Targeting retroviral and virus-like particles for gene and protein delivery

摘要 该MIRA应用程序结合了三个NIGMS奖项R01GM070837、1R01GM110639、 和5R01GM108487，研究了小鼠白血病病毒(MLV)的捆绑和整合。 前整合复合体(PIC)伽玛逆转录病毒需要细胞进行有丝分裂 整合并优先整合在活性启动子区域内，靠近 转录起始点(TSS)和CpG岛。对有丝分裂的要求限制了对 这些向量导致细胞分裂。整合部位与细胞的致癌潜能有关。 病毒及其致病机制，并用作基因输送载体。 研究集中在三个有针对性的领域。P12蛋白是拴系细胞所必需的 图为有丝分裂染色体。最近对MLV p12系留特性的分析揭示了 抑制定位于p12蛋白N末端的拴系的新机制。一个 提出了一个模型，该模型结合了从前体Gag蛋白到 这是在PIC中发现的，该PIC通过其中系留域是 被N-和C-末端电荷相互作用抑制。测试该模型的实验将涉及 生化和结构方法，包括p12系留域的核磁共振分析。 我们的合作研究已经确定了宿主BET蛋白家族，特别是Brd 2，3和4蛋白作为MLV IN的相互作用因子。C-MLV分子间的相互作用 末端尾肽和ET结构域影响TSS和CpG岛的整合。 建议的研究建立在这种新定义的病毒-宿主相互作用的基础上。在解决方案的基础上构建 CTD中MLV的结构来自我们实验室，实验的目的是确定 包括CTD：Brd3Et络合物的MLV在内的较大络合物的结构数据，以及 与DNA结合的IN结构域。MLV IN蛋白与已知ET竞争的能力 将研究域交联体。将使用下一代测序和生物信息学 为了探测一组IN蛋白质的靶点偏好，这两个位点都在本地 人类基因组中的整合以及全球整合。靶DNA的识别 在核小体结构内进行了检查。 这些研究将与我们实验室的两个新兴领域结合起来。第一 开发基于MLV的病毒样颗粒(VLP)，用于运送蛋白质，而不是基因进入 细胞。第二个研究领域开发了一种改进的环境系统，允许通过 单链抗体和单体靶向部分。该系统具有一定的通用性和可扩展性 以解决广泛的应用和疾病。

Abstract This MIRA application combines three NIGMS awards R01GM070837, 1R01GM110639, and 5R01GM108487, studying the tethering and integration the murine leukemia virus (MLV) preintegrative complex (PIC). Gammaretroviruses require cells to undergo mitosis for integration and preferentially integrate within active promoters regions, proximal to transcriptional start sites (TSS) and CpG islands. The requirement for mitosis limits the use of these vectors to dividing cells. The site of integration is linked to the oncogenic potential of the virus and thus its pathogenesis and use as a gene delivery vector. Studies are focused on three targeted areas. The p12 protein is required for tethering the PIC to mitotic chromosomes. Recent analysis of MLV p12 tethering properties has revealed a novel mechanism of suppression of tethering localizing in the N-terminus of the p12 protein. A model is proposed that incorporates an orientation reversal from the precursor Gag protein to that found in the PIC that transitions through an intermediate in which the tethering domain is inhibited by N- and C-terminal charge interactions. Experiment testing this model will involve biochemical and structural approaches, including NMR analysis of the p12 tethering domain. Our collaborative research has identified the host BET family of proteins, specifically the Brd 2,3, and 4 proteins as interactors with the MLV IN. The interaction between the MLV IN C- terminal tail peptide and the ET domain influences the integration into TSS and CpG islands. Studies proposed build on this newly defined viral-host interaction. Building on the solution structure of the MLV IN CTD from our laboratory, experiments are aimed at defining the structural data of larger complexes including the MLV IN CTD:Brd3 ET complex, and the domains of IN bound to DNA. The ability of the MLV IN protein to compete with known ET domain interactors will be studied. Next-generation sequencing and bioinformatics will be used to probe a panel of IN proteins for their target-site preferences, both locally at the site of integration as well as globally within the human genome. The recognition of the target DNA within the nucleosome structure is examined. These studies will be combined with two emerging areas in our laboratory. The first develops MLV based virus-like particles (VLP) for the delivery of proteins, rather than genes into cells. The second area of research develops a modified Env system that allows entry through scFvs and monobodies as targeting moieties. This system could be generalized and expanded to address a wide range of applications and diseases.