CHARACTERIZATION OF THE NONSTRUCTURAL PROTEINS OF ADENO ASSOCIATED VIRUS

I.代表78的自我联系。新城疫病毒复制启动蛋白 AAV-2、Rep78和Rep68是结合病毒的必需基因产物 病毒复制起点(ORI)，在某个位置截取ORI DNA-和 链特异性方式(因此提供了3‘-OH基团 启动病毒DNA合成)，并随后解开病毒基因组 在前进的聚合酶复合体前面的双链。我们已经调查了 Rep78在体外和体内的自我结合能力。 利用定点Rep78突变体鉴定了两个必需的基序 到寡聚化。这些是i)一个假定的螺旋，由一个 AA残基中的3，4-疏水七肽重复序列(或卷曲结构域) 160-185，以及ii)先前已知的核苷三磷酸 (NTP)结合基序出现在AA残基332-346中。该组件 凝胶过滤确定Rep78低聚物的状态 在有或没有ORI底物的情况下和通过 化学交联分析。这些实验证明， 在缺乏DNA的情况下，Rep78主要以单体形式存在 底物；然而，在AAV ORI序列存在的情况下，Rep78形成 一种斯托克斯半径约6.3 nm的六聚体蛋白质复合体。 这些结果表明Rep78是一种六聚体解旋酶。 异源分子间相互作用。的瞬时表达 Rep有一种戏剧性的表型影响，从抑制细胞 基因表达对细胞停滞和细胞死亡的影响。Rep诱导的表型 可能部分归因于Rep的DNA结合活性， 然而，由于缺少Rep结合序列启动子也 由于受到压制，需要另一种解释。那么很有可能， 这些效应是由于代表和细胞之间的相互作用 蛋白质。使用酵母双杂交筛选系统，几个cDNA 被分离出来，编码与代表相互作用的蛋白质。 这些候选蛋白是一种与Rep 52结合的新型细胞激酶。代表 52和Rep 78很容易与激酶共沉淀，而 Rep 68只有微弱的免疫共沉淀。命题分析 确认Rep 52和Rep 78的羧基末端是必需的 进行互动。它的反式和自体磷酸化活性 蛋白激酶被Rep 52抑制。因此，我们已经确定了一种细胞 与未剪接和剪接的差异相互作用的激酶 代表的形式，即分别代表78、52和68、40。

I. Self-association of Rep 78. The replication initiator proteins of AAV-2, Rep78 and Rep68, are essential viral gene products that bind the viral origin of replication (ori), nick the ori DNA in a site- and strand-specific fashion (thus providing a 3' -OH group with which to prime viral DNA synthesis), and subsequently unwind the viral genomic duplex ahead of the advancing polymerase complex. We have investigated the ability of Rep78 to self-associate in vitro and in vivo. Site-directed Rep78 mutants were used to identify two motifs essential to oligomerization. These are i) a putative helix consisting of a 3,4-hydrophobic heptad repeat (or coiled-coil domain) within aa residues 160-185, and ii) a previously recognized nucleoside triphosphate (NTP)-binding motif occurring within aa residues 332-346. The assembly state of Rep78 oligomers was ascertained by gel filtration chromatography in the presence or absence of ori substrate and by chemical cross-linking analyses. These experiments demonstrated that Rep78 exists predominantly as a monomeric species in the absence of DNA substrate; however, in the presence of AAV ori sequences, Rep78 forms a hexameric protein complex with a Stokes radius of approximately 6.3nm. These results demonstrate that Rep78 is a hexameric helicase. II. Heterologous intermolecular interactions. Transient expression of Rep has a dramatic phenotypic affect ranging from repression of cellular gene expression to cytostasis and cell death. The Rep induced phenotype may be attributable in part to the DNA binding activities of Rep, however, since promoters which lack a Rep binding sequence are also repressed, an alternative explanation is required. It is likely then, that these effects are due to interactions between Rep and cellular proteins. Using a yeast two hybrid screening system, several cDNAs have been isolated which encode proteins that interact with Rep. One of these candidates is a novel cellular kinase that binds to Rep 52. Rep 52 and Rep 78 are readily co-immunoprecipitated with the kinase whereas Rep 68 is only weakly co-immunoprecipitated. Deletional analysis confirms that the carboxy terminus of Rep 52 and Rep 78 are necessary for the interaction. Trans- and auto- phosphorylation activity of this kinase is inhibited by Rep 52. Thus, we have identified a cellular kinase that differentially interacts with the unspliced and spliced forms of Rep, i.e. Rep 78, Rep 52 and Rep 68, Rep 40 respectively.