Isolation and characterization of HeLa cell lines blocked at different steps in the poliovirus life cycle.
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.
Kaplan
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Department of Microbiology, Columbia University College of Physicians & Surgeons, New York, New York 10032.
Levy
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A
Racaniello
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D015152:Blotting, Northern; D002455:Cell Division; D002470:Cell Survival; D002999:Clone Cells; D003588:Cytopathogenic Effect, Viral; D004797:Enzyme-Linked Immunosorbent Assay; D006367:HeLa Cells / Q000382:microbiology* / Q000502:physiology; D006801:Humans; D017955:Poliovirus / Q000235:genetics / Q000254:growth & development* / Q000502:physiology; D011233:Precipitin Tests; D012367:RNA, Viral / Q000032:analysis / Q000502:physiology; D012397:Rosette Formation; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis; D014779:Virus Replication
D012367:RNA, Viral; D014764:Viral Proteins