DISRUPTING VIRAL GENES WITH GROUP II INTRONS

描述：本提案的总体目标是评估 移动的II组内含子破坏哺乳动物细胞中的基因，重点是HIV和 CCR 5基因作为靶基因。第二组内含子的移动是由一个 核糖核蛋白（RNP）由一个切除的内含子RNA酶和一个 内含子编码的蛋白质，具有逆转录酶、DNA内切酶和RNA 剪接成熟酶活性。乳酸乳球菌II群的迁移率 内含子通过两步过程发生。首先，内含子催化 将其自身反向剪接到DNA靶位点，然后将内含子RNA 由内含子编码的蛋白质逆转录成DNA。插入部位 DNA上的内含子RNA通过约14个碱基对识别， 内含子编码的蛋白质最近，我们证明了第二组内含子可以 被改造为位点特异性插入HIV- 1聚合物中的靶向DNA位点 和CCR 5基因通过修饰存在于内含子RNA上的DNA结合位点。 这些重定向的内含子保留了插入和破坏它们的能力。 当它们被瞬时转染到哺乳动物细胞中时， 与它们的内含子编码蛋白复合。具体目标是 建议是：1）继续探索II组内含子的能力， 通过位点特异性DNA插入破坏HIV和CCR 5基因， 将重建的II组RNP转染到哺乳动物细胞中，2） 评估哺乳动物细胞中II组内含子剪接和迁移率， 内含子RNA和内含子编码的蛋白质的细胞内表达，3） 描述乳酸乳球菌II组内含子的序列要求 在哺乳动物细胞中的移动性。4)评价II组RNP的能力， 通过位点特异性地插入病毒DNA来消除HIV前病毒， 感染的细胞并使细胞对HIV感染和复制具有抗性。 这些研究也将为移动的II组的开发奠定基础 在哺乳动物细胞中用于靶向基因破坏的内含子， 研究应用。

DESCRIPTION: The overall goal of this proposal is to evaluate the ability of mobile group II introns to disrupt genes in mammalian cells focusing on HIV and CCR5 genes as targets. Group II intron mobility is mediated by a ribonucleoprotein (RNP) composed of an excised intron RNA lariat and an intron-encoded protein that has reverse transcriptase, DNA endonuclease and RNA splicing maturase activities. Mobility of the Lactococcus lactis group II intron occurs through a two-step process. First the intron catalyzes the reverse-splicing of itself into a DNA target site and then the intron RNA is reverse transcribed into DNA by the intron encoded protein. The insertion site on DNA is recognized by both the intron RNA, through about l4 base pairs, and the intron-encoded protein. Recently, we demonstrated that group II introns can be engineered to site specifically insert into targeted DNA sites in HIV- 1 pol and CCR5 genes by modifying the DNA binding sites present on the intron RNA. These retargeted introns retain the ability to insert into and disrupt their targeted DNA sites when they are transiently transfected into mammalian cells in complex with their intron-encoded protein. The specific aims of this proposal are: 1) To continue to explore the ability of group II introns to disrupt HTV and CCR5 genes via site specific DNA insertion following transfection of reconstituted group II RNPs into mammalian cells, 2) To evaluate group II intron splicing and mobility in mammalian cells following intracellular expression of the intron RNA and the intron-encoded protein, 3) To delineate the sequence requirements for Lactococcus lactis group II intron mobility in mammalian cells. 4) To evaluate the ability of group II RNPs to inactivate HIV proviruses by site-specifically inserting into viral DNA in infected cells and to render cells resistant to HIV infection and replication. These studies will also lay the foundation for development of mobile group II introns for targeted gene disruption in mammalian cells for therapeutic arid research applications.