A chimeric protein containing the N terminus of the adeno-associated virus rep protein recognizes its target site in an in vivo assay
A chimeric protein containing the N terminus of the adeno-associated virus rep protein recognizes its target site in an in vivo assay
复制标题
DOI：
10.1128/jvi.74.5.2372-2382.2000
复制
发表时间：
2000-03-01
影响因子：
5.4
通讯作者：
Weitzman, MD
中科院分区：
文献类型：
--
作者：
Cathomen, T;Collete, D;Weitzman, MD
2 型腺相关病毒 (AAV) 的 Rep78 和 Rep68 蛋白参与 DNA 复制、基因表达调节和靶向位点特异性整合。它们与病毒反向末端重复序列和人类 19 号染色体上的 AAVS1 整合位点中发现的特定 Rep 识别序列 (RRS) 结合。之前的体外研究表明 Rep 的 N 末端片段参与 DNA 识别，而其他结构域可能会稳定结合并介导多聚化。为了确定 Rep 识别其在人类基因组中的靶位点的最低要求，我们开发了一种单杂交测定法，在体内检测 DNA-蛋白质相互作用。嵌合蛋白由与不同寡聚化基序融合的 Rep N 末端和转录激活结构域组成，分析了寡聚化、DNA 结合和报告基因表达的激活。报告基因的表达由克隆在最小启动子上游的 RRS 基序驱动，并在哺乳动物细胞中通过转染的质粒进行检测，在酿酒酵母中通过整合到酵母基因组中的报告盒进行检测。我们的结果首次表明，含有 Rep 氨基末端 244 个残基的嵌合蛋白在掺入人工多聚体时能够在体外和体内靶向 RRS。这些研究表明嵌合蛋白可用于利用 AAV 的独特靶向特征进行基因治疗应用。
The Rep78 and Rep68 proteins of adeno-associated virus (AAV) type 2 are involved in DNA replication, regulation of gene expression, and targeting site-specific integration. They bind to a specific Rep recognition sequence (RRS) found in both the viral inverted terminal repeats and the AAVS1 integration locus on human chromosome 19. Previous in vitro studies implied that an N-terminal segment of Rep is involved in DNA recognition, while additional domains might stabilize binding and mediate multimerization. In order to define the minimal requirements for Rep to recognize its target site in the human genome, we developed one-hybrid assays in which DNA-protein interactions are detected in vivo. Chimeric proteins consisting of the N terminus of Rep fused to different oligomerization motifs and a transcriptional activation domain were analyzed for oligomerization, DNA binding, and activation of reporter gene expression. Expression of reporter genes was driven from RRS motifs cloned upstream of minimal promoters and examined in mammalian cells from transfected plasmids and in Saccharomyces cerevisiae from a reporter cassette integrated into the yeast genome. Our results show for the first time that chimeric proteins containing the amino-terminal 244 residues of Rep are able to target the RRS in vitro and in vivo when incorporated into artificial multimers. These studies suggest that chimeric proteins may be used to harness the unique targeting feature of AAV for gene therapy applications.