Structural analysis of viral replicative intermediates isolated from adenovirus type 2-infected HeLa cell nuclei

Abstract

Deoxyribonucleoprotein complexes released 17 h postinfection from adenovirus type 2 (Ad2)-infected [human cervical carcinoma] HeLa cell nuclei were shown by EM to contain filaments much thicker (about 200 .ANG. [20 nm]) than double-stranded DNA (about 20 .ANG. [2 nm]). The complexes were partially purified through a linear sucrose gradient, concentrated and further purified in a metrizamide gradient. The major protein present in the complexes was identified as the 72,000 dalton (72K), adenovirus-coded single-stranded DNA-binding protein (72K DBP). Three types of complexes were visualized by EM. Some linear complexes were uniformly thick, and their length corresponded roughly to that of the adenovirus genome. Other linear genome-length complexes appeared to consist of a thick filament connected to a thinner filament with the diameter of double-stranded DNA. Forked complexes consisting of 1 thick filament connected to a genome-length, thinner double-stranded DNA filament were also visualized. Both thick and thin filaments were sensitive to DNase and not to RNase, but only the thick filaments were digested by the single-strand-specific Neurospora crassa nuclease, indicating that they correspond to a complex of 72K DBP and Ad2 single-stranded DNA. Experiments with anti-72K DBP immunoglobulins indicated that these nucleoprotein complexes, containing the 72K DBP, correspond to replicative intermediates. Both strands of the Ad2 genome were found associated to the 72K DBP. Apparently, the in vivo association of the 72K DBP with adenovirus single-stranded DNA, as previously suggested from in vitro studies, and support a strand displacement mechanism for Ad2 DNA replication, in which both strands can be displaced. Late in infection, histones are not bound to adenovirus DNA in the form of a nucleosomal chromatine-like structure.