Laboratory of Molecular Virology, Institut de Recherches Cliniques de Montréal, Montreal, Quebec, Canada.
J Virol. 2012 Jan;86(1):94-107. doi: 10.1128/JVI.00751-11. Epub 2011 Oct 19.
Human papillomaviruses (HPVs) are the etiological agents of cervical cancer and other human malignancies. HPVs are classified into high- and low-risk genotypes according to their association with cancer. Host cell transformation by high-risk HPVs relies in part on the ability of the viral E6 protein to induce the degradation of p53. We report the development of a cellular assay that accurately quantifies the p53 degradation activity of E6 in vivo, based on the fusion of p53 to Renilla luciferase (RLuc-p53). This assay was used to measure the p53 degradation activities of E6 proteins from 29 prevalent HPV types and variants of HPV type 16 (HPV16) and HPV33 by determining the amount of E6 expression vector required to reduce by half the levels of RLuc-p53 (50% effective concentration [EC₅₀]). These studies revealed an unexpected variability in the p53 degradation activities of different E6 proteins, even among active types whose EC₅₀s span more than 2 log units. Differences in activity were greater between types than between variants and did not correlate with differences in the intracellular localization of E6, with most being predominantly nuclear. Protein and mRNA expression of the 29 E6 proteins was also examined. For 16 high-risk types, spliced transcripts that encode shorter E6*I proteins of variable sizes and abundances were detected. Mutation of the splice donor site in five different E6 proteins increased their p53 degradation activity, suggesting that mRNA splicing can limit the activity of some high-risk E6 types. The quantification of p53 degradation in vivo represents a novel tool to systematically compare the oncogenic potentials of E6 proteins from different HPV types and variants.
人乳头瘤病毒（HPV）是宫颈癌和其他人类恶性肿瘤的病因。根据与癌症的关联，HPV 分为高危型和低危型基因型。高危型 HPV 引起宿主细胞转化部分依赖于病毒 E6 蛋白诱导 p53 降解的能力。我们报告了一种基于 p53 与海肾荧光素酶（RLuc-p53）融合的体内准确量化 E6 降解 p53 活性的细胞测定方法的开发。该测定法用于通过确定降低 RLuc-p53 水平一半所需的 E6 表达载体的量（50%有效浓度 [EC₅₀]）来测量 29 种常见 HPV 类型和 HPV 16 型（HPV16）和 HPV33 型的 E6 蛋白的 p53 降解活性。这些研究揭示了不同 E6 蛋白的 p53 降解活性存在意外的可变性，即使在 EC₅₀ 跨度超过 2 个对数单位的活性类型之间也是如此。活性差异在类型之间大于变体之间，并且与 E6 的细胞内定位差异无关，大多数主要是核定位。还检查了 29 种 E6 蛋白的蛋白和 mRNA 表达。对于 16 种高危型，检测到编码具有不同大小和丰度的较短 E6*I 蛋白的剪接转录本。在五种不同的 E6 蛋白中突变剪接供体位点增加了它们的 p53 降解活性，表明 mRNA 剪接可以限制一些高危型 E6 类型的活性。体内 p53 降解的定量代表了一种新的工具，可以系统地比较来自不同 HPV 类型和变体的 E6 蛋白的致癌潜力。
