NOVEL CELL CYCLE REGULATION BY ADENOVIRUS E1B 55K

腺病毒E1B55-kDa(55K)蛋白在病毒生长和病毒介导的细胞转化过程中发挥作用。在病毒生长过程中，55K蛋白与E4orf6蛋白结合，调节mRNA的生物合成。在转化过程中，55K蛋白与肿瘤抑制基因p53结合，并阻止p53反应基因的转录，包括那些促进细胞凋亡和诱导G1期生长停滞的基因。然而，感染55K突变病毒的大多数细胞不会发生凋亡，不会停滞在G1期，也不会失败地复制突变病毒DNA。因此，55K在病毒生长和转化中的作用之间的联系仍然不清楚。这项工作将阐明这一联系，作为更大目标的一部分，即了解腺病毒靶向细胞生长控制机制以促进病毒生长的方法。由于55K突变病毒只生长在S早期感染的HeLa细胞中，而不是在G1期，55K蛋白克服了细胞周期对病毒生长的限制。这一限制以及克服这一限制的55K函数将由三个具体目标决定。首先，通过比较相关的G1限制性腺病毒突变体的生长和对病毒生长的允许和限制的感染细胞之间的mRNA运输的控制，对腺病毒必须篡夺对细胞周期无关的生长的mRNA的控制的假设进行评估。其次，允许和限制细胞的异核体将被用来阐明这种限制的细胞学基础，并确定限制表型的显性。第三，将确定55K/E4orf6复合体针对细胞周期无关病毒生长的细胞蛋白，并确定这些蛋白对细胞周期的调节。这项研究将确定55K蛋白如何颠覆细胞周期控制病毒的生长。这可能代表了55K蛋白在裂解生长和病毒转化之间的第一个联系。这些研究将加深我们对病毒致病机制、病毒致癌机制以及细胞生长控制的基本机制的理解。此外，本研究还将确定在S期细胞中限制生长的腺病毒突变体。这种具有复制能力的病毒可以用作溶瘤剂来治疗快速增长的人类肿瘤。

The adenovirus E1B 55-kDa (55K) protein functions during virus growth and virus-mediated cellular transformation. During virus growth, the 55K protein binds the E4orf6 protein and regulates mRNA biogenesis. During transformation, the 55K protein binds the tumor suppressor p53 and blocks transcription from p53-responsive genes including those that promote apoptosis and induce G1 growth arrest. However, most cells infected with the 55K-mutant virus do not undergo apoptosis, do not arrest in G1, and do not fail to replicate mutant viral DNA. Consequently, the link between 55K function in virus growth and transformation remains unclear. This work will elucidate this link as part of the larger goal of understanding the means by which adenovirus targets mechanisms of cellular growth control for virus growth. Because 55K-mutant viruses grow only in HeLa cells that were infected in early S phase but not in G1, the 55K protein overcomes a restriction imposed on virus growth by the cell cycle. This restriction and the 55K function that overcomes this restriction will be determined by three specific aims. First, the hypothesis that adenovirus must usurp the control of mRNA for cell cycle-independent growth will be evaluated by comparing the growth of related G1-restricted adenovirus mutants and the control of mRNA transport among infected cells that are permissive and restrictive for virus growth. Second, heterokaryons of permissive and restrictive cells will be used to elucidate the cellular basis for this restriction and determine the dominance of the restrictive phenotype. Third, cellular proteins targeted by 55K/E4orf6 complex for cell cycle-independent virus growth will be identified and the cell cycle- regulation of these proteins determined. This study will determine how the 55K protein subverts cell cycle controls for virus growth. This may represent the first link between the roll of the 55K protein in lytic growth and viral transformation. These studies will increase our understanding of the mechanisms of viral pathogenesis and viral oncogenesis as well as fundamental mechanisms of cellular growth control. Furthermore, this study will identify adenovirus mutants that are restricted for growth in S phase cells. Such replication competent viruses can be used as oncolytic agents to treat rapidly growing human tumors.