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.
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.
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DOI：
10.1186/s12985-018-1086-4
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发表时间：
2018-11-16
期刊：
影响因子：
4.8
通讯作者：
Azzimonti B
中科院分区：
文献类型：
--
作者：
Squarzanti DF;Sorrentino R;Landini MM;Chiesa A;Pinato S;Rocchio F;Mattii M;Penengo L;Azzimonti B
尽管采取了疫苗接种和筛查措施，但主要由HPV 16癌蛋白促进的肛门生殖器癌仍然是妇女的第四大肿瘤和第二大死亡原因。细胞复制保真度是宿主DNA损伤反应（DDR）的结果。与许多通过DDR失活促进其生命周期的DNA病毒不同，HR-HPV在DDR开启的情况下仍促进细胞增殖，其发生的原因和方式仅部分阐明。在HPV 16感染期间，E6通过与E6 AP LXXLL序列结合来连接并降解p53;不幸的是，E6在DDR反应中的直接作用尚未明确确定。类似地，E7通过与E2 F1-pRb相互作用竞争来增加DDR，从而导致pRb的失活，并通过与也含有LXXLL序列的pRb样蛋白CBP/p300和p107结合，并通过几种DDR蛋白的相互作用和激活，E2 F1介导的DDR基因翻译的促进。为了获得关于E6和E7在DDR激活中的贡献的信息，我们产生了体外3D HPV 16-E6 E7感染的上皮，已经巩固了HPV的研究模型，并通过评估H&E染色和BrdU、HPV 16 DNA、E6 E7蛋白和γH2A.X/53 BP 1双链断裂（DSBs）传感器表达来验证它;然后将E6和E7与cyclin E2和B1进行免疫共定位。由于53 BP 1与E6和E7一样，也与p53和pRb结合，我们推测它们可能直接结合。为了探索这一假设，我们用53 BP 1进行了E6和E7的双重免疫荧光，53 BP 1在其BRCT 2结构域内的序列分析，然后在CaSki，E6 E7 HPV 16 NHEK和3D模型内进行了原位PLA。体外上皮类似于体内感染组织的组织学和典型事件。E6 E7 HPV 16在基底层和分化层均表达，并诱导H2A.X磷酸化和53 BP 1向核灶中的增加。在突出显示E6和E7与53 BP 1和53 BP 1 BRCT 2结构域内的LKVLL序列的共表达后，我们通过PLA技术证明了结合。我们的研究结果加强了E6和E7在细胞功能控制中的作用，为这种肿瘤病毒的活性提供了潜在的新见解。
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. 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. 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. Our results reinforce E6 and E7 role in cellular function control providing potentially new insights into the activity of this tumor virus.
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