cDNA in the presence of 0uM, 0.1uM, 0.5uM, 1.0uM and 2.0uM TAE226. Infection was measured by FACS analysis for GFP positive cells. Error bar represents the standard deviations of the mean from three separate experiment, *p<0.02, **p<0.015 (Student's t test). (B) HPV16 PsV infection level was decreased with TAE226, but not by the Src Family kinase inhibitor PP2. The inactive PP2 analogue PP3 was used as control. Cells were infected with HPV16 GFP reporter PsVs, and percent infection was analyzed by flow cytometry. Error bar represents the standard deviations of the mean from three separate experiments (in triplicate). Significance was determined by Student's t test and p-value was set at *p< 0.05. (C) FAK expressing (DU17) and FAK knockout DU3 MEFs cells infected in the presence or absence of the FAK kinase inhibitor TAE226 (2uM). Cells were infected with HPV16 GFP reporter PsVs and percent infection was measured by flow cytometry. Error bar represents the standard deviations of the mean from three separate experiments (in triplicate). Significance was determined by Student's t test and p-value was set at *p<0.05. (D) FAK expressing MEFs (DU17) (uninfected control in lane 1) were infected with HPV16 PsV (lane 2), incubated with 20% FBS (lane 3), or infected with HPV16 in the presence of an anti-α6 funtional blocking integrin antibody (lane 4). No FAK expression was detected in FAK knockout cells (lanes 5-8). Lanes 1-8, NP-40 lysates were run on an 8% SDS-PAGE and levels of phosphorylated FAK-Tyr³⁹⁷ (Top bands), total FAK (middle band) and actin blots (lower band). Loading values were normalized based on total FAK expression, which was normalized to actin values. Uninfected cells FAK phosphorylation levels are assigned a value of 1 (lane 1) for comparison.

Fig 7. TAE226 inhibits gap closure in HaCaT wound closure model

(A-I) HaCaT monolayers grown to near confluence were incubated in the presence or absence of 2.0uM TAE226 +/- HPV16 PsV at the time of wounding. DMSO was used as a vehicle control. Images were obtained by light microscopy with a 4X objective lens. (J) Percentage gap closure represented as a bar graph (black bars represent gap closure at 24 hours, grey bars represent gap closure at 36 hours). Error bars represent the standard deviations of the percent gap closure measured within a marked area of at least 4 data points. Gap closure was calculated by the percentage change in area at greater than four data points and calculated by the formula: (area of scratch wound at time zero) – (area of scratch wound after 24 or 36 hours)/100. Significance was determined by Student's t test and p-value set at p<0.05, * significance at 24 hours, **significance at 36 hours.

Fig 8. TAE226 inhibits filopodia formation but not virus binding

(A and B) Mock infected HaCaT cells in the absence and presence of 2.0uM TAE226 respectively. (C and E) HaCaT cells infected with virus for 10 and 30 minutes in the absence of inhibitor, respectively. (D and F) HaCaT cells pre-incubated with 2.0uM TAE226 infected for 10 and 30 minutes respectively. Actin is viewed using phalloidin-488 (green); HPV PsVs are stained with anti-HPV16.V5 antibody; actin overlapping with HPV PsVs is observed (yellow). Filopodia extensions (white arrows) and virus colocalizing with the phalloidin stained actin (yellow arrows) are shown. Nuclei are visualized with TOPRO-3 (blue).

Fig 9. TAE226 prevents HPV16 trafficking to the early endosome

Control uninfected HaCaT cells without (A) or with TAE226 (B). HPV16 binding to HaCaT cells (C) is not affected by TAE226 (D). PsVs internalization in the absence (E, G, I, K) or presence (F, H, J, L) of TAE226 at the corresponding time. Cells were fixed, permeabilized and stained for the early endosome marker EEA1 (green), and HPV16 PsV with anti-HPV16.V5 antibody. Nuclei are visualized with TOPRO-3 (blue). HPV16 PsVs (red arrows) did not colocalize with EEA1 (green arrows) at 10, 30, 60 minutes or 4 hours in TAE226 treated cells. Confocal images were obtained using a 60X oil objective.

Fig 10. siRNA Knockdown of α6 integrin expression decrease HPV16 infection in KH-SV cells

(A) IgG isotype staining of control uninfected KH-SV cells; (B) Flow cytometry analysis of control uninfected KH-SV cells; (C-F) KH-SV cells were double transfected with α6 siRNA or control siRNA, after 72 hours cells were infected with HPV16 PsVs and level of infection measured by flow cytometry. Panels: (C) α6 integrin expression level in control cells; (D) level of HPV16 infection in control cells; (E) α6 integrin expression level in α6 siRNA expressing cells; (F) level of HPV16 infection in α6 siRNA expressing cells; (G) real time RT-PCR analysis for the expression of GFP mRNA (HPV16 PsV encapsidate GFP cDNA plasmid). Graph shows real-time GFP RT-PCR of infected and uninfected cells after 48 hours. HPV16 GFP expression in the absence of siRNA treatment was considered as 100%. No signal was detected in α6 siRNA transfected cells in the absence of HPV16 PsV infection (Last bar). Each condition was performed in triplicates.