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Title: Default assembly of early adenovirus chromatin

Abstract

In adenovirus particles, the viral nucleoprotein is organized into a highly compacted core structure. Upon delivery to the nucleus, the viral nucleoprotein is very likely to be remodeled to a form accessible to the transcription and replication machinery. Viral protein VII binds to intra-nuclear viral DNA, as do at least two cellular proteins, SET/TAF-I{beta} and pp32, components of a chromatin assembly complex that is implicated in template remodeling. We showed previously that viral DNA-protein complexes released from infecting particles were sensitive to shearing after cross-linking with formaldehyde, presumably after transport of the genome into the nucleus. We report here the application of equilibrium-density gradient centrifugation to the analysis of the fate of these complexes. Most of the incoming protein VII was recovered in a form that was not cross-linked to viral DNA. This release of protein VII, as well as the binding of SET/TAF-I{beta} and cellular transcription factors to the viral chromatin, did not require de novo viral gene expression. The distinct density profiles of viral DNA complexes containing protein VII, compared to those containing SET/TAF-I{beta} or transcription factors, were consistent with the notion that the assembly of early viral chromatin requires both the association of SET/TAF-1{beta} and the releasemore » of protein VII.« less

Authors:
 [1]
  1. Department of Microbiology and Immunology, Intercollege Graduate Degree Program in Genetics, and Cellular and Molecular Biology Program, Pennsylvania State University College of Medicine, Hershey, PA 17033 (United States). E-mail: dspector@psu.edu
Publication Date:
OSTI Identifier:
20976994
Resource Type:
Journal Article
Resource Relation:
Journal Name: Virology; Journal Volume: 359; Journal Issue: 1; Other Information: DOI: 10.1016/j.virol.2006.09.005; PII: S0042-6822(06)00648-9; Copyright (c) 2006 Elsevier Science B.V., Amsterdam, The Netherlands, All rights reserved; Country of input: International Atomic Energy Agency (IAEA)
Country of Publication:
United States
Language:
English
Subject:
60 APPLIED LIFE SCIENCES; ADENOVIRUS; CENTRIFUGATION; CHROMATIN; CROSS-LINKING; DNA; FORMALDEHYDE; GENES; TRANSCRIPTION; TRANSCRIPTION FACTORS

Citation Formats

Spector, David J. Default assembly of early adenovirus chromatin. United States: N. p., 2007. Web. doi:10.1016/j.virol.2006.09.005.
Spector, David J. Default assembly of early adenovirus chromatin. United States. doi:10.1016/j.virol.2006.09.005.
Spector, David J. Thu . "Default assembly of early adenovirus chromatin". United States. doi:10.1016/j.virol.2006.09.005.
@article{osti_20976994,
title = {Default assembly of early adenovirus chromatin},
author = {Spector, David J.},
abstractNote = {In adenovirus particles, the viral nucleoprotein is organized into a highly compacted core structure. Upon delivery to the nucleus, the viral nucleoprotein is very likely to be remodeled to a form accessible to the transcription and replication machinery. Viral protein VII binds to intra-nuclear viral DNA, as do at least two cellular proteins, SET/TAF-I{beta} and pp32, components of a chromatin assembly complex that is implicated in template remodeling. We showed previously that viral DNA-protein complexes released from infecting particles were sensitive to shearing after cross-linking with formaldehyde, presumably after transport of the genome into the nucleus. We report here the application of equilibrium-density gradient centrifugation to the analysis of the fate of these complexes. Most of the incoming protein VII was recovered in a form that was not cross-linked to viral DNA. This release of protein VII, as well as the binding of SET/TAF-I{beta} and cellular transcription factors to the viral chromatin, did not require de novo viral gene expression. The distinct density profiles of viral DNA complexes containing protein VII, compared to those containing SET/TAF-I{beta} or transcription factors, were consistent with the notion that the assembly of early viral chromatin requires both the association of SET/TAF-1{beta} and the release of protein VII.},
doi = {10.1016/j.virol.2006.09.005},
journal = {Virology},
number = 1,
volume = 359,
place = {United States},
year = {Thu Mar 01 00:00:00 EST 2007},
month = {Thu Mar 01 00:00:00 EST 2007}
}
  • The authors investigated the structure of adenovirus deoxyribonecleic acid (DNA)-protein complexes in nuclei of infected cells by using micrococal nuclease. Parental (infecting) DNA was digested into multimers which had a unit fragment size that was indistinguishable from the size of the nucleosomal repeat of cellular chromatin. This pattern was maintained in parental DNA throughout infection. Similar repeating units were detected in hamster cells that were nonpermissive for human adenovirus and in cells pretreated with n-butyrate. Late in infection, the pattern of digestion of viral DNA was determined by two different experimental approaches. Nuclear DNA was electrophoresed, blotted, and hybridized withmore » labeled viral sequences; in this procedure all virus-specific DNA was detected. This technique revealed a diffuse protected band of viral DNA that was smaller than 160 base pairs, but no discrete multimers. All regions of the genome were represented in the protected DNA. To examine the nuclease protection of newly replicated viral DNA, infected cells were labeled with (/sup 3/)thymidine after blocking of cellular DNA synthesis but not viral DNA synthesis. With this procedure they identified a repeating unit which was distinctly different from the cellular nucleosomal repeat. The authors found broad bands with midpoints at 200, 400, and 600 base pairs, as well as the limit digest material revealed by blotting. High-resolution acrylamide gel electrophoresis revealed that the viral species comprised a series of closely spaced bands ranging in size from less than 30 to 250 base pairs.« less
  • We have investigated the relationships among the major proteins encoded by early region 1A of Ad2 by comparative analysis of their (/sup 35/S)methionine-labeled tryptic peptides using reverse phase high-performance liquid chromatography. In some instances the purified peptides were subjected to automatic sequential Edmund degradation in order to determine the position at which the methionine residues occur within the peptides. These peptides have then been located in the amino acid sequence predicted from the DNA sequence of the leftmost 4.5% of the viral genome. The data show that the 50,000, 46,000, and 42,000 molecular weight E1A proteins (all of which focusmore » at ca. pH 6.0) obtained from Ad2-infected HeLa cells are derived from the 1.1 kb region 1A mRNA. Similarly, it is shown that two polypeptides, 58,000 (58K) and 48,000 (48K), result from cell-free translation of the 1.1 kb mRNA. The data are also consistent with the hypothesis that the in vivo 46,000, 42,000, and 38,000 molecular weight E1A proteins (all of which focus at ca pH 5.9) are derived from the 0.9 kb region 1A mRNA. Cell-free translation of this mRNA yields two polypeptides, 54,000 (54K) and 42,000 (42K).« less
  • RNA present in cells derived from cervical carcinoma that contained human papillomavirus 18 genomes was initiated in the 1.053-kilobase BamHI fragment that covered the complete noncoding region of this virus. When cloned upstream of the chloramphenicol acetyltransferase gene, this viral fragment directed the expression of the bacterial enzyme only in the sense orientation. Initiation sites were mapped around the ATG of open reading frame E6. This promoter was active in some human and simian cell lines, and its expression was modulated positively by simian virus 40 large T antigen and negatively by adenovirus type 5 E1a antigen.
  • Adenoviruses are generally weak interferon inducers, triggering chicken embryo fibroblast cells by a UV-resistant viral component, probably the capsid or capsid elements, to produce 50 to 100 IU of interferon per ml. Adenovirus types 12, 18, and 31, however, can induce 10 to 20 times more interferon than other types do by a UV-sensitive mechanism. By using mutant and recombinant adenoviruses, we demonstrated that early region 1A was responsible for the enhanced interferon production of chicken cells infected with adenovirus type 12.
  • Six independent rat hybridoma cell lines producing monoclonal antibodies to human subgroup C adenovirus early region 1A (E1A) proteins were isolated. Competition binding experiments revealed that each of the monoclonal antibodies was directed against the same epitope or overlapping cluster of epitopes on the E1A proteins. Viral E1A deletion mutants and deleted forms of E1A proteins expressed in Escherichia coli were used to localize the antibody recognition sites to sequences between amino acids 23 and 120, encoded within the first exon of the E1A gene. Similarly, polyclonal antisera raised against the trpE-E1A fusion protein, as well as against the native,more » biologically active E1A protein, were also directed primarily against this immunodominant region.« less