Adeno-Associated Virus Gene Therapy Vectors: Molecular Interactions on Cell Entry

项目说明 腺相关病毒(AAV)是一种主要的基因治疗载体，用于传递DNA以纠正遗传错误或 疾病的易感性。我们的目标是了解病毒与宿主之间的相互作用 进入。这个基础将支持广泛寻求的目标，即操纵细胞特异性，有效地转导 所需的细胞和减少偏离目标的副作用。这一目标推动了广泛的遗传和表型 特征，它的结构，为对病毒进入的一般洞察提供了一个特殊的范例。 我们的结合和siRNA研究对已发表的辅助受体提出了质疑，促使在 单倍体细胞系。在抗甲型肝炎病毒突变细胞中最常见的基因编码了一种蛋白质，我们将其命名为 AAVR与NM亲和力结合，抑制转导，对所有AAV血清型和细胞类型都是必不可少的 测试过。我们已经表达了AAV结合域，并将通过冷冻-DNA分析确定该复合体的结构。 在通过诱变进一步表征相互作用之前，进行电子显微镜(EM)检查。 屏幕上还牵涉到了其他基因。我们将确定哪些编码蛋白与AAV或 AAVR通过光诱导交联、下拉和质谱学相互作用鉴定 域名。这些将被表达用于结合分析和混合x射线/EM结构。基因陷阱表明 潜在的合作伙伴，不仅在细胞表面，而且在AAV向核周围的跨高尔基体贩运期间 这将阐明AAV的磷脂酶A2结构域是如何被释放以实现内体逃逸的。 将开发适用于AAV的EM方法，使其具有通用性。对于混合结构的改进，我们的 地图拟合优化将包含对模型灵活性的简约约束，以避免常见的问题 在中等分辨率下的过度拟合。通过基于模型的方法，改进差分地图分析 校准，以提高小配体和微妙构象变化的灵敏度 用高分辨电子显微镜进行分析。这将适用于AAV与胞外多聚糖结合的研究， 或与其他实验室的离子通道、核糖体或酶复合体的配体结合有关。 在MIRA框架内，将继续为蛋白质动力学的合作研究作出贡献。这些是 将晶体结构与核磁共振弛豫色散和剩余偶极耦合相结合来表征速率。 当精氨酸激酶翻转时，限制其毫秒/微秒的蛋白质运动。我们的模型系统很有启发性 对内在运动和构象选择的一般原理知之甚少。这些基本原则 问题是主要的目标，但研究也反馈到方法的发展。核磁共振 松弛交换数据允许进行验证测试，即我们的简约模型参数化能捕获真实 构象变化。总而言之，我们将继续致力于挑战结构生物学， 推动开发广泛适用的方法论的基本和应用目标。

Project Description Adeno-associated virus (AAV) is a leading gene therapy vector for delivery of DNA to correct genetic errors or predispositions to disease. Our objective is an understanding of the virus-host interactions that mediate cell entry. This foundation will support the widely sought goal of manipulating cell specificity, efficiently transducing desired cells and reducing off-target side effects. This goal has motivated extensive genetic and phenotypic characterization, which, with structure, provide an exceptional paradigm for general insights into viral entry. Our binding and siRNA studies cast doubt on published co-receptors, motivating a gene trap screen in a haploid cell line. The gene most frequently hit in AAV-resistant mutant cells, encoded a protein that we named AAVR which binds with nM affinity, inhibits transduction, and is essential for all AAV serotypes and cell types tested. We have expressed the AAV-binding domains, and will determine a structure of the complex by cryo- electron microscopy (EM) before further characterizing the interactions through mutagenesis. Other genes were implicated by the screen. We will determine which encoded proteins interact with AAV or AAVR through photo-induced cross-linking, pull-down, and mass spectrometric identification of interacting domains. These will be expressed for binding analysis, and hybrid x-ray/EM structure. The gene trap indicates potential partners, not just at the cell surface, but during AAV's trafficking to the peri-nuclear trans Golgi network, and which will illuminate how AAV's phospholipase A2 domain is released for endosomal escape. EM methods, applicable to AAV, will be developed for general use. For refinement of hybrid structures, our map-fitting optimization will incorporate parsimony restraints on model flexibility to avoid the common problem of over-fitting at intermediate resolution. Difference map analysis will be improved, through model-based calibration, to enhance the sensitivity with which small ligands and subtle conformational changes can be analyzed by high resolution EM. This will be applicable to studies of AAV's attachment to extracellular glycans, or to the binding of ligands to ion channels, ribosomes or enzyme complexes in other laboratories. Within the MIRA framework, contributions to collaborative studies of protein dynamics will continue. These are integrating crystal structure with NMR relaxation dispersion and residual dipolar couplings to characterize rate- limiting milli-/micro-second protein motions in arginine kinase as it turns over. Our model system is illuminating poorly understood general principles of intrinsic motions and conformational selection. These fundamental questions are the primary goal, but the research also feeds back into methods development. The NMR relaxation exchange data allows validation tests that our parsimonious model parameterization captures real conformational changes. In summary, we will continue to work on challenging structural biology with both fundamental and applied goals that drive the development of widely applicable methodology.