Raeber A J, Borchelt D R, Scott M, Prusiner S B
Department of Neurology, University of California, San Francisco 94143-0518.
J Virol. 1992 Oct;66(10):6155-63. doi: 10.1128/JVI.66.10.6155-6163.1992.
The scrapie prion protein (PrPSc) is derived from a cellular isoform (PrPC) that acquires protease resistance posttranslationally. We have used several different experimental approaches in attempts to reconstitute in vitro the processes leading to protease-resistant PrPSc molecules. In the first study, we performed mixing experiments by adding mouse PrP 27-30 (MoPrP27-30), the protease-resistant core of PrPSc, to PrPC and then incubating the mixture to investigate the possibility of heterodimer formation as a first step in prion replication. We used epitopically tagged PrP molecules, synthesized in murine neuroblastoma (N2a) cells transfected with the chimeric mouse/Syrian hamster MHM2 PrP construct, which are recognized by the Syrian hamster-specific monoclonal antibody 3F4. After as long as 24 h of incubation, the reaction mixture was assayed for heterodimeric intermediates of MHM2 PrPC and MoPrPSc and for protease-resistant 3F4-reactive PrP. We were unable to identify any aggregates of MHM2 PrPC and MoPrPSc on immunoblots; furthermore, we did not observe de novo formation of protease-resistant MHM2 PrP. In a second study, MoPrPC was metabolically radiolabeled in scrapie prion-infected N2a cultured cells, and then the cell extract was homogenized and incubated under various conditions to allow for the formation of protease-resistant MoPrPSc. We observed no radiolabeled MoPrPSc by immunoprecipitation after as long as 24 h of in vitro incubation. In a third approach, Syrian hamster PrP (SHaPrP) was synthesized in a cell-free translation system supplemented with microsomal membranes derived from either normal or scrapie prion-infected cultured cells. We found that all SHaPrP species translocated across microsomal membranes from scrapie prion-infected cells were protease sensitive in the presence of detergents and displayed the same topology as those generated by microsomes from normal cells or from dog pancreas. We also studied PrP molecules that encode the codon 102 mutation that causes the rare human prion disease Gerstmann-Sträussler-Scheinker (GSS) syndrome. On the basis of our data, GSSPrP appears to yield topological forms similar to those of the wild-type PrP when processed by either normal or scrapie prion-derived microsomes.
瘙痒病朊病毒蛋白（PrPSc）源自一种细胞异构体（PrPC），该异构体在翻译后获得蛋白酶抗性。我们采用了几种不同的实验方法，试图在体外重建导致产生蛋白酶抗性PrPSc分子的过程。在第一项研究中，我们通过将小鼠PrP 27-30（MoPrP27-30，PrPSc的蛋白酶抗性核心）添加到PrPC中进行混合实验，然后孵育混合物，以研究异二聚体形成作为朊病毒复制第一步的可能性。我们使用了在转染了嵌合小鼠/叙利亚仓鼠MHM2 PrP构建体的鼠神经母细胞瘤（N2a）细胞中合成的表位标记PrP分子，这些分子可被叙利亚仓鼠特异性单克隆抗体3F4识别。孵育长达24小时后，检测反应混合物中MHM2 PrPC和MoPrPSc的异二聚体中间体以及蛋白酶抗性3F4反应性PrP。我们在免疫印迹上无法鉴定出MHM2 PrPC和MoPrPSc的任何聚集体；此外，我们没有观察到蛋白酶抗性MHM2 PrP的从头形成。在第二项研究中，在感染瘙痒病朊病毒的N2a培养细胞中对MoPrPC进行代谢性放射性标记，然后将细胞提取物匀浆并在各种条件下孵育，以形成蛋白酶抗性MoPrPSc。体外孵育长达24小时后，通过免疫沉淀我们未观察到放射性标记的MoPrPSc。在第三种方法中，在补充有源自正常或感染瘙痒病朊病毒培养细胞的微粒体膜的无细胞翻译系统中合成叙利亚仓鼠PrP（SHaPrP）。我们发现，在去污剂存在下，所有从感染瘙痒病朊病毒的细胞转运穿过微粒体膜的SHaPrP物种对蛋白酶敏感，并且显示出与正常细胞或狗胰腺微粒体产生的拓扑结构相同。我们还研究了编码导致罕见人类朊病毒疾病格斯特曼-施特劳斯勒-谢inker（GSS）综合征的第102位密码子突变的PrP分子。根据我们的数据，当由正常或瘙痒病朊病毒来源的微粒体处理时，GSSPrP似乎产生与野生型PrP相似的拓扑形式。
