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

摘要 此MIRA应用程序结合了三个NIGMS奖项R01GM070837，1R01GM110639， 和5R01GM108487，研究小鼠白血病病毒（MLV）的系留和整合 前整合复合体（PIC）。γ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，邻近 转录起始位点（TSS）和CpG岛。有丝分裂的要求限制了 这些载体到分裂的细胞。整合的位点与肿瘤细胞的致癌潜力有关。 病毒及其致病机理以及作为基因递送载体的用途。 研究集中在三个目标领域。p12蛋白是连接细胞所必需的。 有丝分裂染色体的PIC。最近对MLV p12拴系特性的分析揭示了 抑制p12蛋白N-末端束缚定位的新机制。一 提出了一种模型，该模型将前体Gag蛋白的方向反转纳入到 在PIC中发现的，通过中间体过渡，在该中间体中， 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 生物化学和结构的方法，包括NMR分析的p12拴系结构域。 我们的合作研究已经确定了宿主BET蛋白质家族，特别是Brd 2、3和4蛋白作为与MLV IN的相互作用物。MLV IN C-之间的相互作用 末端尾肽和ET结构域影响整合到TSS和CpG岛中。 提出的研究建立在这种新定义的病毒-宿主相互作用的基础上。以解决方案为基础 从我们实验室的MLV IN CTD的结构，实验的目的是定义 包括MLV IN CTD：Brd3 ET复合物在内的较大复合物的结构数据，以及 与DNA结合的IN结构域。MLV IN蛋白与已知ET竞争的能力 将研究域相互作用者。将使用下一代测序和生物信息学 为了探测一组IN蛋白的靶位点偏好， 整合，以及全球范围内的人类基因组。目标DNA的识别 在核小体结构内进行检查。 这些研究将与我们实验室的两个新兴领域相结合。第一 开发了基于MLV的病毒样颗粒（VLP），用于将蛋白质而不是基因递送到 细胞第二个研究领域开发了一种改进的Env系统， scFv和单体作为靶向部分。该系统可以推广和扩展 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.