Chromatin-like organization of the adenovirus chromosome.

Abstract

This result is discussed in terms of a higher order folding of viral DNA within the virus particle. Although the capsid architecture of the adenovirus particle is known in great detail, no fundamental understanding of the internal structure exists. Recent proposals for the structure of eukaryotic chromatin (1, 2) suggest a powerful model for the organization of the adenovirus core. The DNA in chromatin is wound around (3) specific histone complexes (4, 5) to form repeating subunits (6-8). The chromosomes of at least two animal viruses, polyoma and simian virus 40, are also condensed into subunits (9-11) within virus particles by complexing with cellular histones (12, 13). The adenovirus core does not contain histones, but we show in this paper that the core has a chromatin-like design. MATERIALS AND METHODSCell Culture and Synchronization. HeLa S3 cells were grown in suspension culture using medium F-13 (Grand Island Biological Co.) (17). The first two methods yielded "cornerless" particles which were not separated from free capsomers. Pyridine treatment stripped off the entire capsid. The resulting cores were further purified from capsomers by sediAbbreviation: bp, base pairs.* To whom reprint requests should be sent. mentation in a sucrose gradient. All preparations were adjusted to 1 mM CaCl2 and pH 7.5.Isolation of Nuclei. Cells were centrifuged at 500 X g for 5 min and resuspended at a concentration of 107 cells per ml in buffer containing 0.3 M sucrose, 10 mM Tris-cacodylate, 3 mM CaCl2, 0.5% Nonidet P-40, pH 7.8. Cells were lysed at 20 by repeatedly drawing (about 20 times) the suspension into a pasteur pipet. After 5 min, the nuclei were pelleted at 500 X g for 10 min and resuspended at 107 nuclei per ml in 0.3 M sucrose, 10 mM Tris-cacodylate, 1 mM CaCl2, pH 7.8.Nuclease Digestion. Staphylococcal nuclease (nucleate 3'-oligonucleotidohydrolase; EC 3.1.4.7) was purchased from Worthington Biochemical Corp. Reaction mixtures were incubated at 370 and reactions were terminated by adjusting the solutions to 10 mM EDTA, 1 mg of Pronase per ml, 2% Sarkosyl, and 10% glycerol. After 1 hr at 370, samples were applied directly to gels. One unit of nuclease corresponds to a change of 1 A260 unit at 37°.Gel Electrophoresis. Agarose (Sigma) gels containing 0.5 ,ug of ethidium bromide per ml were cast in 6 mm X 30 cm Plexiglas tubes. The running buffer, E buffer (18), contained 0.4 ,tg of ethidium bromide per ml. Electrophoresis was at 5 mA per gel for the indicated times. Gels were cut into 0.2 inch (0.5 cm) slices. Each slice was placed with 0.25 ml of H20 into 1 dram vials, autoclaved, and mixed with 2.5 ml of Aquasol. The radioactivity was determined by scintillation counting. RESULTS Nuclease digestion of disrupted adenovirus particlesWe have used staphylococcal nuclease (EC 3.1.4.7) to probe the structure of the adenovirus core. Adenovirus particles dialyzed against 5 mM Tris-maleate buffer at pH 6.4 lose pentons and neighboring hexons (17). The rest of the capsid, a shell of 180 hexons, still surrounds the...

Current Advances and Future Challenges in Adenoviral Vector Biology and Targeting

“…Early studies on the structure of the nucleoprotein core have resulted in different models for core organization. Nuclease digestion experiments suggest that core proteins may form nucleosome-like structures, protecting 150-200 bp fragments of DNA from enzymatic digestion [Corden et al, 1976]. Other studies suggest a model whereby the core is organized into 12 lobes present under each of the vertices of the capsid, as visualized by ionetching and electron microscopy of sarkosyl treated cores [Brown et al, 1975, Newcomb et al, 1984.…”

NAP‐I is a functional homologue of TAF‐I that is required for replication and transcription of the adenovirus genome in a chromatin‐like structure

“…The Ad genome DNA in the virion and in host cells is during early stages of infection, complexed with viral basic proteins V and VII in a nucleosome-like structure (Russell et al 1971;Brown et al 1975;Corden et al 1976). The Ad genome is a linear DNA of about 35 000 bp.…”

Adenovirus Protein VII Condenses DNA, Represses Transcription, and Associates with Transcriptional Activator E1A

“…DNA within the capsid is in a highly compact configuration, and electron microscopy studies of purified viral core reveal structures reminiscent of beads on a string, or higherorder chromatin compaction, depending on the method of preparation (8,18,45,49,50,60,63). Nuclease digestion of core preparations results in discrete populations of protected DNA fragments, suggesting a chromatin-like configuration but without a characteristic ladder of repeating units (15,45,58,60). Nuclease sensitivity of viral DNA extracted from infected cells has been reported to change to a state similar to cellular chromatin, although an association of viral DNA with cellular histones has not been demonstrated (16,17,56,57).…”