Gbala Ifeoluwa, Kavcic Nezka, Banks Lawrence
Tumour Virology Laboratory, International Centre for Genetic Engineering and Biotechnology, Trieste, Italy.
J Virol. 2025 Apr 15;99(4):e0220324. doi: 10.1128/jvi.02203-24. Epub 2025 Mar 25.
High-risk human papillomaviruses (HR HPV)-16 and -18, and other closely related subtypes, are associated with at least 90% of human cervical cancers. Cervical cancers and derived cell lines continuously express high levels of the HPV oncoprotein E7, known to degrade the tumor suppressor retinoblastoma protein (pRB). This E7-pRB interaction is important for the maintenance and progression of malignancy. In the case of HPV E6, substrate recognition has been reported to play an important role in stabilizing the viral oncoprotein; however, such regulation of E7 has so far not been investigated. Using biochemical, immunostaining, and clonogenic assays, we describe an intriguing role for pRB in the stabilization of E7 oncoprotein in HPV-positive cervical-cancer-derived cell lines. The knockdown of pRB expression by RNA interference results in a significant decrease in the levels of E7 protein in CaSki, SiHa, HeLa, and C-4 I cells. We show that pRB knockdown regulates HPV E7 at the transcription and protein levels, and significantly reduces the half-life of E7 protein by at least twofold in SiHa and HeLa cells. We also demonstrate that the destabilization of E7 caused by pRB knockdown results in significant inhibition of cell proliferation and colony formation of HPV-16 and -18 E7-positive SiHa and HeLa cells. Furthermore, the expression of wild-type pRB in pRB-depleted cells significantly restored E7 levels. Therefore, we propose that pRB, in addition to being a degradation target for HPV E7, is crucial for its stabilization.IMPORTANCEThe human papillomavirus (HPV) viral proteins E6 and E7 cooperatively contribute to tumorigenesis by disrupting cellular targets. These oncoproteins are degraded via the proteasome pathway; however, they are continuously expressed in cervical cancer cell lines. The retinoblastoma protein, pRB, is a degradation target of high-risk (HR) HPV E7 oncoprotein. Several studies have shown that the binding of E7 to pRB is important for its E7-mediated inactivation and demonstrated how pRB protein levels respond to the presence and absence of E7. However, the modulatory role of pRB on E7 protein levels has so far not been reported. Here, we report a novel regulatory relationship between E7 and pRB. We found that the continuous expression of pRB is critical for E7 stabilization. We demonstrate that this pRB-related E7 destabilization occurs in part through enhanced protein turnover. Thus, our findings provide new insights into the importance of the E7-pRB interaction in driving tumorigenesis.
高危型人乳头瘤病毒（HR HPV）-16和-18以及其他密切相关的亚型与至少90%的人类宫颈癌相关。宫颈癌及其衍生的细胞系持续高水平表达HPV癌蛋白E7，已知该蛋白可降解肿瘤抑制因子视网膜母细胞瘤蛋白（pRB）。这种E7-pRB相互作用对恶性肿瘤的维持和进展至关重要。就HPV E6而言，据报道底物识别在稳定病毒癌蛋白中起重要作用；然而，迄今为止尚未对E7的此类调节进行研究。我们通过生化、免疫染色和克隆形成试验，描述了pRB在HPV阳性宫颈癌衍生细胞系中对E7癌蛋白稳定化的有趣作用。通过RNA干扰敲低pRB表达会导致CaSki、SiHa、HeLa和C-4 I细胞中E7蛋白水平显著降低。我们表明，敲低pRB在转录和蛋白水平上调节HPV E7，并在SiHa和HeLa细胞中使E7蛋白的半衰期显著缩短至少两倍。我们还证明，敲低pRB导致的E7不稳定会显著抑制HPV-16和-18 E7阳性SiHa和HeLa细胞的细胞增殖和集落形成。此外，在pRB缺失的细胞中表达野生型pRB可显著恢复E7水平。因此，我们提出pRB除了是HPV E7的降解靶点外，对其稳定化也至关重要。
重要性
人乳头瘤病毒（HPV）的病毒蛋白E6和E7通过破坏细胞靶点共同促进肿瘤发生。这些癌蛋白通过蛋白酶体途径降解；然而，它们在宫颈癌细胞系中持续表达。视网膜母细胞瘤蛋白pRB是高危（HR）HPV E7癌蛋白的降解靶点。多项研究表明，E7与pRB的结合对其E7介导的失活很重要，并展示了pRB蛋白水平如何对E7的存在和缺失做出反应。然而，迄今为止尚未报道pRB对E7蛋白水平的调节作用。在此我们报道了E7与pRB之间一种新的调节关系。我们发现pRB的持续表达对E7稳定化至关重要。我们证明这种与pRB相关的E7不稳定部分是通过增强蛋白周转发生的。因此，我们的发现为E7-pRB相互作用在驱动肿瘤发生中的重要性提供了新的见解。
