Richardson C D, Vance D E
J Biol Chem. 1976 Sep 25;251(18):5544-50.
The data from chemical studies and electron microscopy suggest that Semliki Forest virus obtains its envelope by budding into the medium from the plasma membrane of the host cell. Biochemical evidence for this phenomenon, however, has not been published. Therefore, we undertook a series of pulse-chase studies so that we might quantitatively evaluate the importance of the budding mechanism in the morphogenesis of Semliki Forest virus. Baby hamster kidney cells (clone 13) were grown in culture and infected with Semliki Forest virus. The cells were exposed to [4,5-3H] leucine for 20 min and the subsequent incorporation of the label into virus proteins associated with cytoplasmic membranes and extracellular virus was determined. Initial experiments were conducted with microsomes and a precursor-product relationship was demonstrated between viral proteins in the microsomes and in extracellular virus. Further studies were performed with endoplasmic reticulum and plasma membrane preparations. Maximal incorporation of [3H] leucine was observed in the viral proteins located in the endoplasmic reticulum at the end of a 20-min pulse period; greater than 50% of this activity had disappeared within 2 h. The plasma membrane fraction contained no radioactivity at the end of the pulse period; subsequently, maximal labeling of the viral proteins in the plasma membrane occurred 4 h into the chase period and these labeled proteins had disappeared from this membrane 11 h after the pulse. At this time maximal incorporation of the labeled proteins into extracellular virus was observed. These data are consistent with a precursor-product relationship between the viral proteins in the endoplasmic reticulum which migrate to the plasma membrane and are subsequently incorporated into extracellular virus. All the radioactivity in the extracellular virus appears to have been derived from viral proteins associated with the plasma membrane of the cell. Therefore, mechanisms for the morphogenesis of Semliki Forest virus (in baby hamster kidney cells), other than budding from the plasma membrane, are unlikely to be of quantitative importance.
化学研究和电子显微镜的数据表明，辛德毕斯病毒通过从宿主细胞质膜出芽进入培养基而获得包膜。然而，这一现象的生化证据尚未发表。因此，我们进行了一系列脉冲追踪研究，以便定量评估出芽机制在辛德毕斯病毒形态发生中的重要性。幼仓鼠肾细胞（克隆13）在培养物中生长并感染辛德毕斯病毒。将细胞暴露于[4,5-³H]亮氨酸20分钟，然后测定随后标记物掺入与细胞质膜和细胞外病毒相关的病毒蛋白中的情况。最初的实验是用微粒体进行的，结果证明微粒体中的病毒蛋白与细胞外病毒中的病毒蛋白之间存在前体-产物关系。进一步的研究是用内质网和质膜制剂进行的。在20分钟脉冲期结束时，在内质网中的病毒蛋白中观察到[³H]亮氨酸的最大掺入量；在2小时内，超过50%的这种活性消失了。在脉冲期结束时，质膜部分没有放射性；随后，在追踪期4小时时质膜中的病毒蛋白出现最大标记，并且这些标记蛋白在脉冲后11小时从该膜中消失。此时观察到标记蛋白最大程度地掺入细胞外病毒中。这些数据与内质网中的病毒蛋白之间的前体-产物关系一致，这些蛋白迁移到质膜并随后掺入细胞外病毒中。细胞外病毒中的所有放射性似乎都来自与细胞的质膜相关的病毒蛋白。因此，辛德毕斯病毒（在幼仓鼠肾细胞中）除了从质膜出芽之外的形态发生机制在数量上不太可能具有重要意义。
