Hruby D E, Guarino L A, Kates J R
J Virol. 1979 Feb;29(2):705-15. doi: 10.1128/JVI.29.2.705-715.1979.
Using cytochalasin B-induced enucleation techniques, we examined the ability of vaccinia virus to replicate in the absence of the host-cell nucleus in several mammalian cell lines. It was found that virus-infected enucleated cells (cytoplasts) prepared from BSC-40, CVC, and L cells were incapable of producing infectious progeny virus. The nature of this apparent nuclear involvement was studied in detail in BSC-40 cells. Modulations designed to maximize cytoplast integrity and longevity, such as reduction of the growth temperature and initial multiplicity of infection, did not improve virus growth in cytoplasts. Sodium dodecyl sulfate-polyacrylamide gel analysis of the [(35)S]methionine pulse-labeled proteins synthesized in vaccinia virus-infected cytoplasts demonstrated that both early and late viral gene products were being expressed at high levels and with the proper temporal sequence. Vaccinia virus cytoplasmic DNA synthesis, as measured by [(3)H]thymidine incorporation, peaked at 3 h postinfection and was 70 to 90% of control levels in cytoplasts. However, in the cytoplasts this DNA was not converted to a DNase-resistant form late in infection, which was consistent with the failure to isolate physical particles from infected cytoplasts. Treatment of vaccinia virus-infected cells with 100 mug of rifampin/ml from 0 to 8 h to increase the pools of viral precursors, followed by subsequent removal of the drug, resulted in a threefold increase virus yield. This treatment had no effect on virus-infected cytoplasts. Finally, vaccinia virus morphogenesis was studied under an electron microscope in thin sections of virus-infected cells and cytoplasts which had been prepared at various times during a single-step growth cycle. It was apparent that, although early virus morphogenetic forms appeared, there was no subsequent DNA condensation or particle maturation in the cytoplasts. These results suggest that vaccinia virus requires some factor or function from the host-cell nucleus in order to mature properly and produce infectious progeny virus.
利用细胞松弛素B诱导去核技术，我们在几种哺乳动物细胞系中检测了痘苗病毒在无宿主细胞核情况下的复制能力。结果发现，从BSC - 40、CVC和L细胞制备的病毒感染去核细胞（胞质体）无法产生有感染性的子代病毒。我们在BSC - 40细胞中详细研究了这种明显的核参与的性质。旨在最大化胞质体完整性和寿命的调控措施，如降低生长温度和初始感染复数，并未改善病毒在胞质体中的生长。对痘苗病毒感染的胞质体中合成的[(35)S]甲硫氨酸脉冲标记蛋白进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶分析表明，早期和晚期病毒基因产物均以高水平且按正确的时间顺序表达。通过[(3)H]胸腺嘧啶核苷掺入法测定，痘苗病毒胞质DNA合成在感染后3小时达到峰值，且在胞质体中为对照水平的70%至90%。然而，在胞质体中，这种DNA在感染后期并未转化为抗DNA酶的形式，这与无法从感染的胞质体中分离出物理颗粒一致。用100μg/ml利福平在0至8小时处理痘苗病毒感染的细胞以增加病毒前体池，随后去除药物，导致病毒产量增加了三倍。该处理对病毒感染的胞质体没有影响。最后，在电子显微镜下对在单步生长周期不同时间制备的病毒感染细胞和胞质体的薄切片进行痘苗病毒形态发生研究。很明显，尽管出现了早期病毒形态发生形式，但胞质体中随后没有DNA浓缩或颗粒成熟。这些结果表明，痘苗病毒需要宿主细胞核中的某些因子或功能才能正常成熟并产生有感染性的子代病毒。
