Rahkila P, Alakangas A, Väänänen K, Metsikkö K
Biocenter, University of Oulu, Finland.
J Cell Sci. 1996 Jun;109 ( Pt 6):1585-96. doi: 10.1242/jcs.109.6.1585.
We have infected isolated skeletal muscle fibers with the vesicular stomatitis virus or the mutant tsO45, whose glycoprotein is blocked in the endoplasmic reticulum at 39 degrees C. Immunofluorescence analysis for the viral glycoprotein indicated that the fibers were infected over their entire length at a virus dose of 10(9)/ml. When we infected the myofibers with the tsO45 mutant at 39 degrees C, the viral glycoprotein appeared to be localised to the terminal cisternae of the sarcoplasmic reticulum. Upon shifting the cultures to the permissive temperature, 32 degrees C, in the presence of dinitrophenol, which blocks vesicular transport, the viral glycoprotein proceeded to completely fill the sarcoplasmic reticulum. Thus, both the endoplasmic reticulum located at the terminal cisternae of the sarcoplasmic reticulum, and the entire endoplasmic and sarcoplasmic reticulum appeared to be continuous. Shifting the culture temperature from 39 degrees C to 20 degrees C, resulted in prominent perinuclear staining throughout the fibers, accompanied by the appearance of distinct bright dots between the nuclei. Electron microscopic immunoperoxidase labeling indicated that these bright structures represented the Golgi apparatus. When either the tsO45-infected or wild-type virus-infected fibers were incubated at 32 degrees C, the viral glycoprotein showed a staining pattern that consisted of double rows of punctate fluorescence. Immunogold labeling showed that the viral glycoprotein was present in both the transverse tubules as well as the endoplasmic/sarcoplasmic reticulum endomembranes. In addition, extensive viral budding was observed in the transverse tubules. Metabolic labeling experiments revealed that only half of the glycoprotein was processed in the Golgi, and this processed form had become incorporated into the budding viral particles. Thus, the processed viral glycoprotein was targeted to the transverse tubules. The other half of the glycoprotein remained endoglycosidase H-sensitive, suggesting its retention in the endoplasmic/sarcoplasmic reticulum endomembranes.
我们用水泡性口炎病毒或突变体tsO45感染分离的骨骼肌纤维，其糖蛋白在39℃时在内质网中被阻断。对病毒糖蛋白的免疫荧光分析表明，在病毒剂量为10(9)/ml时，纤维在其整个长度上均被感染。当我们在39℃用tsO45突变体感染肌纤维时，病毒糖蛋白似乎定位于肌浆网的终池。在存在阻断囊泡运输的二硝基苯酚的情况下，将培养物转移至允许温度32℃后，病毒糖蛋白开始完全充满肌浆网。因此，位于肌浆网终池的内质网以及整个内质网和肌浆网似乎是连续的。将培养温度从39℃转变为20℃，导致纤维中出现明显的核周染色，并伴有核间出现明显的亮点。电子显微镜免疫过氧化物酶标记表明，这些明亮结构代表高尔基体。当tsO45感染的或野生型病毒感染的纤维在32℃孵育时，病毒糖蛋白呈现出由双排点状荧光组成的染色模式。免疫金标记表明，病毒糖蛋白存在于横管以及内质网/肌浆网内膜中。此外，在横管中观察到广泛的病毒出芽。代谢标记实验表明，只有一半的糖蛋白在高尔基体中进行了加工，并且这种加工形式已被整合到出芽的病毒颗粒中。因此，加工后的病毒糖蛋白被靶向到横管。另一半糖蛋白对内切糖苷酶H仍敏感，表明其保留在内质网/肌浆网内膜中。
