DOUBLE STRAND BREAK REPAIR--INHIBITION BY ADENOVIRUS E4

双链断裂修复——腺病毒 E4 的抑制

• 批准号: 6689600
• 负责人: Gary W Ketner
• 金额: $ 24.18万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-19 至 2005-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6689600
• 关键词: Adenoviridae; DNA damage; DNA repair; DNA replication; apoptosis; enzyme activity; genetic recombination; immunoglobulin genes; immunoprecipitation; microorganism culture; oncoproteins; protein kinase; protein protein interaction; virus DNA; virus genetics; virus infection mechanism; virus protein; virus replication

DESCRIPTION: Adenovirus early region 4 (E4) encodes proteins critical in a variety of processes required for a successful viral infection. While E4 is not required for DNA replication, three E4 proteins (E4 11k, product of E4 ORF3; E4 34k, product of E4 ORF6; and the product of E4 ORF4) regulate replication in infected cells. In addition, most of the viral DNA produced by E4 mutants lacking E4 11k and E4 34k is structurally abnormal, consisting of concatemers of the viral genome up to 6 or more monomers in length. Concatemeric viral DNA is not observed in cells infected by wild-type virus. Eukaryotic cells possess efficient double strand break repair (DSBR) systems for rejoining DNAs broken by radiation and other agents. To test the hypothesis that the concatenated viral DNA seen in E4 mutant infections arises by end-to-end joining of linear intracellular viral DNAs by DSBR, concatemer formation was examined in cells lacking the DNA-dependent protein kinase (DNA PK), an essential component of the cellular DSBR system. No concatemers were observed in E4 mutant infections of DNA PK- cells, consistent with the hypothesis that concatemers arise by DSBR and suggestion that in wild-type infections, E4 prevents concatenation by inhibiting DSBR. Further, E4 34k inhibited V(D)J recombination, a process that requires DSBR, in a plasmid-based assay. Finally, immunoprecipitation experiments showed that both the E4 11k and E4 34k proteins associate physically with DNA PK. Together, these data strongly suggest that both E4 proteins inhibit DSBR, possibly by a mechanism that involves binding to DNA PK. It is likely that suppression of concatemer formation increases the efficiency of viral DNA replication. Additionally, since DNA PK is a proximal element in the pathway that induces p53 activity in response to DNA, inhibition of DNA PK may be anti-apoptotic in infected cells. Thus, the interaction of E4 with DSBR may contribute in two distinct and novel ways to the success of an adenoviral infection. The goal of the work proposed here is to develop an understanding of the physical nature of the interactions between these E4 products and DNA PK, and to determine the significance to the viral life cycle of this newly-recognized aspects of E4 function.

描述：腺病毒早期4（e4）编码至关重要的蛋白质 成功的病毒感染所需的多种过程。虽然E4不是 DNA复制所需的三种E4蛋白（E4 11K，E4 ORF3的产物； E4 34K，E4 ORF6的产物； E4 ORF4的乘积）调节复制 感染细胞。另外，E4突变体产生的大多数病毒DNA 缺少E4 11K和E4 34K在结构上是异常的，由助剂组成 病毒基因组的长度高达6个或更多。串联病毒DNA 在被野生型病毒感染的细胞中观察到。真核细胞拥有 有效的双链断修复（DSBR）系统，用于重新加入DNA 通过辐射和其他代理。测试串联的假设 在E4突变感染中看到的病毒DNA是通过线性端到端连接而产生的 细胞内的细胞内病毒DNA在细胞中检查了condemer形成 缺少DNA依赖性蛋白激酶（DNA PK），这是 细胞DSBR系统。在E4突变体感染中未观察到辅助者 DNA PK-细胞的含量与dsbr产生的confemers的假设一致 并建议在野生型感染中，E4阻止了通过 抑制DSBR。此外，E4 34K抑制V（d）J重组，这一过程 在基于质粒的测定中需要DSBR。最后，免疫沉淀 实验表明，E4 11K和E4 34K蛋白均 与DNA PK物理。这些数据共同表明两个E4 蛋白质抑制DSBR，可能是通过涉及与DNA PK结合的机制。 抑制辅导者形成可能会提高效率 病毒DNA复制。此外，由于DNA PK是近端元素 诱导p53活性响应DNA的途径，抑制DNA PK 在感染细胞中可能是抗凋亡。因此，E4与DSBR的相互作用 可能以两种不同的新颖方式贡献腺病毒的成功 感染。这里提出的工作的目的是建立对 这些E4产品与DNA PK之间相互作用的物理性质， 并确定对此病毒生命周期的重要性 E4功能的新认识的方面。