Widmer C, Robb J A
J Virol. 1974 Dec;14(6):1530-46. doi: 10.1128/JVI.14.6.1530-1546.1974.
We have used immunofluorescence in parallel with transmission and scanning electron microscopy to characterize the unusual cytoplasmic and nucleolar accumulation of Simian virus 40 (SV40) virion protein (C antigen) at restrictive temperatures (39 to 41 C) in monkey cells infected with a temperature-sensitive mutant of SV40 defective in virion assembly, tsB11. Cytoplasmic and nucleolar accumulation of C antigen did not occur in wild-type-infected cells at any temperature. Wild-type- and tsBll-infected cells were not distinguishable at 33 C by immunofluorescence or electron microscopy. Temperature-shift experiments using metabolic inhibitors of DNA (cytosine arabinonucleoside, 20 mug/ml), RNA (actinomycin D, 5 mug/ml), and protein synthesis (cycloheximide, 2 x 10(-4) to 10 x 10(-4) M) were used to investigate the requirements for ongoing DNA, RNA, and protein synthesis in the distribution of virion protein between the nucleus, nucleolus, and cytoplasm. The transport of C antigen from the nucleolus and cytoplasm into the nucleus was complete after a temperature shift-down (41 and 39 to 33 C). Limited virus particle formation occurred after the shift-down in the presence of actinomycin D and cycloheximide, indicating some of the 39 to 41 C synthesized virion protein could be used for capsid assembly at 33 C in the absence of further virion protein synthesis. Nucleolar and cytoplasmic accumulations of C antigen occurred in the absence of drugs after a shift-up (33 to 39 C and 41 C) indicating a continuous requirement for the tsB11 mutant function. Furthermore, the virion protein synthesized at 33 C remained confined to the nucleus when the cells were shifted to 39 and 41 C in the presence of actinomycin D or cycloheximide. In the presence of cytosine arabinonucleoside, however, the virion protein accumulated in large aggregates in the nucleus and nucleolus after the shift-up, but did not migrate into the cytoplasm as it did in drug-free tsB11-infected control cells. Colchicine (10(-3) M) had no effect on the abnormal accumulation of C antigen during shift-up or shift-down experiments suggesting that microtubular transport plays little if any role in the abnormal transport of tsB11 virion protein from cytoplasm to nucleus. Although virus particles were never observed by electron microscopy and V antigen was not detected by immunofluorescence at 39 or 41 C in tsB11-infected cells, dense amorphous accumulations were formed in the nucleoli and cytoplasm. We suggest that the tsB11 function is continuously required for the normal transport of SV40 virion protein between the cytoplasm, nucleolus, and nucleus and for the assembly of capsids and virions. Several possible mechanisms for the altered tsB11 function or protein are discussed. One of the virion proteins may also be involved in some presently undetermined nucleolar function during SV40 productive infection.
我们运用免疫荧光技术，并结合透射电子显微镜和扫描电子显微镜，对感染了病毒体装配缺陷的温度敏感突变株tsB11的猴细胞在限制温度（39至41摄氏度）下猿猴病毒40（SV40）病毒体蛋白（C抗原）在细胞质和核仁中的异常积累情况进行了表征。在任何温度下，野生型感染细胞中均未出现C抗原在细胞质和核仁中的积累。通过免疫荧光或电子显微镜观察，在33摄氏度时，野生型感染细胞和tsB11感染细胞并无差异。利用DNA（阿糖胞苷，20微克/毫升）、RNA（放线菌素D，5微克/毫升）和蛋白质合成（环己酰亚胺，2×10⁻⁴至10×10⁻⁴摩尔）的代谢抑制剂进行温度转换实验，以研究在病毒体蛋白在细胞核、核仁与细胞质之间分布过程中，持续进行的DNA、RNA和蛋白质合成的需求情况。温度下调（从41和39摄氏度降至33摄氏度）后，C抗原从核仁和细胞质向细胞核的转运完成。在放线菌素D和环己酰亚胺存在的情况下，温度下调后出现了有限的病毒颗粒形成，这表明在39至41摄氏度合成的一些病毒体蛋白在33摄氏度且无进一步病毒体蛋白合成的情况下可用于衣壳装配。温度上调（从33摄氏度升至39和41摄氏度）后，在无药物的情况下出现了C抗原在核仁和细胞质中的积累，这表明对tsB11突变体功能存在持续需求。此外，当细胞在放线菌素D或环己酰亚胺存在的情况下转移至39和41摄氏度时，在33摄氏度合成的病毒体蛋白仍局限于细胞核内。然而，在阿糖胞苷存在的情况下，温度上调后病毒体蛋白在细胞核和核仁中聚集成大的聚集体，但并未像在无药物的tsB11感染对照细胞中那样迁移至细胞质中。秋水仙碱（10⁻³摩尔）在温度上调或下调实验中对C抗原的异常积累没有影响，这表明微管运输在tsB11病毒体蛋白从细胞质到细胞核的异常运输中作用甚微或几乎没有作用。尽管在tsB11感染细胞中，在39或41摄氏度时通过电子显微镜从未观察到病毒颗粒，且通过免疫荧光未检测到V抗原，但在核仁和细胞质中形成了密集的无定形聚集体。我们认为，tsB11功能对于SV40病毒体蛋白在细胞质、核仁与细胞核之间的正常转运以及衣壳和病毒体的装配是持续必需的。讨论了几种tsB11功能或蛋白改变的可能机制。在SV40生产性感染过程中，其中一种病毒体蛋白可能还参与了目前尚未确定的某些核仁功能。
