DOUBLE STRAND BREAK REPAIR--INHIBITION BY ADENOVIRUS E4

双链断裂修复——腺病毒 E4 的抑制

• 批准号: 6626707
• 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/6626707
• 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：腺病毒早期区域 4 (E4) 编码在 成功的病毒感染所需的各种过程。虽然E4不是 DNA 复制所需的三个 E4 蛋白（E4 11k，E4 ORF3 的产物；E4 34k，E4 ORF6的产物；和E4 ORF4的产物）调节复制 被感染的细胞。此外，E4突变体产生的大部分病毒DNA 缺少 E4 11k 和 E4 34k 结构异常，由多联体组成 病毒基因组长度可达6个或更多单体。多联体病毒DNA 在被野生型病毒感染的细胞中未观察到这种现象。真核细胞具有 高效的双链断裂修复 (DSBR) 系统，用于重新连接断裂的 DNA 通过辐射和其他物质。检验串联的假设 E4 突变体感染中的病毒 DNA 是由线性序列的端对端连接产生的 通过 DSBR 检测细胞内病毒 DNA，检查细胞中多联体的形成 缺乏 DNA 依赖性蛋白激酶 (DNA PK)，这是 蜂窝 DSBR 系统。在 E4 突变体感染中未观察到串联体 DNA PK-细胞，与 DSBR 产生多联体的假设一致 并建议在野生型感染中，E4 通过以下方式防止串联 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 物理上与DNA PK。总之，这些数据强烈表明 E4 蛋白质可能通过与 DNA PK 结合的机制抑制 DSBR。 抑制串联体形成可能会提高效率 病毒DNA复制。此外，由于 DNA PK 是近端元件 诱导 p53 响应 DNA 活性的途径，抑制 DNA PK 可能在受感染的细胞中具有抗凋亡作用。因此，E4 与 DSBR 的相互作用 可能以两种独特且新颖的方式促进腺病毒的成功 感染。这里提出的工作目标是加深对 这些 E4 产品和 DNA PK 之间相互作用的物理性质， 并确定该病毒对病毒生命周期的重要性 E4 功能的新认识的方面。