Electron Microscopy of Adenovirus Cores

Nermut, M. V.; Harpst, J.A.; Russell, W. C.

doi:10.1099/0022-1317-28-1-49

Cited by 17 publications

(21 citation statements)

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“…Polypeptide VII (174 aa for Ad2) is highly basic and binds tightly to DNA (Russell & Precious, 1982) and since there are over 800 copies per virion these appear to be spread along the length of the virus DNA, although there is some indication of the formation of particulate structures and supercoiling on treatment with nucleases (Goding & Russell, 1983b;Nermut et al, 1975;Wong & Hsu, 1989). Another very basic component of the nucleoprotein core is Mu (36 aa), with about 100 copies per virion.…”

Section: Other Capsid Proteinsmentioning

confidence: 99%

Adenoviruses: update on structure and function

Russell

2009

Journal of General Virology

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268

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Section: Other Capsid Proteinsmentioning

confidence: 99%

Adenoviruses: update on structure and function

Russell

2009

Journal of General Virology

Self Cite

339

268

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“…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).…”

mentioning

confidence: 99%

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

Johnson

Osheim

Xue

et al. 2004

J Virol

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Adenovirus protein VII is the major protein component of the viral nucleoprotein core. It is highly basic, and an estimated 1070 copies associate with each viral genome, forming a tightly condensed DNA-protein complex. We have investigated DNA condensation, transcriptional repression, and specific protein binding by protein VII. Xenopus oocytes were microinjected with mRNA encoding HA-tagged protein VII and prepared for visualization of lampbrush chromosomes. Immunostaining revealed that protein VII associated in a uniform manner across entire chromosomes. Furthermore, the chromosomes were significantly condensed and transcriptionally silenced, as judged by the dramatic disappearance of transcription loops characteristic of lampbrush chromosomes. During infection, the protein VII-DNA complex may be the initial substrate for transcriptional activation by cellular factors and the viral E1A protein. To investigate this possibility, mRNAs encoding E1A and protein VII were comicroinjected into Xenopus oocytes. Interestingly, whereas E1A did not associate with chromosomes in the absence of protein VII, expression of both proteins together resulted in significant association of E1A with lampbrush chromosomes. Binding studies with proteins produced in bacteria or human cells or by in vitro translation showed that E1A and protein VII can interact in vitro. Structure-function analysis revealed that an N-terminal region of E1A is responsible for binding to protein VII. These studies define the in vivo functions of protein VII in DNA binding, condensation, and transcriptional repression and indicate a role in E1A-mediated transcriptional activation of viral genes.The adenovirus nucleoprotein core consists of doublestranded genomic DNA, three highly basic viral proteins VII, V, and (mu), as well as protein IVa2 and the 55-kDa terminal protein (1,8,33,42,(52)(53)(54)61). Protein VII is the major protein component of the core, with an estimated 1,070 copies present per virion (20). Along with , it is bound noncovalently to the DNA in a sequence-independent manner (2, 6, 36, 55). Protein V contacts the DNA as well and also acts as a bridge between protein VII and the outer capsid (19,55). Protein IVa2 makes sequence-specific contacts with the viral DNA packaging sequence and is thought to play a role in DNA packaging (64). Salt-extracted preparations of the core contain only DNA and protein VII, suggesting that this protein is the most tightly DNA bound of all core proteins (60). Similarly, Sarkosyl preparations of the core contain predominantly DNA and protein VII (8).Structural features of the DNA-protein complex within the adenovirus capsid and during infection remain largely unknown. 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 prote...

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“…It does not bind to DNA as tightly as polypeptide VII (30,31). Cores lacking polypeptide V are less compact in electron micrographs (32). The minor core protein may therefore crosslink neighboring units, as postulated for histone fi in chromatin (1).…”

Section: Nuclease Digestion Of Disrupted Adenovirus Particlesmentioning

confidence: 94%

Chromatin-like organization of the adenovirus chromosome.

Corden¹,

Engelking²,

Pearson³

1976

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Electron Microscopy of Adenovirus Cores

Cited by 17 publications

References 2 publications

Adenoviruses: update on structure and function

Adenoviruses: update on structure and function

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

Chromatin-like organization of the adenovirus chromosome.

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