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Journal of Virology, March 2007, p. 2592-2604, Vol. 81, No. 6
0022-538X/07/$08.00+0     doi:10.1128/JVI.01643-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

High-Frequency Epigenetic Repression and Silencing of Retroviruses Can Be Antagonized by Histone Deacetylase Inhibitors and Transcriptional Activators, but Uniform Reactivation in Cell Clones Is Restricted by Additional Mechanisms{triangledown}

Richard A. Katz,1* Emily Jack-Scott,1,{dagger} Anna Narezkina,1,§ Ivan Palagin,1,§ Pamela Boimel,1,¶ Joseph Kulkosky,2 Emmanuelle Nicolas,1 James G. Greger,1,{ddagger} and Anna Marie Skalka1

Fox Chase Cancer Center, Institute for Cancer Research, Philadelphia, Pennsylvania 19111,1 Chestnut Hill College, Philadelphia, Pennsylvania 191182

Received 1 August 2006/ Accepted 13 December 2006

Integrated retroviral DNA is subject to epigenetic gene silencing, but the viral and host cell properties that influence initiation, maintenance, and reactivation are not fully understood. Here we describe rapid and high-frequency epigenetic repression and silencing of integrated avian sarcoma virus (ASV)-based vector DNAs in human HeLa cells. Initial studies utilized a vector carrying the strong human cytomegalovirus (hCMV) immediate-early (IE) promoter to drive expression of a green fluorescent protein (GFP) reporter gene, and cells were sorted into two populations based on GFP expression [GFP(+) and GFP(–)]. Two potent epigenetic effects were observed: (i) a very broad distribution of GFP intensities among cells in the GFP(+) population as well as individual GFP(+) clones and (ii) high-frequency GFP reporter gene silencing in GFP(–) cells. We previously showed that histone deacetylases (HDACs) can associate with ASV DNA soon after infection and may act to repress viral transcription at the level of chromatin. Consistent with this finding, we report here that treatment with the histone deacetylase inhibitor trichostatin A (TSA) induces GFP activation in GFP(–) cells and can also increase GFP expression in GFP(+) cells. In the case of the GFP(–) populations, we found that after removal of TSA, GFP silencing was reestablished in a subset of cells. We used that finding to enrich for stable GFP(–) cell populations in which viral GFP reporter expression could be reactivated by TSA; furthermore, we found that the ability to isolate such populations was independent of the promoter driving the GFP gene. In such enriched cultures, hCMV IE-driven, but not the viral long terminal repeat-driven, silent GFP reporter expression could be reactivated by the transcriptional activator prostratin. Microscopy-based studies using synchronized cells revealed variegated reactivation in cell clones, indicating that secondary epigenetic effects can restrict reactivation from silencing. Furthermore we found that entry into S phase was not required for reactivation. We conclude that HDACs can act rapidly to initiate and maintain promoter-independent retroviral epigenetic repression and silencing but that reactivation can be restricted by additional mechanisms.

* Corresponding author. Mailing address: 333 Cottman Avenue, Philadelphia, PA 19111. Phone: (215) 728-3668. Fax: (215) 728-2778. E-mail: Richard.Katz{at}fccc.edu.

{triangledown} Published ahead of print on 3 January 2007.

{dagger} Present address: Yale University, New Haven, CT.

§ Present address: Smolensk State Medical Academy, Smolensk, Russia.

Present address: Albert Einstein College of Medicine, New York, NY.

{ddagger} Present address: Wyeth Research, Collegeville, PA.

Journal of Virology, March 2007, p. 2592-2604, Vol. 81, No. 6
0022-538X/07/$08.00+0     doi:10.1128/JVI.01643-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Senigl, F., Plachy, J., Hejnar, J. (2008). The Core Element of a CpG Island Protects Avian Sarcoma and Leukosis Virus-Derived Vectors from Transcriptional Silencing. J. Virol. 82: 7818-7827 [Abstract] [Full Text]  
  • Poleshko, A., Palagin, I., Zhang, R., Boimel, P., Castagna, C., Adams, P. D., Skalka, A. M., Katz, R. A. (2008). Identification of Cellular Proteins That Maintain Retroviral Epigenetic Silencing: Evidence for an Antiviral Response. J. Virol. 82: 2313-2323 [Abstract] [Full Text]  


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