Circular adenovirus DNA-protein complexes from infected HeLa cell nuclei

Abstract

Adenovirus (Ad) DNA was extracted by the use of Sarkosyl from Ad5-infected HeLa cell nuclei at 15 hr after infection. Mature and replicating viral DNA molecules in the Sarkosyl extract were purified by sucrose gradient centrifugation followed by equilibrium centrifugation in preformed metrizamide gradients. Part of the material was digested with the Hpa I restriction endonuclease and analyzed by electrophoresis through 1% agarose gels. Both terminal fragments D and E did not enter the gel unless first treated with Pronase, suggesting that intracellular viral DNA molecules were linked to proteins by their termini. Examination under the electron microscope of mature intracellular Ad2 or Ad5 DNA molecules extracted with Sarkosyl showed the presence of a majority of circular unit-length molecules. Most of these were attached to a prominent globular structure which disappeared, together with the circularization of the molecule, after treatment with Pronase. We interpret these structures as protein links responsible for the circularization of the molecule. The mature linear DNA molecules observed also showed evidence for linkage structures attached to one or both of their ends. Examination of replicating Ad5 DNA molecules extracted with Sarkosyl revealed the presence of various types of forked, circular molecules, most of which showed evidence for the presence of linkage structures. It is suggested that replicating and newly made Ad DNA molecules are circular inside the cell, perhaps through linkage with proteins, and the possible role of circularization in the replication of viral DNA is discussed.