Relationship between higher-order structure of adenovirus genome and gene expression, and involvement of topoisomerases in the life cycle of adenovirus

The genome of human adenovirus is a linear, double-stranded DNA approximately 36 kilobase pairs long. DNA inside the virion particle is complexed with virus-encoded proteins (mostly VII and V). First, electron microscopic analysis of bis-psoralen crosslinked adenovirus DNA shows that linear adenovirus DNA is organized into supercoiled domains. The supercoiled conformation of DNA inside the virion is also demonstrated by sensitivity of the adenovirus core to supercoiling-dependent endonucleolytic activity of Bal31 nuclease. Our data suggest that each supercoiled domain is equal to about 12% of adenovirus genome and that 8 loop-domains can be formed in the viral particles. However, regions at two ends (2%) of viral genome are not included in supercoiled domains.;The conformation change of viral nucleoprotein complexes following virus entry into the host was studied. Sucrose gradient sedimentation and electron microscopic analyses indicate that adenovirus genome gradually unfolds during the early phase of infection. Unfolding of viral templates of the E1a deletion mutants, dl312, is delayed in HeLa cells, while their unfolding proceeds normally in 293 cells, which expresses endogenous E1a gene products. The result indicate that E1a gene products are required for efficient unfolding of the highly condensed, supercoiled DNA.;We tested the possible roles of DNA topoisomerases in the life cycle of adenovirus in HeLa cells. Using different topoisomerases inhibitors, it was found that different topoisomerases activities are involved in replication, transcription, and packaging of the linear adenovirus genome. Topoisomerase I and II inhibitors induce single- and double-stranded cleavages, respectively, at specific sites of adenovirus DNA in early times of infection. These results suggest that topoisomerases are associated with early viral templates at specific sites. Topoisomerase I, not II, is needed for adenovirus replication. Both topoisomerase I and II inhibitors block adenovirus transcription; topoisomerase I inhibitor causes premature termination of major late transcription. The packaging of adenovirus DNA into the virion is blocked by topoisomerase II inhibitor. This result suggests that folding of linear virus DNA into supercoiled conformation is mediated by topoisomerase II. Electron microscopic data provide direct visualization of topologically independent supercoiled domains of intracellular adenovirus DNA-protein complexes. (Abstract shortened with permission of author.).