Transforming Properties of Beta-3 Human Papillomavirus E6 and E7 Proteins

Abstract

The beta human papillomaviruses (HPVs) are subdivided into 5 species (beta-1 to beta-5), and they were first identified in the skin. However, the beta-3 species appears to be more highly represented in the mucosal epithelia than in the skin. Functional studies have also highlighted that beta-3 HPV49 shares some functional similarities with mucosal high-risk (HR) HPV16. Here, we describe the characterization of the in vitro transforming properties of the entire beta-3 species, which includes three additional HPV types: HPV75, HPV76, and HPV115. HPV49, HPV75, and HPV76 E6 and E7 (E6/E7), but not HPV115 E6 and E7, efficiently inactivate the p53 and pRb pathways and immortalize or extend the life span of human foreskin keratinocytes (HFKs). As observed for HR HPV16, cell cycle deregulation mediated by beta-3 HPV E6/E7 expression leads to p16^INK4a accumulation, whereas no p16^INK4a was detected in beta-2 HPV38 E6/E7 HFKs. As shown for HPV49 E6, HPV75 and HPV76 E6s degrade p53 by an E6AP/proteasome-mediated mechanism. Comparative analysis of cellular gene expression patterns of HFKs containing E6 and E7 from HR HPV16, beta-3 HPV types, and beta-2 HPV38 further highlights the functional similarities of HR HPV16 and beta-3 HPV49, HPV75, and HPV76. The expression profiles of these four HPV HFKs show some similarities and diverge substantially from those of beta-3 HPV115 E6/E7 and beta-2 HPV38 E6/E7 HFKs. In summary, our data show that beta-3 HPV types share some mechanisms with HR HPV types and pave the way for additional studies aiming to evaluate their potential role in human pathologies.IMPORTANCE Human papillomaviruses are currently classified in different genera. Mucosal HPVs belonging to the alpha genus have been clearly associated with carcinogenesis of the mucosal epithelium at different sites. Beta HPV types have been classified as cutaneous. Although findings indicate that some beta HPVs from species 1 and 2 play a role, together with UV irradiation, in skin cancer, very little is known about the transforming properties of most of the beta HPVs. This report shows the transforming activity of E6 and E7 from beta-3 HPV types. Moreover, it highlights that beta-3 HPVs share some biological properties more extensively with mucosal high-risk HPV16 than with beta-2 HPV38. This report provides new paradigms for a better understanding of the biology of the different HPV types and their possible association with lesions at mucosal and/or cutaneous epithelia.

Keywords: beta-3 and mucosal HPV types; keratinocyte immortalization; p53 and pRb regulated pathways.

FIG 1

Stable expression of E6/E7 of HPV49 and HPV76 immortalizes primary HFKs. (A) Total RNA was extracted from HFKs transduced with the indicated retroviruses and subjected to retrotranscription. PCR was performed using different cDNAs as templates with HPV E7-specific primers. (B) Growth curve of HFKs (donor 1) expressing E6/E7 from the indicated HPVs. (C) Morphology of HFKs transduced with the indicated recombinant retroviruses at the indicated PDs. The same magnification (×20) was used for all the microphotographs. (D) Total RNA extracted from cells at the same passage was retrotranscribed and used as a template for RT–real-time PCR analysis of hTERT gene expression, normalized to GAPDH. Each result shown in the histogram represents the mean of results from three independent experiments performed in two donors (*, P < 0.05; ***, P < 0.005). (E) Total DNA was extracted from HFKs transduced with the indicated retroviruses and subjected to PCR using specific pLXSN primers recognizing regions immediately before and after the viral genes (top panel). Portions of these PCR products were used for a second PCR performed with HPV16 E7 primers (bottom panel).

FIG 2

Beta-3 E6/E7 alter pRb function in cell cycle control. (A) Protein extracts from HFKs expressing or not expressing E6/E7 of the indicated HPV types were incubated with or without λPP and analyzed by IB using pRb, phosphorylated pRb (Pi-pRB) (Ser795), and β-actin antibodies. (B) Retrotranscribed total RNA was used as a template for real-time PCR with primers specific for cdc2 and CDK2 genes or GAPDH. cdc2 and CDK2 expression levels were normalized to GAPDH levels. Results shown in the histogram are the means of data from 6 independent experiments performed using HFKs from 2 independent donors. (C and D) Protein extracts from HFKs or transduced HFKs expressing E6/E7 of the different beta-3 types were analyzed by IB using the indicated antibodies (Cdc2, CycA, and β-actin [C] and p16^INK4a and β-actin [D, left panel]). Band intensities were quantified, normalized to β-actin levels, and then calibrated against the HFKs (D, right panel). The error bars represent the standard deviations of results from 5 independent experiments performed in HFKs from 2 independent donors (*, P < 0.05; **, P < 0.01; ***, P < 0.005). (E) PHFs were transduced with empty retrovirus (pLXSN) or with retroviruses expressing E6 and E7 of the 4 beta-3 HPV types. Cell were grown in DMEM with 10% or 0.5% fetal calf serum for 48 h, and the cell cycle profile was analyzed by flow cytometry. The levels of inhibition of the S phase by serum starvation are given at the bottom of the figure.

FIG 3

Beta-3 E6/E7 alter p53 function by a mechanism similar to that seen with HR HPV16. (A) HFKs transduced with E6/E7 from beta-3 types with HPV16 E6/E7 or nontransduced HFKs were treated for 8 h with the DNA-damaging agent doxorubicin (DOXO) used at the final concentration of 2 μg/ml or with DMSO as a control and were collected. Levels of p53 and β-actin were determined by IB using specific antibodies. Band intensities were quantified, normalized to β-actin levels, and then calibrated against the corresponding protein band levels obtained in the scramble samples (right). Results shown are the means of data from 3 independent experiments performed in primary HFKs from 2 donors (*, P < 0.05; **, P < 0.01). (B) Total RNA extracted from cells treated as described for panel A was retrotranscribed and used as a template for RT real-time PCR analysis of PUMA and p21 gene expression, normalized to GAPDH levels. Results shown in the histogram are the means of data from 3 independent experiments performed in one donor. (C) Nontransduced HFKs or HPV16, HPV49, HPV75, and HPV76 E6/E7-transduced HFKs were treated for 4 h with a proteasome inhibitor (MG132). Levels of p53 and β-actin were determined by IB using specific antibodies. Band intensities were quantified, normalized to β-actin levels, and then calibrated against the corresponding protein band levels obtained in the scramble samples (right). Results shown are the means of data from 2 (HFKs) independent experiments performed in primary HFKs from 2 donors (*, P < 0.05; **, P < 0.01). (D) HPV16, HPV49, HPV75, and HPV76 E6/E7-transduced HFKs were transiently transfected with siRNA directed against E6AP (siRNA) or with scramble (Scr). Levels of p53, E6AP, and β-actin were determined by IB using specific antibodies (left). Band intensities were quantified, normalized to β-actin levels, and then calibrated against the corresponding protein band levels obtained in the scramble samples (right). Results shown are the means of data from 2 (HPV16) or 3 independent experiments performed in primary HFKs from 2 donors (*, P < 0.05; **, P < 0.01; ***, P < 0.005). (E) The indicated GST or GST/E6 fusion proteins were incubated with 1.5 mg of HNC136 total protein extract. Levels of MAML1, E6AP, and GST were determined by IB using specific antibodies.

FIG 4

Generation of HPV76 E6 mutants. E6s from different alpha and beta HPV types were aligned. The arrows indicate the positions mutated, and the corresponding amino acid residues mutagenized in HPV76 are indicated on the right. Black arrows indicate amino acids directly involved in p53 binding, and the light blue arrow indicates the amino acid involved in binding the LxxLL motif of E6AP.

FIG 5

Mutation of HPV76 E6 in amino acid residues corresponding to p53 and E6AP binding sites in HPV16 E6 altered its biological activities. (A) Growth curve of primary HFKs expressing E6/E7 with WT E6 or mutated E6. PDs are reported on the vertical axis. (B) Morphology of HFKs transduced with the indicated recombinant retroviruses at PD2. The same magnification (×20) was used for all the microphotographs. (C) NIKs transduced with E7 and with WT or mutated E6, or with the empty vector (pLXSNØ) as a control, were treated with the DNA-damaging agent doxorubicin (Doxo) used for 8 h at the final concentration of 10 μg/ml. Levels of p53 and GAPDH were determined by IB using specific antibodies. (D) (Left) NIKs transduced as described in the Fig. 4D legend were treated with a protein synthesis inhibitor (cycloheximide, at the final concentration of 10 μg/ml) and collected at the indicated time points. Twenty micrograms of total protein extracts from NIKs expressing pLXSNØ, WT, and E39R E6 mutant were run on a Western blot separately from NIKs expressing Y42R, D44A, and F45E E6 mutants (upper and lower panels, respectively). Levels of p53 and GAPDH were determined by IB using specific antibodies. (Right) Graph showing quantification of p53 signal, normalized to GAPDH levels and calibrated against the levels of p53 at 2 h of treatment (set as 100%). Results shown are the means of data from 3 independent experiments. (E) Total RNA was extracted from the indicated NIKs and retrotranscribed. hTERT gene expression was determined by quantitative PCR and normalized to GAPDH. The histogram represents the quantification of the results from three independent experiments. ***, P < 0.005.

FIG 6

Expression of E6/E7 from the immortalizing beta-3 HPVs altered the whole expression profile of HFKs similarly to expression of HPV16 E6/E7. (A) Heat map of the significantly deregulated genes in the HFKs expressing E6/E7 from the indicated HPVs. The colors represent expression levels of the genes: red represents higher expression than that seen in empty vector HFKs, and green represents lower expression. (B) Pathway analysis (Enrichr software) of the significantly deregulated genes in cells expressing E6/E7 of the indicated HPVs. The length of the bar and the brightness of the color represent the significance of the specific pathway (combined score ranges were as follows: for HPV16, 8.25 to 72.34; for HPV38, 1.27 to 1.76; for HPV49, 6.88 to 72.91; for HPV75, 5.06 to 14.60; for HPV76, 2.57 to 6.99; for HPV115, 3.27 to 5.94). The size of each of the gene sets is indicated by the number above each histogram. AGE-RAGE, advanced glycation end product/receptor for advanced glycation end product; HIF-1, hypoxia-inducible factor 1; HTLV-1, human T-cell leukemia virus type 1; MAPK, mitogen-activated protein kinase. (C) Venn diagram of the genes deregulated in the cells expressing E6/E7 from HPV16, HPV38, and HPV76. (D) Percentages of genes of the indicated beta-3 type that are also deregulated in HPV16 or HPV38. Common genes with opposite deregulation trends have been excluded from the analysis.