Human papillomavirus type 16 E6 and E7 oncoproteins interact with the nuclear p53-binding protein 1 in an in vitro reconstructed 3D epithelium: new insights for the virus-induced DNA damage response

Abstract

Background: Despite vaccination and screening measures, anogenital cancer, mainly promoted by HPV16 oncoproteins, still represents the fourth tumor and the second cause of death among women. Cell replication fidelity is the result of the host DNA damage response (DDR). Unlike many DNA viruses that promote their life cycle through the DDR inactivation, HR-HPVs encourage cells proliferation despite the DDR turned on. Why and how it occurs has been only partially elucidated. During HPV16 infection, E6 links and degrades p53 via the binding to the E6AP LXXLL sequence; unfortunately, E6 direct role in the DDR response has not clearly identified yet. Similarly, E7 increases DDR by competing with E2F1-pRb interaction, thus leading to the inactivation of pRb, and promotion, E2F1 mediated, of DDR genes translation, by binding to the pRb-like proteins CBP/p300 and p107, that also harbour LXXLL sequence, and via the interaction and activation of several DDR proteins.

Methods: To gain information regarding E6 and E7 contribution in DDR activation, we produced an in vitro 3D HPV16-E6E7 infected epithelium, already consolidated study model for HPVs, and validated it by assessing H&E staining and BrdU, HPV16 DNA, E6E7 proteins and γH2A.X/53BP1 double-strand break (DSBs) sensors expression; then we made an immuno-colocalization of E6 and E7 with cyclin E2 and B1. Since 53BP1, like E6 and E7, also binds p53 and pRb, we supposed their possible direct binding. To explore this hypothesis, we performed a double immunofluorescence of E6 and E7 with 53BP1, a sequence analysis of 53BP1 within its BRCT2 domain and then an in situ PLA within CaSki, E6E7HPV16 NHEKs and the 3D model.

Results: The in vitro epithelium resembled the histology and the events typical of in vivo infected tissues. E6E7HPV16 were both expressed in basal and differentiated strata and induced H2A.X phosphorylation and 53BP1 increment into nuclear foci. After highlighting E6 and E7 co-expression with 53BP1 and a LKVLL sequence within the 53BP1 BRCT2 domain, we demonstrated the bindings via the PLA technique.

Conclusions: Our results reinforce E6 and E7 role in cellular function control providing potentially new insights into the activity of this tumor virus.

Keywords: Cancer; DNA damage response; Double-strand break; E6-associated protein; Genomic instability; High-risk Human Papillomavirus; In vitro 3D epithelial model; Proximity ligation assay; World Health Organization.

Fig. 1

H&E staining. A) Control in vitro reconstructed 3D epithelial culture made up with normal keratinocytes and B) E6E7 HPV16 transduced keratinocytes. C) In vivo normal epithelia obtained from tumor-free mucosa margins. D) HPV16 positive anal intraepithelial neoplasia (AIN), grade III. Bar scales in 20x fields = 100 μm

Fig. 2

In vitro 3D epithelial cultures. HPV16 DNA (FISH) staining and immunofluorescent detection of BrdU, E6 and E7 of control normal and E6E7 HPV16 infected reconstructed in vitro 3D epithelial cultures. Viral genome amplification positivity is evidenced in red in the mid and upper strata of the infected epithelium only (A, B). To show cell nuclei, sections are counterstained with DAPI (blue). S phase cells positivity to the BrdU marker is limited to the basal and basal/parabasal strata of normal (C) and infected epithelia (D) respectively. Direct analysis of E6 and E7 proteins in normal uninfected (E and G for E6 and E7 respectively) and E6E7 HPV16 positive culture sections (F-F′ for E6 and H-H′ for E7). Positive cells are shown in red, while cells nuclei are counterstained in blue with DAPI. Bar scales in 20x fields = 100 μm. Immunoblotting. Western blot analysis of E6, E7 and tubulin protein extracts derived by peeling off the epithelial layers of 3D reconstructed epithelial cultures (control normal and E6E7HPV16) from the collagen-based dermis. After run on polyacrylamide gels, proteins were blotted onto nitrocellulose or PVDF membranes (I)

Fig. 3

DDR characterization of the 3D in vitro reconstructed E6E7 HPV16 infected epithelia. Double immunofluorescent and western blot (B) analysis of the DNA break sensors. γH2A.X (in red) and 53BP1 (in green) in control normal (A) and E6E7HPV16 infected epithelia (C-C′-C″). γH2A.X and 53BP1 are expressed in HPV infected 3D samples

Fig. 4

Double immunofluorescent analysis of E6 and E7 with cyclin E2 and B1. E6 and E7 HPV16 pushes a subset of differentiating cells into G1/S (cyclin E2 positive cells, B and F) and S/G2 checkpoint phases (cyclin B1 positive cells, C and G). Double immunofluorescent analysis of E6 and E7 with 53BP1. CaSki cells have a high number of discrete small 53BP1 positive nuclear foci inside most of the same cells that were E6 (A-A’-A”) and E7 (E, E’, E”) positive; in HPV16 epithelial models E6/53BP1 (D) and E7/53BP1 (H) positivity is throughout the epithelium, both in undifferentiated and differentiated layers. Nuclei were always visualized in blue with DAPI counterstaining. Bar scales in 20x fields = 100 μm

Fig. 5

E6 and E7 HPV16 oncoproteins interaction with 53BP1 proteins visualized with PLA technique. The red perinuclear punctuate dots visible in the CaSki (A-D) and E6E7 HPV16 NHEKs (B-E) are also present in the HPV16 reconstructed infected epithelium (C and F). Nuclei were always visualized in blue with DAPI counterstaining. Bar scales in 20x fields = 100 μm