Intracellular forms of adenovirus dna: VI. Quantitation and characterization of the four size-classes of adenovirus type 2 DNA in human KB cells

Abstract

In human KB cells productively infected with adenovirus type 2, four size-classes of newly synthesized viral DNA have been resolved by zone velocity sedimentation in alkaline sucrose density gradients. The >100 and the 50–90 S DNA classes represent in part viral DNA linked to cellular DNA. The bulk of the newly synthesized viral DNA is found in the 34 S genome-size adenovirus type 2 DNA peak, and the <20 S DNA class contains fragments of viral DNA. Using the technique of reassociation kinetics, the total amount of viral DNA in infected cells has been determined at various times after infection. Under optimal conditions more than 460,000 adenovirus type 2 DNA copies per cell are produced in this system. KB cells growing in monolayers which are approaching confluence synthesize considerably smaller quantities (10%) of viral DNA than do dividing cells. The number of viral genome copies per cell has been determined in each size-class. As early as 5 hr postinfection, 1000–3000 adenovirus type 2 DNA copies are present in the high molecular weight forms of viral DNA (>100 and 50–90 S DNA size-classes), indicating that parental viral DNA may have been linked to cellular DNA. The amount of viral DNA in high molecular weight DNA remains relatively constant throughout the course of infection. Reconstitution experiments demonstrate that the high molecular weight forms of viral DNA cannot be due to artifactual inclusion of free viral DNA in the mass of cellular DNA. Restriction enzyme fragments generated by the restriction endonucleases EcoRI and Bam H-I have been used in reassociation-kinetics experiments to measure which parts of the viral genome are represented in individual DNA size-classes. In the 34 S unit-genome-length DNA, all parts of the viral DNA are about equally represented. The 50–90 S DNA, however, contains the right molecular end in at least 10- to 15-fold greater abundance than the left terminus. Similarly, in the <20 S DNA size-class, the right-end sequences are more frequently represented than those from the left end, although the factor of overrepresentation is only four to five. The >100 S DNA appears to exhibit the inverse pattern of representation, in that the left end of adenovirus type 2 DNA is more abundant in that size-class. A model is presented to explain the observed patterns of representation of certain segments of viral DNA in individual size-classes of DNA. It is postulated that recombinational events may be responsible for the attachment of specific parts of the viral genome to replicating cellular DNA in productively infected cells. The significance of these events for viral replication and their consequences for the cell will have to be elucidated.