Persistence of heterologous nucleic acids after uptake by mammalian cells.
Plasmids containing the complete genome of poliovirus-1 are transcribed at random in transfected cells and give rise to infectious RNA molecules. These generate viruses which can be detected easily in a plaque assay. Using this system, we analyzed the persistence of the biologically active portion of transfected poliovirus cDNA by determining its infectious activity in mammalian cells. Transfection and the cultivation of the cells for up to 16 days were performed under the influence of guanidine or other drugs which inhibit plaque formation. Removal of the drug then allowed viral development to start at defined time points. We thus ensured that the reduction of plaques correlated with the decay of the transfected polio cDNA or the viral RNA synthesized exclusively from that DNA. We showed that the intracellular cDNA kept its full capacity to generate viruses for as long as 8 to 10 days post-transfection. After this time, this capacity declined, and no viruses were detected after 14 to 16 days. The plaque-producing activity depended primarily on the stability of the cDNA and its ability to be transcribed and not on the stability of the RNA transcripts, which decayed within hours.
Graeber
I
I
Institut für Wasser- Boden- und Lufthygiene, Umweltbundesamt, Berlin, Germany.
Tischer
J
J
Heinrich
J
J
Hachula
G
G
López-Pila
J M
JM
D000818:Animals; D002460:Cell Line; D002522:Chlorocebus aethiops; D018076:DNA, Complementary / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism*; D010957:Plasmids / Q000378:metabolism; D017955:Poliovirus / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D012367:RNA, Viral / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D010948:Viral Plaque Assay; D019065:Virus Assembly
D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral