Essential Roles for Soluble Virion-Associated Heparan Sulfonated Proteoglycans and Growth Factors in Human Papillomavirus Infections
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.