Mechanisms of prion strain dynamics

朊病毒应变动力学机制

基本信息

批准号：
10450690
负责人：
Jason C Bartz
金额：
$ 37.06万
依托单位：
CREIGHTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-30 至 2024-06-30
项目状态：
已结题

项目摘要

Prion diseases are inevitably fatal neurodegenerative zoonotic disorders of animals, including humans, with no known treatment or cure. Prions are comprised largely, if not entirely, of PrPSc, a misfolded form of the normal non-infectious prion protein PrPC. Prion strains are operationally defined by differences in neuropathology that breed true under controlled conditions that are encoded by strain-specific conformations of PrPSc. The long- term goal of this work is to understand the dynamics of prion strains. The objective of this application is to determine if prion strains exist as a mixture of substrains or as a homogeneous population. We hypothesize that prions strains are a dynamic mixture of prion substrains (i.e. quasispecies). This would fundamentally impact our understanding of the mechanism of interspecies transmission, adaptation of prions in response to anti-prion drugs, the ecology of prion transmission in natural settings and may be relevant to other protein misfolding diseases that share prion strain-like features (e.g. Alzheimer’s). Since the structure of PrPSc is poorly defined and technologies do not exist to measure the structure of an individual protein in a mixture, we will test the hypothesis based on the predicted properties of quasispecies that are experimentally feasible. First we will determine the effect of repeated limiting dilutions on prion fitness. Repeated bottleneck passage of a quasispecies leads to a reduction in fitness (i.e. Muller’s ratchet) since, on average, the fitness of an individual infectious unit is lower compared to the fitness of the overall population. We anticipate that repeated transmission of prions at limiting dilutions in PMCA or in cell culture will result in a reduction in the fitness of the prion agent compared to agent passaged at high titer. Second we will establish if biologically cloned prion strains contain substrains. Quasispecies hypothesis predicts that any given prion strain is comprised of a dynamic population of substrains. We will test this by amplifying PrPSc in vitro or passaging in cells under conditions that favor the selection of a minor substrain. In a second series of experiments, we will select for subpopulations of PrPSc that have common shared biochemical features to seed PMCA reactions or prion susceptible RK13 cells. Serial repeated rounds of selection followed by transmission in animals will determine if biologically cloned prion strains contain additional strains and if biochemical selective pressure results in the emergence of strains with the selected properties. The results of these experiments will directly test the hypothesis that prions are quasispecies.

朊病毒病是包括人类在内的动物不可避免的致命性神经退行性人畜共患疾病，没有已知的治疗方法或治愈方法主要是（如果不是全部的话）PrPSc，这是正常蛋白的错误折叠形式。非感染性朊病毒蛋白 PrPC 在操作上是通过神经病理学差异来定义的。在由 PrPSc 菌株特异性构象编码的受控条件下真正繁殖。这项工作的长期目标是了解朊病毒菌株的动态。确定朊病毒株是作为亚株的混合物还是作为同质群体存在。朊病毒株是朊病毒亚株的动态混合物（即准种）。影响我们对种间传播机制的理解，朊病毒的适应抗朊病毒药物、自然环境中朊病毒传播的生态学，可能与其他蛋白质有关具有类似朊病毒株特征的错误折叠疾病（例如阿尔茨海默病），因为 PrPSc 的结构是由于定义不明确并且不存在测量混合物中单个蛋白质结构的技术，我们首先将根据实验上可行的准种的预测特性来检验假设。我们将确定重复有限稀释对朊病毒适应性的重复瓶颈通过的影响。准物种会导致适应度下降（即穆勒棘轮），因为平均而言，个体的适应度与总体人口的适应度相比，我们预计这种情况会重复出现。朊病毒在 PMCA 或细胞培养物中的有限稀释度下的传播将导致细胞适应性的降低其次，我们将确定朊病毒是否是生物克隆的。菌株包含亚菌株。准种假说预测任何给定的朊病毒菌株都包含一个亚菌株。我们将通过体外扩增 PrPSc 或在细胞中传递来测试这一点。在第二系列实验中，我们将选择有利于选择次要亚菌株的条件。 PrPSc 亚群具有共同的生化特征，可引发 PMCA 反应或朊病毒可疑的 RK13 细胞将通过一系列重复的选择然后在动物中传播来确定。生物克隆的朊病毒菌株是否含有其他菌株，并且生化选择压力是否导致这些实验的结果将直接检验具有选定特性的菌株的出现。朊病毒是准种的假设。