Characterization Of Adeno Associated Virus Non-structura

人类依赖病毒腺相关病毒（AAV）需要与非相关DNA病毒（如腺病毒）共同感染才能有效感染细胞。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体外感染细胞可能导致病毒基因组的整合。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，AAVS1位点。靶向整合在真核病毒中是独特的，需要顺式和反式作用的元件。AAV的非结构蛋白Rep是靶向整合所必需的。在图位5的启动子Rep 78和Rep 68上表达的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底物的存在下多聚并可形成六聚体。酪氨酸156是核酸内切酶活性所必需的，与DNA形成共价酪氨酸-磷酸二酯作为中间体。通过迄今为止尚未表征的反应，我们已经证明Rep 78可以将缺口DNA的5‘端连接到另一个DNA底物的3’端。因此，已知p5 Rep蛋白的活性可以满足AAV靶向整合的生化要求。过表达p5 Rep蛋白对体外细胞有不良影响。细胞生长速度减慢，死亡细胞比例增加。我们最近证明Rep 78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。为了鉴定Rep在诱导细胞凋亡中的作用，我们在细胞中过量表达了一组Rep 78衍生物。结果出乎意料：野生型Rep 78和内切酶缺陷型Rep 78突变体Y156F之间没有差异。由p19表达的Rep 52缺乏p5 Rep蛋白的氨基端，其具有DNA结合和诱导酶的功能，几乎具有细胞毒性。Rep 78 K340H在保守的atp酶基序中有一个突变，在细胞毒性中处于中间位置。因此，Rep介导的细胞凋亡似乎是由几个Rep编码的活性引起的：解旋酶/ atp酶活性和DNA结合，而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有助于细胞毒性。

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.