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Mechanisms of prion strain dynamics

朊病毒应变动力学机制

基本信息

批准号：
9789977
负责人：
Jason C Bartz
金额：
$ 38.75万
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 2023-06-30
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.

Prion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病，没有已知的治疗方法或治愈方法。如果不是全部的话，PrPSc是由PrPSc组成的，PrPSc是正常蛋白质的一种错误折叠形式非传染性病毒蛋白PrPC。Pron菌株在操作上是由神经病理学的差异来定义的在由PrPSc的菌株特定构象编码的受控条件下繁殖True。长的- 这项工作的学期目标是了解Pron菌株的动力学。此应用程序的目标是确定Pron菌株是以亚种的混合物形式存在，还是以同质种群的形式存在。我们假设 Pron菌株是Prion亚种(即准种)的动态混合。这将从根本上影响了我们对物种间传播机制的理解，适应了Pron对抗病毒药物、自然环境中病毒传播的生态学以及可能与其他蛋白质有关错误折叠的疾病，具有类似蛋白菌株的特征(例如阿尔茨海默氏症)。由于PrPSc的结构是定义不明确，而且没有技术来测量混合物中单个蛋白质的结构，我们将根据实验上可行的准物种的预测特性来检验这一假设。第一我们将确定反复限制稀释对Pron适合性的影响。反复出现的瓶颈通道准种会导致适合度的降低(即穆勒棘轮)，因为平均而言，个体的适合度与总体人口的适合度相比，感染单位处于较低的水平。我们预计这种情况会反复发生在PMCA中或在细胞培养中以有限稀释度传播普恩病毒将导致 Pron试剂与高滴度传代的试剂相比。第二，我们将确定是否通过生物克隆的普恩病毒菌株含有亚株。准物种假说预测，任何给定的普恩病毒株都由一个亚株的动态种群。我们将通过在体外扩增PrPSc或在细胞中传代来测试这一点有利于选择次要亚种的条件。在第二系列实验中，我们将选择具有共同生化特征的PrPSc亚群，可导致PMCA反应或PrPSC 易感RK13细胞。经过一系列重复的选择，然后在动物身上传递，将确定如果生物克隆的Prion菌株包含额外的菌株，并且如果生化选择压力导致出现具有所选特性的菌株。这些实验的结果将直接考验普恩是准种的假说。