Regulation of Transcription and Translation by Human Polyomaviruses

描述(申请人提供)：多核病毒科的一些成员在实验动物或其自然宿主中诱发肿瘤。在这些情况下，动物的肿瘤形成或细胞培养中的转化需要一种或多种称为肿瘤抗原的病毒蛋白的表达。猴病毒SV40及其近亲BKV和JCV编码一种作为主要癌蛋白的大肿瘤抗原(LT)。SV40诱导的转化和肿瘤发生已经得到了广泛的研究，需要LT与三种类型的细胞蛋白相互作用：(1)Rb蛋白(pRb、p107和p130)、hsc70和p53。SV40LT与Rb蛋白的结合，再加上hsc70的作用，触发了细胞周期基因依赖于E2F的转录，这一作用足以驱动静止的细胞增殖。SV40 LT ALS与细胞肿瘤抑制基因P53的DNA结合域结合，从而抑制其结合和激活P53反应基因启动子的能力。因此，细胞周期停滞或细胞死亡诱导蛋白的表达被阻断。与SV40一样，人类多瘤病毒BKV和JCV编码的LTS也结合并抑制RB蛋白和P53。然而，这些LT诱导转化的效率比SV40 LT低得多。一些研究表明，BKV和JCV的较弱转化是由于病毒启动子转录较低所致。我们通过驱动SV40、BKV和JCV LTS在同一共同的异源启动子上的表达来比较它们的转化能力。虽然BKV和JCV LTS的稳态水平比SV40 LT低得多，但这三种LT都有同样的能力扰乱PRB途径并诱导细胞增殖。然而，在许多检测中，BKV和JCV LTS的转化效率远远低于SV40 LT。每个LT可能结合和失活的P53的量在每个病例中都有很大的不同，并且与每个特定LT的量相关。因此，我们假设SV40诱导的转化增加是由LT-P53复合体的“功能增益”来解释的。我们预测LT不会简单地阻止P53依赖基因的表达--阻止P53与启动子的关联--它还利用P53激活结构域刺激细胞基因的转录，从而对转化做出积极贡献。我们将通过一系列的p53芯片序列实验来验证这一假设。我们还发现，SV40 LT增加并控制调节因子CBP/p300的翻译，这是LT分子以前未知的功能。因为所有的SV40、BKV和JCV LT都有很高的同源性，我们假设翻译控制是多发性LTS的共同特征。我们将通过对核糖体相关的mRNAs进行RNA-SEQ实验来检验这一假设。这些研究将填补我们对多瘤病毒诱导的肿瘤发生的理解的重要空白。

DESCRIPTION (provided by applicant): Some members of the Polyomaviridae induce tumors in laboratory animals or in their natural hosts. In these cases, tumorigenesis in animals or transformation in cell culture requires the expression of one or more viral proteins called tumor antigens. The monkey virus SV40 and its close relatives BKV and JCV encode a large tumor antigen (LT) that acts as the main oncoprotein. Transformation and tumorigenesis induced by SV40 has been studied extensively and requires the interaction of LT with three types of cellular proteins: (1) the Rb-proteins (pRb, p107, and p130), hsc70, and p53. The association of SV40 LT with Rb-proteins, coupled with the action of hsc70, triggers E2F-dependent transcription of cell cycle genes and this action is sufficient to drive quiescent cells to proliferate. SV40 LT als binds to the DNA-binding domain of the cellular tumor suppressor p53 and thus inhibits its ability to bind and activate the promoters of p53-responsive genes. Consequently, the expression of cell-cycle arrest or cell death-inducing proteins is blocked. Like SV40, the LTs encoded by the human polyomaviruses BKV and JCV also bind and inhibit Rb-proteins and p53. However, these LTs induce transformation much less efficiently than SV40 LT. Several studies have suggested that the weaker transformation seen by BKV and JCV is due to lower transcription from the viral promoters. We have compared the transforming abilities of the SV40, BKV, and JCV LTs by driving their expression from the same common heterologous promoter. While BKV and JCV LTs accumulated to much lower steady state levels than SV40 LT, all three LTs were equally capable of disrupting the pRb pathway and inducing cell proliferation. However, BKV and JCV LTs transformed much less efficiently than SV40 LT in a number of assays. The amount of p53 - presumably bound and inactivated by each LT- varied considerably in each case and correlated to the amount of each specific LT present. We thus hypothesize that the increased transformation induced by SV40 is explained by a "gain-of-function" attributed to LT-p53 complexes. We predict that LT does not simply block the expression of p53-dependent genes - preventing p53 association with promoters - but that it also uses the p53 activation domain to stimulate the transcription of cellular genes thus contributing positively to transformation. We will test this hypothesis by a series of p53 ChIP-seq experiments. We also found that SV40 LT increases and controls the translation of the regulators CBP/p300, a function previously unknown to LT molecules. Because all SV40, BKV and JCV LT share a high degree of homology, we hypothesize that translational control is a common property of polyoma LTs. We will test this hypothesis by performing RNA-seq experiments on ribosome associated mRNAs. These studies will fill important gaps in our understanding of polyomavirus-induced tumorigenesis.