Adenovirus type 12 DNA firmly associates with mammalian chromosomes early after virus infection or after DNA transfer by the addition of DNA to the cell culture medium.
Human adenovirus type 12 (Ad12) infects human cells productively and leads to viral replication, whereas infection of hamster cells remains abortive, with total blocks in viral DNA replication and late viral gene transcription. The intranuclear fate of Ad12 DNA in productively infected human cells and in abortively infected hamster cells was monitored by using the fluorescent in situ hybridization (FISH) technique. Human HeLa cells, primary human umbilical cord fibroblasts, hamster BHK21 cells, primary embryonal hamster cells, and the Ad12-transformed T637 hamster cell line were studied. As early as 2 h after infection, extensive association of Ad12 DNA with metaphase chromosomes was demonstrated by FISH in all of these cells. Chromosomal association continued until late (24 to 28 h) after infection, when about 100% of the human cell nuclei and 70 to 80% of the hamster cell nuclei showed distinct FISH signals. This chromosomal association of Ad12 DNA in infected cells seemed to be rather firm, since it proved to be resistant to mechanically stretching the chromosomes and to different types of chemical treatment. Moreover, laser scan microscopy of mechanically stretched chromosomes from Ad12-infected HeLa cells and from the Ad12-transformed T637 cell line, with about 20 copies of Ad12 DNA provably integrated, revealed identical FISH patterns. Therefore, it was likely that even in infected cells the chromosomal association of Ad12 DNA was very similar to the integrated state. Late in productively infected cells, large nuclear areas were taken over by viral DNA replication, as visualized by FISH in interphase nuclei. Chromosomal association at many sites was frequently limited to one chromatid, but signals in adjacent positions on both chromatids were also seen. Upon the long-term cultivation and passage of abortively infected BHK21 cells for 96 h after infection, a gradual decrease of viral DNA association with chromosomes was observed. Integration of Ad12 DNA in hamster cells early after infection was previously documented, and recombination between viral and cellular DNAs in human cells was also shown. The FISH data on extensive chromosomal association of Ad12 DNA suggest a means to study the pathway of Ad12 DNA from early steps in viral infection via chromosomal interactions to integration events. In a different approach, Ad12 DNA, Ad12 DNA with the terminal protein covalently linked to its ends (Ad12 DNA-TP), or Ad2 DNA was simply added to the culture medium of HeLa or BHK21 cells. Precipitation or selection procedures were avoided. Depending on the experimental conditions, up to 25 to 30% of the interphase nuclei of HeLa cells and 9 to 19% of the interphase nuclei of BHK21 cells showed positive FISH signals at 24 h after the addition of DNA. Viral DNA also became associated in some cases with both chromatids. The uptake of Ad12 DNA-TP appeared to be 10 to 20 times more efficient than that of Ad12 DNA completely freed of proteins. Control bacteriophage lambda, M13, or plasmid DNA could not be detected in the nuclei under these conditions.
Schröer
J
J
Institute of Genetics, University of Cologne, Germany.
Hölker
I
I
Doerfler
W
W
D000260:Adenoviruses, Human / Q000235:genetics / Q000502:physiology*; D000818:Animals; D001665:Binding Sites; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D002875:Chromosomes / Q000502:physiology*; D002877:Chromosomes, Human / Q000502:physiology*; D006224:Cricetinae; D004279:DNA, Viral / Q000302:isolation & purification / Q000378:metabolism*; D005347:Fibroblasts; D006367:HeLa Cells; D006801:Humans; D007624:KB Cells; D007621:Karyotyping; D008322:Mammals; D008677:Metaphase; D014162:Transfection / Q000379:methods; D014470:Umbilical Cord / Q000166:cytology
D004279:DNA, Viral