Integration of Murine Retroviral Vectors

描述（由申请人提供）：由于逆转录病毒插入激活，基因治疗方案存在潜在的基因激活风险。如果对逆转录病毒整合缺乏基本了解，则无法成功实现重新定位或限制逆转录病毒整合位点的能力。该提案研究了小鼠白血病病毒（MuLV）的整合，这是基因治疗方案中最常见的逆转录病毒载体。整合是病毒生命周期中的关键步骤，其导致病毒基因组在整个宿主DNA中的稳定插入。不存在切除整合的前病毒的机制，并且整合可导致细胞基因的破坏和/或活化。 该建议利用生物化学和遗传学方法来定义协调蛋白质-DNA整合复合物组装的相互作用。提出了三个具体的目标，其中审查了一体化综合体的各个组成部分。第一个目标集中在病毒整合酶（IN）蛋白，催化酯交换过程。这些研究旨在确定与病毒和靶DNA组装突触复合物所需的蛋白质结构域。第二个特定目的定义了病毒底物的结构和/或序列要求，即LTR末端的反向末端重复序列。已经开发了在MuLV LTR末端的小沟内序列特异性结合的新型聚酰胺。将检查这些化合物对MuLV LTR结构的影响。第三个特定目的修饰MuLV IN蛋白，以包括被认为是转录失活的指导整合到异染色质的序列。这种方法将使酵母Ty 5系统鉴定的机制适用于逆转录病毒颗粒。将检查Ty 5标签靶向逆转录病毒整合的潜力。迄今为止，还没有已知的逆转录病毒整合酶与靶或病毒DNA的结构。这些研究检查了蛋白质-DNA识别的关键方面，这些方面是识别稳定的突触复合物以进行结构和功能分析所需的。 这种逆转录病毒整合的知识对于进一步操纵以靶向和/或抑制病毒生命周期中的这一必要步骤是必要的。

DESCRIPTION (provided by applicant): Gene therapy protocols risk the potential of gene activation due to retroviral insertional activation. The ability to retarget or limit the sites of retroviral integration though cannot be successfully achieved in the absence of the basic understanding of retroviral integration. This proposal studies the integration of murine leukemia virus (MuLV), the most common retroviral vector used in gene therapy protocols. Integration is a critical step in the viral life cycle which results in the stable insertion of the viral genome throughout the host DNA. There is no mechanism of excision of an integrated provirus and integration can lead to the disruption and/or activation of cellular genes. The proposal utilizes biochemical and genetic approaches to define the interactions which orchestrate the assembly of protein-DNA integrative complex. Three specific aims are proposed which examine individual components of the integration complex. The first aim focuses on the viral Integrase (IN) protein, which catalyzes the transesterification process. These studies aim at defining the domains of the protein required to assemble a synaptic complex with the viral and target DNAs. The second specific aim defines the structural and/or sequence requirements of the viral substrate, the inverted terminal repeats at the ends of the LTRs. Novel polyamides which sequence specifically bind within the minor groove of the MuLV LTR termini have been developed. The effect of these compounds on the structure of the MuLV LTR will be examined. The third specific aim modifies the MuLV IN protein to include sequences found to direct integration to heterochromatin, considered to be transcriptionally inactive. This approach will adapt the mechanism identified for the yeast Ty5 system to the retroviral particles. The potential of the Ty5 tag to target retroviral integration will be examined. To date, there is no known structure of a retroviral integrase with either the target or viral DNA. These studies examine critical aspects of the protein-DNA recognition needed to identify a stable synaptic complex for structural and functional analysis. This knowledge of retroviral integration is necessary for further manipulation to target and/or inhibit this requisite step in the viral life cycle.