EVALUATION OF THE INTERACTION AMONG HPV16 E6 AND E7 ONCOPROTEINS AND THE DNA DAMAGE SENSOR 53BP1 IN 2D AND 3D EPITHELIAL CULTURES BY THE PROXIMITY LIGATION ASSAY

INTRODUCTION. Human papillomaviruses (HPV) group several viruses able to infect squamous stratified epithelia and cause benign papillomas, warts and anogenital lesions. They also correlate to oropharyngeal and anogenital malignancies, mainly promoted by the high risk (HR) α-HPV16 E6E7 oncoproteins. Despite scientific progresses and the development of vaccines, these tumors are still common and represent one of the major causes of women’s death. Host’s cell replication fidelity depends by the DNA Damage Response (DDR). Unlike from other DNA viruses, HR-HPVs encourage cells proliferation without inactivating the DDR: the mechanisms at the basis haven’t been clarified yet. During HPV16 infection, E6 binds and degrades p53 through the E6AP LXXLL domain. Similarly, E7 competes with E2F1-pRb interaction, thus inactivating pRb, and promoting the linking the pRb-like proteins CBP/p300 and p107, that also harbor a LXXLL sequence. Unfortunately, E6 E7 role in the DDR activation is not elucidated yet. EXPERIMENTAL MODEL. To gain new information, we reproduced an in vitro 3D HPV16-E6E7 infected epithelium, already characterized for HPVs studies, and checked for cellular and viral markers, among them HPV16E6E7 oncoproteins and the double strand breaks (DSB) sensor 53BP1; we then made a Co-IF for E6 and E7 with 53BP1. Since E6 and E7 both interact with LXXLL containing proteins, we analyzed 53BP1 BRCT2 domain and we explored the binding hypothesis via the in situ PLA technique in 2D in CaSki and E6E7HPV16 keratinocytes and in the 3D model. RESULTS. The in vitro infected epithelium resembles the in vivo tissue. E6E7HPV16, both in basal and differentiated strata, induce a 53BP1 increase in nuclear foci. After highlighting E6 and E7 co-expression with 53BP1 and a LKVLL sequence within the 53BP1 BRCT2 domain, we demonstrated the binding in all the employed cellular models. CONCLUSION. Our results add new information on HPV16 oncoproteins capability in overcome cellular defense strategies.