NOVEL CELL CYCLE REGULATION BY ADENOVIRUS E1B 55K

描述(申请人提供)：溶瘤ELB-55K突变型腺病毒已进入第三阶段临床试验。然而，它们在肿瘤细胞中选择性复制的基础还知之甚少。本实验室的工作表明，ELB-55K突变病毒在S期细胞中选择性复制并比G1期细胞杀伤效果更好，这可能是肿瘤选择性复制的原因。本研究试图通过了解E1B-55K突变体的S相选择性和G1限制性性质来了解其对肿瘤的选择性。前期研究结果表明，G1细胞中晚期病毒基因的表达在翻译水平上受到抑制，这显然是对E4orf1或E4orf2基因的反应。ELB-55K突变病毒感染的G1细胞和S期晚期病毒的多聚体分布表明，病毒晚期翻译在G1细胞中受核糖体分流水平的限制。野生型病毒的E1B-55K蛋白与E4or1“6蛋白和宿主细胞因子一起促进病毒翻译。一个候选宿主细胞因子已被定位到RUNX1基因。在这项申请中提出了三个具体目标。目的1通过比较G1期和S期感染细胞中病毒基因的合成/编辑，以及同步感染和感染细胞中关键翻译步骤的比率，来确定病毒晚期翻译在G1感染细胞中被抑制的程度。这将检验在感染G1的细胞中核糖体分流减少的假设。目的2阐明E4orf1和E4orf2在抑制病毒晚期翻译和抑制G1期病毒生长中的作用。将制备E4orlT或E4orf2缺陷病毒以及E1B-55K基因，并就细胞周期限制进行评估。在细胞周期受限和非限制性突变病毒感染的细胞中，将比较核糖体扫描和核糖体分流指导的翻译，以检验E4orf1在核糖体分流水平上抑制翻译的假设，这是G1限制的基础。AIM 3将鉴定对E1B-55K功能重要的RUNX1亚型，并确定其在感染过程中的活性是否依赖于细胞周期。候选异构体将被过度表达，以确定它们是否弥补E1B-55K功能的丧失；RNA干扰将被用于去除RUNX1的表达，以确定RUNX1对腺病毒感染的重要性。

DESCRIPTION (provided by applicant): Oncolytic ElB-55K-mutant adenoviruses have entered phase III clinical trials. However, the basis for their selective replication in tumor cells is poorly understood. Work of this lab has shown that ElB-55K-mutant viruses selectively replicate in and kill S phase cells better than G1 cells; this is likely to account for the selective replication in tumors. This research seeks to understand the tumor-selective nature of the E1B- 55K-mutant by understanding its S phase-selective and G1-restricted nature. Results from the previous period showed that late viral gene expression is suppressed in G1 cells at the level of translation, apparently in response to the E4orf1 or E4orf2 genes. The polysomal distribution of late viral mRNA in S phase and G1 cells infected with ElB-55K-mutant viruses suggests that late viral translation is restricted in G1 cells at the level of ribosome shunting. The E1B-55K protein of the wild-type virus promotes viral translation in association with the E4orl"6 protein and host cell factors. A candidate host cell factor has been mapped to the RUNX1 gene. Three specific aims are proposed in this application. Aim 1 seeks to determine how late viral translation is suppressed in G1-infected cells by comparing viral mRNA synthesis/ed in G1- and S phase-infected cells and by measuring the rates of key translational steps in synchronized and infected cells. This will test the hypothesis that ribosome shunting is decreased in the G1-infected cell. Aim 2 will elucidate the role of E4orfl and E4orf2 in attenuating late viral translation and restricting virus growth in G1. Viruses defective in either E4orlT or E4orf2 and the E1B-55K genes will be prepared and evaluated with respect to the cell cycle-restriction. Translation directed by ribosome scanning and ribosome shunting will he compared in cells infected with cell cycle-restricted and non-restricted mutant viruses to test the hypothesis that E4orf 1 attenuates translation at the level of ribosome shunting and that this is the basis for the G1-restriction. Aim 3 will identify the RUNX1 isoform important for E1B-55K function and determine if its activity during an infection is cell cycle-dependent. Candidate isoforms will be over expressed to determine if they compensate for the loss of E1B-55K function and RNA interference will be used to ablate RUNX1 expression to determine the importance of RUNX1 to adenovirus infection.