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

BackgroundDespite 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.MethodsTo 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.ResultsThe 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.ConclusionsOur results reinforce E6 and E7 role in cellular function control providing potentially new insights into the activity of this tumor virus.

Genomic profiling reveals the pivotal role of hrHPV driving copy number and gene expression alterations, including mRNA downregulation of TP53 and RB1 in penile cancer

“…oncoprotein E6 interferes directly with the p53 pathway, while E7 binds to the pRB, mediating ubiquitination and degradation. 53,54 This disruption leads to loss of critical cell cycle checkpoints, induction of cell proliferation, inhibition of apoptosis, and progressive genetic instability. 55 Interestingly, we found that the TP53 and RB1 genes were downregulated in the PeCa cases of our study.…”

“…It is relevant to note that among eleven cytobands that showed association with clinical and histopathological parameters, seven were sites for HPV integration, from which two, 4q13.2 and 20q13.32‐q13.33, showed association with HPV positivity. The genomic instability in these cytobands, could be caused by the viral DNA insertion into the host genome, which occurs more frequently near to E2 gene of the hrHPV subtypes, causing impairment of the negative feedback control of E6 and E7 oncogenes; oncoprotein E6 interferes directly with the p53 pathway, while E7 binds to the pRB, mediating ubiquitination and degradation 53,54 . This disruption leads to loss of critical cell cycle checkpoints, induction of cell proliferation, inhibition of apoptosis, and progressive genetic instability 55 …”

Human papillomavirus E7 oncoprotein targets RNF168 to hijack the host DNA damage response

“…S2F). However, HPV16 E7 was recently reported to colocalize with 53BP1 using proximityligation assays, both in differentiated HPV + keratinocytes and in CaSki (48), arguing that both proteins are near each other during viral replication and cellular transformation. Consistently, we found that E7 and the CR3 domain alone are recruited to foci of RNF168 Fig.…”

<p>DNMT1 Enhances the Radiosensitivity of HPV-Positive Head and Neck Squamous Cell Carcinomas via Downregulating SMG1</p>

“…12 E6 and E7 can degrade the expression products of the tumor suppressor genes p53 and pRb. 13 HPV E6 interacts with P53, which degrades P53 protein and loses its anti-cancer effect, increasing the chance of host cell malignant transformation. 13 HPV E7 acts on Rb to inactivate it.…”

“…13 HPV E6 interacts with P53, which degrades P53 protein and loses its anti-cancer effect, increasing the chance of host cell malignant transformation. 13 HPV E7 acts on Rb to inactivate it. 14 The inactivated Rb gene can reversely activate multiple transcription factors.…”