Emergence of drug resistance in prion populations

摘要 朊病毒是引起传染性海绵状脑病的病原体，其独特之处在于 它们主要（如果不是完全）由 PrPSc 的多聚体组成，PrPSc 是宿主的构象异构体 编码蛋白质PrPC。朊病毒以多种菌株的形式存在，其不同之处在于 表型特性，但其 PrPSc 具有相同的氨基酸序列。据信 菌株身份是由 PrPSc 的构象编码的，并且有许多不同的 存在应变时的构象。 我们观察到，一种朊病毒株 22L，当从大脑转移到细胞培养物时，改变了其 特征，但当重新引入大脑时逐渐恢复它们。而且，当 在存在抑制药物苦马豆素（朊病毒群）的情况下在细胞培养物中繁殖 获得了对该药物的耐药性，但长时间传播后失去了耐药性 没有它的时期。考虑到细菌和病毒获得 耐药性是其遗传物质随机突变的结果，而朊病毒 不包含信息核酸。 我们的工作假设是，朊病毒株群体并非由具有独特特征的 PrPSc 组成。 构象，但具有相关构象的多种物种（“亚株”）。这是 类似于我们三十年来首次为 RNA 噬菌体建立的“准种”概念 以前。我们假设亚株的复制率可能因宿主细胞而异。在 在特定的环境中，这些物种之一可能代表主要成分和输出 复制其他构象异构体，但同时不断产生变异构象异构体 以较低的速率（因为相互转化的活化能垒很低）。之上 转移到不同的宿主或环境发生变化，不同的构象异构体可能会成为 人口的主要组成部分。 我们希望确定 PrPSc 是否与耐药性和药物敏感性朊病毒相关 显示出可能反映不同构象的物理化学差异，以及是否 两种类型的朊病毒在宿主细胞中以不同的速度复制。我们将调查是否存在朊病毒 可以对其他抑制药物产生耐药性。重要的是，我们将确定是否 从未接触过苦马豆素的朊病毒群体已经具有耐药性 正如“准种”假说所预期的那样。我们打算从生物学角度 克隆药物敏感的朊病毒并确定在过程中是否出现苦马豆素抗性变体 在没有药物的情况下繁殖，如果有的话，繁殖速度是多少。最后，我们将确定是否 可以选择对离液剂或离液剂更具抵抗力的朊病毒“亚株” 温度，这将是有利于朊病毒种群异质性的进一步证据。 使这项研究成为可能的主要技术创新是细胞组分析 (CPA)， 它基于标准瘙痒细胞测定 (SSCA)，两者均于 2017 年开发。 我们的实验室可以快速区分朊病毒株。 朊病毒可以获得耐药性的发现对设想的策略产生影响 朊病毒病的治疗方法。

Abstract Prions, the pathogens causing transmissible spongiform encephalopathies, are unique in that they consist mainly, if not entirely of multimers of PrPSc, a conformational isomer of the host- encoded protein PrPC. Prions occur in the form of diverse strains, which differ in various phenotypic properties but whose PrPSc has the same amino acid sequence. It is believed that strain identity is encoded by the conformation of PrPSc and that there are as many distinct conformations as there are strains. We have observed that a prion strain, 22L, when transferred from brain to cell culture, changed its characteristics, but gradually regained them when re-introduced into brain. Moreover, when propagated in cell culture in the presence of an inhibitory drug, swainsonine, the prion population acquired resistance against the drug, but lost the resistance when propagated for prolonged periods in its absence. This is quite remarkable, considering that bacteria and viruses acquire drug resistance as a consequence of random mutations in their genetic material, whereas prions do not contain informational nucleic acids. Our working hypothesis is that a prion strain population is comprised not of PrPSc with a unique conformation but of a multitude of species ("sub-strains") with related conformations. This is analogous to the concept of "quasispecies" we first established for RNA phages three decades ago. We postulate that the replication rates of sub-strains may vary depending on the host cell. In a particular environment one of these species may represent the major component and out- replicate other conformers, but at the same time, variant conformers are continuously generated at some low rate (because the activation energy barriers for interconversion are low). Upon transfer to a different host or a change in the environment a different conformer may become the major constituent of the population. We wish to determine whether PrPSc associated with drug resistant and drug sensitive prions show physico-chemical differences that may reflect different conformations, and whether the two types of prions replicate at different rates in host cells. We will investigate whether prions can develop resistance against other inhibitory drugs. Importantly, we will determine whether prion populations that have never been exposed to swainsonine already comprise resistant variants, as would be expected from the "quasispecies" hypothesis. We intend to biologically clone a drug-sensitive prion and determine whether swainsonine-resistant variants arise during propagation in the absence of the drug, and if so, at what rate. Finally, we will ascertain whether it is possible to select prion "sub-strains" that are more resistant to chaotropic agents or to temperature, which would be further evidence in favor of heterogeneity of prion populations. The major technical innovation making this investigation possible is the Cell Panel Assay (CPA), which is based on the Standard Scrapie Cell Assay (SSCA), both of which were developed in our laboratories and which allow the rapid discrimination between prion strains. The finding that prions can acquire drug resistance impacts on the strategies envisaged for therapeutic approaches to prion disease.