NOVEL CELL CYCLE REGULATION BY ADENOVIRUS E1B 55K

描述（由申请人提供）：溶瘤ElB-55 K突变腺病毒已进入III期临床试验。然而，它们在肿瘤细胞中选择性复制的基础知之甚少。该实验室的工作表明，ElB-55 K突变病毒选择性地在S期细胞中复制并杀死S期细胞比G1细胞更好;这可能是肿瘤中选择性复制的原因。本研究旨在通过了解E1 B-55 K突变体的S期选择性和G1限制性来了解其肿瘤选择性。前期的结果表明，晚期病毒基因表达在G1细胞中在翻译水平上受到抑制，这显然是对E4 orf 1或E4 orf 2基因的响应。晚期病毒mRNA在感染ElB-55 K突变病毒的S期和G1细胞中的多核糖体分布表明，晚期病毒翻译在核糖体分流水平上限制在G1细胞中。野生型病毒的E1 B-55 K蛋白促进与E4 or 1 - 6蛋白和宿主细胞因子相关的病毒翻译。候选宿主细胞因子已被定位到RUNX 1基因。在本申请中提出了三个具体目标。目的1旨在通过比较G1和S期感染细胞中的病毒mRNA合成/艾德以及通过测量同步化和感染细胞中的关键翻译步骤的速率来确定晚期病毒翻译在G1感染细胞中是如何被抑制的。这将检验核糖体分流在G1感染的细胞中减少的假设。目的2将阐明E4 orf 1和E4 orf 2在减弱晚期病毒翻译和限制G1期病毒生长中的作用。将制备E4 orlT或E4 orf 2和E1 B-55 K基因缺陷的病毒，并就细胞周期限制进行评价。将在感染细胞周期限制性和非限制性突变病毒的细胞中比较由核糖体扫描和核糖体分流指导的翻译，以检验E4 orf 1在核糖体分流水平上减弱翻译并且这是G1限制的基础的假设。目的3将鉴定对E1 B-55 K功能重要的RUNX 1亚型，并确定其在感染期间的活性是否依赖于细胞周期。候选亚型将被过度表达以确定它们是否补偿E1 B-55 K功能的丧失，并且RNA干扰将被用于消除RUNX 1表达以确定RUNX 1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.