Intracellular forms of Adenovirus DNA in Productively Infected Cells: Evidence for Integration of the Viral Genome
In order to understand the mechanism of replication of adenovirus DNA in productively infected human cells, the intracellular forms of newly synthesized DNA have been studied. KB cells growing in monolayers were inoculated with CsCl-purified adenovirus type 2 (Ad2) at a multiplicity of 100 PFU/cell. At various times after infection, the cells were labeled with 3H-uridine or 3H-thymidine. In some experiments the cells were prelabeled with 14C-thymidine. The intracellular DNA was extracted after lysis of the cells with SDS or with alkali and was analyzed in dye-buoyant density gradients or by zonal centrifugation in neutral or alkaline sucrose gradients. The results of the experiments can be summarized as follows: 1) There is no evidence that parental or newly synthesized Ad 2 DNA becomes supercoiled. 2) A virus-specific DNA-RNA complex can be isolated in dye-buoyant density gradients. This complex is probably involved in transcription. 3) In CsCl density gradients viral DNA of high buoyant density is observed which is a precursor to virion DNA as judged from pulse-chase experiments. These molecules are in part single-stranded. 4) In alkaline sucrose gradients viral DNA is detected which sediments at a rate of 50–90 S. The evidence suggests that this DNA may represent an integrated form of the viral genome.
KeywordsPost Infection Buoyant Density Equilibrium Sedimentation Intracellular Form Equilibrium Centrifugation
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