Characterization of a new adenovirus type 5 assembly intermediate.

Adenovirus H5 (Ad5) DNA-protein complexes were extracted with ammonium sulphate (0.2 M) from virus-infected HeLa cell nuclei at 18 h after infection. Analysis of the material by centrifugation through discontinuous sucrose gradients in heavy water revealed the existence of several populations of molecules which were identified, in order of increasing buoyant density, as mature DNA-protein complexes, replication complexes, assembly intermediates and virions. When observed under the electron microscope, some of the assembly intermediates showed a capsid with a tail of entirely double-stranded (ds) DNA, or of dsDNA continued by a portion of single-stranded (ss) DNA thickened by a coat of E-72 K DNA binding protein. Singly or doubly-forked Ad5 replicating DNA molecules partially packaged in virus capsids were also observed. It is suggested that these molecules could be assembly intermediates, i.e. one of the first steps of assembly corresponding to virus DNA entering pre-formed capsids or their precursors. The fact that replication was still going on at one end of many of the DNA molecules in the intermediates, while encapsidation was taking place at the other, raises the possibility of a coupled DNA replication-packaging process in the formation of adenovirions.