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Journal of Virology, March 2008, p. 2313-2323, Vol. 82, No. 5
0022-538X/08/$08.00+0     doi:10.1128/JVI.01882-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Identification of Cellular Proteins That Maintain Retroviral Epigenetic Silencing: Evidence for an Antiviral Response

Andrey Poleshko, Ivan Palagin, Rugang Zhang, Pamela Boimel, Carolyn Castagna, Peter D. Adams, Anna Marie Skalka, and Richard A. Katz^*

Fox Chase Cancer Center, Institute for Cancer Research, Philadelphia, Pennsylvania 19111

Received 28 August 2007/ Accepted 10 December 2007

Integrated retroviral DNA is subject to epigenetic gene silencing, resulting in loss of expression of viral genes as well as reporter or therapeutic genes transduced by retroviral vectors. Possible mediators of such silencing include the histone deacetylase (HDAC) family of cellular proteins. We previously isolated HeLa cell populations that harbored silent avian sarcoma virus-based green fluorescent protein (GFP) vectors that could be reactivated by treatment with HDAC inhibitors. Here, we developed a small interfering RNA (siRNA)-based approach to identify specific host factors that participate in the maintenance of silencing. Knockdown of HDAC1, the transcriptional repressor Daxx (a binding partner of HDAC1), or heterochromatin protein 1 gamma resulted in robust and specific GFP reporter gene reactivation. Analyses of cell clones and diverse GFP vector constructs revealed that the roles of HDAC1 and Daxx in retroviral silencing are largely independent of the integration site or the promoter controlling the silent GFP reporter gene. Previous findings from our laboratory and those of others have suggested that Daxx and HDAC proteins may act broadly as part of an antiviral response to repress viral gene transcription. Expression of presumptive viral "countermeasure" proteins that are known to inhibit Daxx or HDACs (pp71, IE2, and Gam1) resulted in the reactivation of GFP reporter gene expression. This study has identified individual host factors that maintain retroviral silencing and supports the proposal that these factors participate in an antiviral response. Furthermore, our results indicate that siRNAs can be used as specific reagents to interrupt the maintenance of epigenetic silencing.

Published ahead of print on 19 December 2007.

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

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

Present address: Temple University School of Law, Philadelphia, PA.

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