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Cited by 59Cytomegalovirus and herpesvirus researchGenomics and Chromatin DynamicsHerpesvirus Infections and Treatments

Functional Interaction between Pleiotropic Transactivator pUL69 of Human Cytomegalovirus and the Human Homolog of Yeast Chromatin Regulatory Protein SPT6

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Winkler, Michael, aus dem Siepen, Thomas, Stamminger, Thomas·2000·Journal of Virology
DOI 10.1128/jvi.74.17.8053-8064.2000PMC PMC112338
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Abstract

The phosphoprotein pUL69 of human cytomegalovirus (HCMV), which is a herpesvirus of considerable medical importance in immunosuppressed patients and newborns, has previously been identified as an early-late viral protein that can stimulate several viral and cellular promoters and thus exerts a rather broad activation pattern. To gain insight into the mechanism of this transactivation process, we looked for cellular factors interacting with pUL69 in a yeast two-hybrid screen. Using a B-lymphocyte cDNA library fused to the GAL4 activation domain, we identified 34 clones, 11 of which comprised one distinct gene. Interaction with this gene turned out to be very strong, producing β-galactosidase levels 100-fold greater than the background as measured in an ONPG ( o -nitrophenyl-β- d -galactopyranoside) assay. Sequencing identified this gene as the human homolog of the yeast factor SPT6, which is thought to be involved in the regulation of chromatin structure. A direct interaction of pUL69 and the carboxy terminus of hSPT6 could be demonstrated using in vitro pull-down experiments. After having generated a specific antiserum that is able to detect the endogenous hSPT6 protein, we were able to observe an in vivo interaction of both proteins by coimmunoprecipitation analysis. The interaction domain within pUL69 was mapped to a central domain of this viral protein that is conserved within the homologous proteins of other herpesviruses such as the ICP27 protein of herpes simplex virus. Internal deletions within this central domain, as well as a single amino acid exchange at position C495, resulted in a loss of interaction. This correlated with a loss of the transactivation potential of the respective mutants, suggesting that the hSPT6 interaction of pUL69 is essential for stimulating gene expression. Furthermore, we demonstrate that the carboxy terminus of hSPT6 also binds to histon H3 and that this interaction can be antagonized by pUL69. This allows the deduction of a model by which pUL69 acts as an antirepressor by competing for binding of histones to hSPT6, thereby antagonizing the chromatin remodeling function of this cellular protein.

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  • Spt6 physically interacts with histone H3 and can facilitate nucleosome reassembly.

    “Another factor reported to increase RNA Pol II speed is the transcription elongation factor Spt6, which physically interacts with histone H3 (Bortvin & Winston, 1996; Winkler et al, 2000) and can facilitate nucleosome reassembly (Adkins & Tyler, 2006).”

    From RNA polymerase II speed: a key player in controlling and adapting transcriptome composition (2021) · cites this paper as Winkler et al, 2000

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Research works printed but not linked: 3

  1. [2]Alford C A, Britt W J. Cytomegalovirus. In: Fields B N, Knipe D M, editors. Virology. New York, N.Y: Raven Press, Ltd.; 1990. pp. 1981–2010. [Google Scholar]
  2. [5]Ausubel F, Brent R, Kingston R E, Moore D D, Seidman J G, Smith J A, Struhl K. Current protocols in molecular biology. New York, N.Y: John Wiley & Sons, Inc.; 1989. [Google Scholar]
  3. [65]Winkler M, Schmolke S, Plachter B, Stamminger T. UL69 of HCMV, a homolog of the HSV ICP27, is contained within the tegument of virions and activates the major IE enhancer of HCMV in synergy with the tegument protein pp71. Scand J Infect Dis Suppl. 1995;99:8–9. [Google Scholar]

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Authors
Winkler, M., aus dem Siepen, T., & Stamminger, T.
Affiliations
  • Friedrich-Alexander-Universität Erlangen-Nürnberg (DE)
  • <!--label omitted: 1-->Institut für Klinische und Molekulare Virologie der Universität Erlangen-Nürnberg, 91054 Erlangen, Germany
ORCID
Venue
Journal of Virology
Publisher
American Society for Microbiology
Published
2000-09
Volume
74
Issue
17
Pages
8053-8064
ISSN
  • 0022-538X (ISSN-L)
  • 1098-5514
DOI
10.1128/jvi.74.17.8053-8064.2000
PMC
PMC112338
OpenAlex
W2084366417
Cited by
59
References
67
Citations by year
2021: 2 · 2018: 1 · 2015: 1 · 2014: 1 · 2013: 1 · 2012: 2
FWCI
3.74
Type
journal article
Language
en
Open access
Licence
journals.asm.org/non-commercial-tdm-license (tdm)
Locations
  • Journal of Virology (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion)
  • PubMed Central (publishedVersion)
  • PubMed (publishedVersion)
Funders
  • Deutsche Forschungsgemeinschaft
  • Bundesministerium für Forschung und Technologie
Topics
  • Health Sciences › Medicine › Epidemiology › Cytomegalovirus and herpesvirus research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
  • Health Sciences › Medicine › Epidemiology › Herpesvirus Infections and Treatments
Keywords
human cytomegalovirus, Spt6, viral transactivation, protein-protein interaction, chromatin remodeling, herpesvirus gene expression, ICP27, herpes simplex virus
MeSH terms
Amino Acid Sequence, Animals, Cell Line, Chromatin, Cytomegalovirus, Fibroblasts, Humans, Molecular Sequence Data, Nuclear Proteins, Phosphoproteins, Precipitin Tests, Protein Binding, Saccharomyces cerevisiae, Blotting, Western, Trans-Activators, Gene Expression Regulation, Viral, Conserved Sequence, COS Cells, Two-Hybrid System Techniques, Saccharomyces cerevisiae Proteins, Transcriptional Elongation Factors, Histone Chaperones

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