CHARACTERIZATION OF THE NONSTRUCTURAL PROTEINS OF ADENO ASSOCIATED VIRUS

腺相关病毒非结构蛋白的表征

• 批准号: 2576788
• 负责人: R 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/2576788
• 关键词: DNA binding protein; DNA replication origin; adeno associated virus group; chromatography; gene induction /repression; genetic promoter element; introns; microorganism culture; nucleic acid sequence; open reading frames; protein structure function; transcription factor; virus genetics; virus protein

The four non-structural proteins (Rep) of adeno-associated virus, AAV, are encoded by a single open reading frame, the rep gene, and differ from each other by utilization of an downstream promoter and excision of an intron. The two larger Rep proteins function as replication origin binding and initiation proteins and are required for viral DNA synthesis. Previously, we have demonstrated that the two larger Rep proteins, Rep 68 and Rep 78, are DNA binding proteins that recognize a sequence motif, the prototype is the GAGC repeat within the AAV inverted terminal repeat (ITR). The DNA binding ability of Rep proteins may have explained the regulatory effects of Rep proteins on gene expression that has been observed by several laboratories. However, this effect appears to be independent of the presence of a canonical Rep binding site within the regulatory region of the transcribed gene. Other cellular phenotypes associated with Rep 68 or Rep 78 expression include: loss of viability, inhibition of cell division, and inhibition of transformation by viral oncogenes. These effects may be induced by Rep association with a cellular protein. Domains of Rep proteins that are necessary for Rep oligomerization have been reported previously. However, the residues that are specifically required for these interactions have not been defined. In order to identify which residues are involved with inter-protein interactions, we have made a series of mutations altering a single residue Rep 78. Our results with analytical chromatography demonstrate that Rep 78 or Rep 68 forms a complex with properties consistent with a hexamer similar to other DNA helicase complexes. Defining how a polypeptide interacts with other proteins enables us to generalize and predict what other interactions may occur. Binding of Rep to epitopes displayed on bacteriophage has resulted in selection of clones with a commonly repeated motif. We are in the process of determining whether cellular proteins containing these epitopes interact with Rep 78 and if so, what are the effects on the cellular processes.

腺相关病毒的四种非结构蛋白(Rep)，AAV， 由单个开放阅读框rep基因编码，不同于 通过利用下游启动子和切除 内含子。两个较大的Rep蛋白起复制起点的作用 结合蛋白和启动蛋白，是病毒DNA合成所必需的。 之前，我们已经证明了两个更大的Rep蛋白，Rep 68和Rep 78是识别序列基序的DNA结合蛋白， 原型是AAV反向末端重复内的GAGC重复 (ITR)。Rep蛋白的DNA结合能力可能解释了 Rep蛋白对已有基因表达的调控作用 被几个实验室观察到的。然而，这种影响似乎是 与规范的Rep结合位点的存在无关 转录基因的调控区。其他细胞表型 与Rep 68或Rep 78表达相关的包括：生存能力丧失， 抑制细胞分裂，抑制病毒转化 致癌基因。这些影响可能是由代表与一个 细胞蛋白质。 Rep寡聚所必需的Rep蛋白结构域有 之前就有过报道。然而，这些残留物特别是 尚未定义这些交互所需的。为了 确定哪些残基参与蛋白质间的相互作用，我们 已经造成了一系列突变，改变了单一残基代表78。我们的 分析层析结果表明，Rep 78或Rep 68 形成一个复合体，其属性与类似于 其他DNA解旋酶复合体。定义多肽如何与之相互作用 其他蛋白质使我们能够概括和预测 可能会发生相互作用。Rep与上显示的表位的结合 噬菌体导致了对克隆的选择，这些克隆通常 重复的主题。我们正在确定细胞是否 含有这些表位的蛋白质与Rep 78相互作用，如果是，是什么 是对细胞过程的影响。