Characterization Of Adeno Associated Virus Non-structura

腺相关病毒非结构的表征

• 批准号: 6690525
• 负责人: Robert Kotin
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6690525
• 关键词: adeno associated virus group; adenosinetriphosphatase; apoptosis; endonuclease; enzyme activity; gene induction /repression; genetic promoter element; helicase; microorganism culture; protein structure function; transcription factor; virus genetics; virus integration; virus protein

The human dependovirus, adeno-associated virus (AAV) requires co-infection with a non-related DNA virus, e.g. adenovirus, to productively infect cells. AAV serotype 2 (AAV2) has been the prototype for this genus. Infection of cells in vitro with AAV2, in the absence of helper virus, may result in a integration of the virus genome. We have demonstrated that AAV2 DNA preferentially integrates into a specific region of the q-arm of human chromosome 19, locus AAVS1. Targeted integration is unique among eukaryotic viruses and requires cis-acting and trans-acting elements. The non-structural proteins of AAV, Rep, are required for targeted integration. The Rep proteins expressed from a promoter at map position 5, Rep 78 and Rep 68, are capable of binding both single stranded and dupex DNA and cleaving duplex and single-stranded DNA. The p5 Rep proteins multimerize and may form hexamers in the presence of a DNA substrate. Tyrosine 156 is required for the endonuclease activity and a covalent tyrosyl-phosphodiester is formed as an intermediate with the DNA. Through an as yet, uncharacterized reaction, we have shown that Rep 78 can then join the 5'-end of the nicked DNA to the 3'-end of another DNA substrate. Therefore the biochemical requirements for AAV targeted integration may be satisfied by the known activities of the p5 Rep proteins. Overexpression of the p5 Rep proteins has adverse effects on cells in vitro. Cell growth rate is retarded and there is increased percentage of dead cells. We have recently demonstrated that Rep 78 induces apoptposis. Cell death was found to occur in G1 and early S phases of the cell cycle. In an attempt to identify the activities of Rep that contribute to apoptosis induction, a set of Rep 78 derivatives was overexpressed in cells. The results were unexpected: there was no difference between wild-type Rep 78 and a endonuclease deficient Rep 78 mutation, Y156F. Rep 52, expressed from p19, lacks the amino terminus of the p5 Rep proteins which contains the DNA binding and enduclease functions, was nearly as cytotoxic. Rep 78 K340H has a mutation within the conserved ATPase motif is intermediate in cytotoxicity. Therefore, it appears that Rep mediated apoptosis results from several Rep encoded activities: helicase/ATPase activities and DNA binding, but not endonuclease activity. Additional experiments have implicated interactions between Rep proteins and cellular proteins as contributing to cytotoxicity.

人类依赖病毒、腺相关病毒 (AAV) 需要与不相关的 DNA 病毒共同感染，例如腺病毒，有效感染细胞。 AAV 血清型 2 (AAV2) 是该属的原型。在没有辅助病毒的情况下，用 AAV2 体外感染细胞可能会导致病毒基因组整合。我们已经证明，AAV2 DNA 优先整合到人类 19 号染色体 q 臂的特定区域，即 AAVS1 基因座。靶向整合在真核病毒中是独一无二的，需要顺式作用和反式作用元件。 AAV 的非结构蛋白 Rep 是靶向整合所必需的。由图谱位置 5 的启动子（Rep 78 和 Rep 68）表达的 Rep 蛋白能够结合单链和双链 DNA 并切割双链和单链 DNA。 p5 Rep 蛋白在 DNA 底物存在下发生多聚化并可能形成六聚体。酪氨酸 156 是核酸内切酶活性所必需的，并且共价酪氨酰磷酸二酯作为 DNA 的中间体形成。通过尚未表征的反应，我们已经证明 Rep 78 可以将带切口的 DNA 的 5' 端连接到另一个 DNA 底物的 3' 端。因此，p5 Rep 蛋白的已知活性可以满足 AAV 靶向整合的生化要求。 p5 Rep 蛋白的过度表达对体外细胞有不利影响。细胞生长速度减慢，死亡细胞百分比增加。我们最近证明 Rep 78 会诱导细胞凋亡。发现细胞死亡发生在细胞周期的 G1 期和早期 S 期。为了确定 Rep 促进细胞凋亡诱导的活性，一组 Rep 78 衍生物在细胞中过表达。结果出人意料：野生型 Rep 78 和核酸内切酶缺陷型 Rep 78 突变 Y156F 之间没有差异。由 p19 表达的 Rep 52 缺乏 p5 Rep 蛋白的氨基末端，而 p5 Rep 蛋白含有 DNA 结合和核酸内切酶功能，几乎具有同样的细胞毒性。 Rep 78 K340H 在保守的 ATPase 基序内有一个突变，具有中等的细胞毒性。因此，看来Rep介导的细胞凋亡是由几种Rep编码的活性引起的：解旋酶/ATP酶活性和DNA结合，但不是核酸内切酶活性。其他实验表明 Rep 蛋白和细胞蛋白之间的相互作用会导致细胞毒性。