Kaplan G, Levy A, Racaniello V R
Department of Microbiology, Columbia University College of Physicians & Surgeons, New York, New York 10032.
J Virol. 1989 Jan;63(1):43-51. doi: 10.1128/JVI.63.1.43-51.1989.
Cotransfection of poliovirus RNA and R1, a poliovirus subgenomic RNA containing a deletion of nearly all of the capsid region, resulted in surviving cells, in contrast to the complete cell death observed after transfection with viral RNA. Cells that survived the cotransfection grew into colonies, produced infectious poliovirus, and underwent cycles of cell lysis (crisis periods) where less than 1% of the cells survived, followed by periods of growth. Poliovirus evolved during the persistent infection as judged by changes in plaque size. After passage for 6 months, a stable line called SOFIA emerged that no longer produced infectious virus and did not contain viral proteins or viral RNA. Cells frozen in liquid N2 while still in crisis and recultured 4 months later (named SOFIA N2) were also stabilized. After infection with poliovirus, SOFIA N2 cells showed a delay in the development of cytopathic effect, viral production, and cellular death when compared with HeLa cells. In contrast, SOFIA cells did not develop cytopathic effect and produced 10,000 times less virus than SOFIA N2 or HeLa cells. Viral production was delayed in SOFIA and SOFIA N2 cells transfected with poliovirus RNA when compared with HeLa cells, suggesting the presence of an intracellular block to poliovirus replication. Analysis of the cellular receptor for poliovirus by virus binding, an enzyme-linked immunosorbent assay, and in situ rosette assays with an antireceptor monoclonal antibody showed that receptors were expressed in SOFIA N2 cells but not in SOFIA cells. Echovirus 6, an enterovirus which uses a different cellular receptor, formed small plaques on SOFIA cells. Vesicular stomatitis virus formed plaques of similar size on SOFIA and HeLa cells, suggesting that the intracellular block was specific for enteroviruses. Cotransfection of the subgenomic replicon R1 with poliovirion RNA therefore resulted in the selection of HeLa cell variants containing blocks to poliovirus replication at the level of receptor and within the cell.
脊髓灰质炎病毒RNA与R1（一种脊髓灰质炎病毒亚基因组RNA，几乎缺失了所有衣壳区域）共转染导致细胞存活，这与用病毒RNA转染后观察到的细胞完全死亡形成对比。共转染后存活的细胞长成集落，产生有感染性的脊髓灰质炎病毒，并经历细胞裂解周期（危机期），在此期间不到1%的细胞存活下来，随后是生长阶段。从空斑大小的变化判断，脊髓灰质炎病毒在持续感染过程中发生了进化。传代6个月后，出现了一个名为SOFIA的稳定细胞系，该细胞系不再产生有感染性的病毒，也不含有病毒蛋白或病毒RNA。在危机期仍处于液氮冷冻状态并在4个月后重新培养的细胞（命名为SOFIA N2）也得到了稳定。与HeLa细胞相比，用脊髓灰质炎病毒感染后，SOFIA N2细胞在细胞病变效应、病毒产生和细胞死亡的发展方面出现延迟。相比之下，SOFIA细胞未出现细胞病变效应，产生的病毒比SOFIA N2或HeLa细胞少10000倍。与HeLa细胞相比，用脊髓灰质炎病毒RNA转染的SOFIA和SOFIA N2细胞中病毒产生延迟，这表明存在对脊髓灰质炎病毒复制的细胞内阻滞。通过病毒结合、酶联免疫吸附测定以及用抗受体单克隆抗体进行的原位玫瑰花结测定对脊髓灰质炎病毒的细胞受体进行分析，结果表明受体在SOFIA N2细胞中表达，但在SOFIA细胞中不表达。埃可病毒6（一种使用不同细胞受体的肠道病毒）在SOFIA细胞上形成小空斑。水疱性口炎病毒在SOFIA和HeLa细胞上形成大小相似的空斑，这表明细胞内阻滞对肠道病毒具有特异性。因此，亚基因组复制子R1与脊髓灰质炎病毒粒子RNA共转染导致选择了在受体水平和细胞内对脊髓灰质炎病毒复制具有阻滞作用的HeLa细胞变体。
