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Journal of Virology, December 2008, p. 12291-12303, Vol. 82, No. 24
0022-538X/08/$08.00+0     doi:10.1128/JVI.01383-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Epigenetic Silencing of Human Immunodeficiency Virus (HIV) Transcription by Formation of Restrictive Chromatin Structures at the Viral Long Terminal Repeat Drives the Progressive Entry of HIV into Latency{triangledown}

Richard Pearson, Young Kyeung Kim, Joseph Hokello, Kara Lassen, Julia Friedman, Mudit Tyagi, and Jonathan Karn*

Department of Molecular Biology and Microbiology, Case Western Reserve University School of Medicine, Cleveland, Ohio

Received 2 July 2008/ Accepted 22 September 2008

The molecular mechanisms utilized by human immunodeficiency virus (HIV) to enter latency are poorly understood. Following the infection of Jurkat T cells with lentiviral vectors that express Tat in cis, gene expression is progressively silenced. Silencing is greatly enhanced when the lentiviral vectors carry an attenuated Tat gene with the H13L mutation. Individual clones of lentivirus-infected cells showed a wide range of shutdown rates, with the majority showing a 50% silencing frequency between 30 to 80 days. The silenced clones characteristically contained a small fraction (0 to 15%) of activated cells that continued to express d2EGFP. When d2EGFP+ and d2EGFP– cell populations were isolated from the shutdown clones, they quickly reverted to the original distribution of inactive and active cells, suggesting that the d2EGFP+ cells arise from stochastic fluctuations in gene expression. The detailed analysis of transcription initiation and elongation using chromatin immunoprecipitation (ChIP) assays confirms that Tat levels are restricted in the latently infected cells but gradually rise during proviral reactivation. ChIP assays using clones of latently infected cells demonstrate that the latent proviruses carry high levels of deacetylated histones and trimethylated histones. In contrast, the cellular genes I{kappa}B{alpha} and GAPDH had high levels of acetylated histones and no trimethylated histones. The levels of trimethylated histone H3 and HP1-{alpha} associated with HIV proviruses fell rapidly after tumor necrosis factor alpha activation. The progressive shutdown of HIV transcription following infection suggests that epigenetic mechanisms targeting chromatin structures selectively restrict HIV transcription initiation. This decreases Tat production below the levels that are required to sustain HIV gene expression.

* Corresponding author. Mailing address: Department of Molecular Biology and Microbiology, Case Western Reserve University, 10900 Euclid Avenue, Room W200, Cleveland, OH 44106-4960. Phone: (216) 368-3915. Fax: (216) 368-3055. E-mail: jonathan.karn{at}case.edu

{triangledown} Published ahead of print on 1 October 2008.

Journal of Virology, December 2008, p. 12291-12303, Vol. 82, No. 24
0022-538X/08/$08.00+0     doi:10.1128/JVI.01383-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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