Epidermal growth factor receptor-dependent stimulation of differentiation by human papillomavirus type 16 E5

Abstract

Human papillomaviruses (HPVs) infect keratinocytes of stratified squamous epithelia, and persistent infection with high-risk HPV types, such as HPV16, may lead to the development of malignancies. HPV evades host immunity in part by linking its gene expression to the host differentiation program, and therefore relies on differentiation to complete its life cycle. Based on previous reports indicating that the HPV16 protein E5 is important in the late stages of the differentiation-dependent life cycle, we found that organotypic cultures harboring HPV16 genomes lacking E5 showed reduced markers of terminal differentiation compared to wild type HPV16-containing cultures. We found that epidermal growth factor receptor (EGFR) levels and activation were increased in an E5-depdendent manner in these tissues, and that EGFR promoted terminal differentiation and expression of the HPV16 L1 gene. These findings suggest a function for E5 in preserving the ability of HPV16 containing keratinocytes to differentiate, thus facilitating the production of new virus progeny.

Keywords: Differentiation; E5; Epidermal growth factor receptor; Papillomaviruses.

Figure 1.

HPV16 increases total protein levels of EGFR independently of E5 in monolayer culture. Cell lines were grown to confluency on cell culture plates, lysed, and analyzed by Western Blotting to detect total levels of EGFR. Band intensities were quantified and normalized to GAPDH (right). N=3.

Figure 2.

HPV16 increases surface localization of EGFR independently of E5. Cells were plated on glass coverslips in 24-well plates, fixed after 24h, then stained with WGA and anti-EGFR antibody. The percentage of total EGFR colocalized with the plasma membrane was calculated using IMARIS and normalized by experiment. HPV16-containing cells were set to 1. Quantifications represent data from at least 15 images from at least 3 experiments.

Figure 3.

HPV16 E5 supports transcription of late viral genes. (a) HPV16 cells were grown in either monolayer culture (mono) or in methylcellulose (MC). Using RT-qPCR, higher transcript levels of E5 were found in cells grown in methylcellulose than those grown in monolayer culture. N=3. (b) HPV16 and E5 Stop rafts were evaluated by RT-qPCR to detect transcript levels of E6–E7, E1Ê4, E5, and L1. N=9.

Figure 4.

HPV16 E5 promotes expression of differentiation markers. Rafts containing HFK, HPV16, and E5 Stop cells were (a) analyzed by RT-qPCR for transcript levels of filaggrin (FLG) and keratin 10 (K10) and (b) stained using anti-filaggrin antibody to detect protein expression. H&E sections from the same rafts are shown below. (c) Staining intensity was quantified using Image J. HFK n=3, HPV16/E5 Stop, n=6.

Figure 5.

HPV16 E5 suppresses proliferation and promotes EGFR activation in differentiated cells. (a) Rafts containing HFK, HPV16, or E5 Stop cells were stained for PCNA and the percent PCNA+ cells were calculated. Basal cells were classified as those touching the basement membrane (dotted line). Suprabasal cells were classified as all cells above the basal layer. HFK n=4, HPV16/E5 Stop, n=6–7. Rafts containing HFK, HPV16, and E5 Stop cells were stained for (b) EGFR and (c) pEGFR; and staining intensities quantified using Image J. Arrows indicate cells positive for pEGFR staining. EGFR: HFK, HPV16 n=13, E5 Stop, n=7; p-EGFR: HFK, HPV16 n=16, E5 Stop n=14.

Figure 6.

EGFR stimulation increases filaggrin staining in cells containing HPV16. (a) Rafts containing HPV16 cells were subjected to treatment with EGF (an EGFR ligand, 100 ng/ml) from day 6–10, harvested, and stained using an anti-filaggrin antibody. (a) Thickness of the corneum was measured and the thickness of the corneum as compared to the total thickness of the raft was quantified (b). Rafts were stained with antibodies against filaggrin (a) and the staining intensity was quantified (c). (d) Levels of L1-containing transcripts were measured by RT-qPCR (n=3).

Figure 7.

Hypothesized functions of E5 the viral life cycle. In the basal layer, low levels of E5 (blue) contribute to suppression of interferon signaling, supporting stable viral genome maintenance[99]. As the viral late promoter is activated in cells committed to differentiation, levels of E5 rise and stimulate EGFR, which enhances cellular differentiation, perhaps through the PKC pathway. Consequently, L1 (red) levels are increased, leading to efficient virion synthesis.