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（如果不是全部）由PrPSc的多聚体组成，PrPSc是宿主的构象异构体， 编码蛋白PrPC。朊病毒以不同菌株的形式存在，其在各种 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，并且有尽可能多的不同的 因为存在应变。 我们观察到朊病毒株22L，当从脑转移到细胞培养物中时， 特征，但当重新引入大脑时逐渐恢复。而且，当 在存在抑制药物苦马豆素的细胞培养中繁殖，朊病毒群体 获得了对药物的耐药性，但随着时间的延长而失去了耐药性 在他缺席的时候。这是相当了不起的，考虑到细菌和病毒获得 由于遗传物质的随机突变而产生耐药性，而朊病毒 不含信息核酸。 我们的工作假设是朊病毒株群体不是由PrPSc组成， 构象，而是具有相关构象的多个物种（“亚株”）。这是 类似于我们三十年来首次为RNA测序建立的“准种”概念， 前我们推测，亚株的复制率可能会有所不同，这取决于宿主细胞。在 在特定的环境中，这些物种中的一种可能代表主要成分， 复制其他构象，但同时，不断产生变体构象 在一些低速率下（因为用于相互转化的活化能势垒低）。后 转移到不同的宿主或环境的变化，不同的构象异构体可能成为 人口的主要组成部分。 我们希望确定PrPSc是否与药物抗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率复制。我们将研究朊病毒 会对其他抑制性药物产生耐药性。重要的是，我们将确定 从未暴露于苦马豆素的朊病毒群体已经包括耐药的 变种，正如“准种”假说所预期的那样。我们打算从生物学角度 克隆一个药物敏感的朊病毒，并确定是否出现苦马豆素耐药变异， 在没有药物的情况下传播，如果是这样，以什么速度。最后，我们将确定是否 有可能选择对离液剂或 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测定（CPA）， 该方法基于标准瘙痒症细胞测定法（SSCA），两者均于 我们的实验室，并允许快速区分朊病毒菌株。 朊病毒可以获得耐药性的发现影响了为预防感染而设想的策略。 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.