Spear P G, Roizman B
J Virol. 1972 Jan;9(1):143-59. doi: 10.1128/JVI.9.1.143-159.1972.
We are reporting a procedure for the purification of herpes simplex enveloped nucleocapsids (virions), an evaluation of the purification procedure and the results of analyses of the virion proteins by high-resolution acrylamide gel electrophoresis. The data may be summarized as follows. (i) The procedure for the purification of virions consists of careful extraction of cytoplasm to prevent nuclear breakage, separation of enveloped nucleocapsids from soluble proteins and membrane vesicles by rate zonal centrifugation of cytoplasmic extracts through dextran 10 gradients, treatment with urea to dissociate virus-debris aggregates, and, lastly, separation of virions from naked nucleocapsids and free membranes by isopycnic flotation in discontinuous sucrose gradients. (ii) Purity was evaluated in three ways, i.e., electron microscopic examination, analysis of purified virions produced in cells labeled with amino acids before infection, and analysis of purified virions from artificial mixtures of infected and labeled, uninfected cells. The extent of purification was 120-to 200-fold with respect to host proteins. Residual contaminants were identified as host and viral constituents of membrane vesicles. Residual host proteins are very likely contaminants and not structural components of the virion. (iii) Analyses by staining and autoradiography of structural proteins of purified virions in 6, 7, 8.5, 9, and 14% acrylamide gels revealed 24 bands of proteins and glycoproteins made and labeled after infection. Co-electrophoresis of viral proteins with six known standards ranging from 25,700 to 220,000 daltons in molecular weight in 6, 7, 8.5, and 9% acrylamide gels indicate that viral proteins range from 25,000 to 275,000 daltons. The sum of the molecular weights of viral proteins is 2,580,000 daltons. Assuming that messenger transcription is asymmetric and noncomplementary, this corresponds to 47% of the genetic information of the virus. (iv) The nonionic detergent NP-40 removes from purified virions some nonglycosylated proteins and a large fraction of the glycosylated proteins. It leaves behind traces of the envelope visible in the electron microscope as well as some glycoproteins thought to be in the envelope.
我们报告了一种单纯疱疹包膜核衣壳（病毒粒子）的纯化方法、该纯化方法的评估以及通过高分辨率丙烯酰胺凝胶电泳对病毒粒子蛋白进行分析的结果。数据可总结如下。（i）病毒粒子的纯化程序包括小心提取细胞质以防止细胞核破裂，通过在葡聚糖10梯度中对细胞质提取物进行速率区带离心，从可溶性蛋白和膜泡中分离包膜核衣壳，用尿素处理以解离病毒碎片聚集体，最后通过在不连续蔗糖梯度中进行等密度浮选从裸露核衣壳和游离膜中分离病毒粒子。（ii）通过三种方式评估纯度，即电子显微镜检查、分析感染前用氨基酸标记的细胞中产生的纯化病毒粒子，以及分析来自感染和标记的未感染细胞的人工混合物中的纯化病毒粒子。相对于宿主蛋白，纯化程度为120至200倍。残留污染物被鉴定为膜泡的宿主和病毒成分。残留的宿主蛋白很可能是污染物而非病毒粒子的结构成分。（iii）在6%、7%、8.5%、9%和14%丙烯酰胺凝胶中对纯化病毒粒子的结构蛋白进行染色和放射自显影分析，显示感染后产生并标记的24条蛋白质和糖蛋白条带。在6%、7%、8.5%和9%丙烯酰胺凝胶中，将病毒蛋白与分子量从25,700到220,000道尔顿的六种已知标准品进行共电泳，表明病毒蛋白的分子量范围为25,000到275,000道尔顿。病毒蛋白的分子量总和为2,580,000道尔顿。假设信使转录是不对称且非互补的，这相当于病毒遗传信息的47%。（iv）非离子去污剂NP - 40从纯化的病毒粒子中去除一些非糖基化蛋白和大部分糖基化蛋白。它留下了在电子显微镜下可见的包膜痕迹以及一些被认为存在于包膜中的糖蛋白。
