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Epigenetic Silencing of HIV Transcription Through Formation of Restrictive Chromatin Structures at the Viral LTR Drives the Progressive Entry of HIV into Latency

Pearson, Richard
http://rave.ohiolink.edu/etdc/view?acc_num=case1223040734

Abstract Details

2009, Doctor of Philosophy, Case Western Reserve University, Molecular Biology and Microbiology.
The clinical relevance of HIV latency is now well understood but the molecular mechanisms that are utilized by HIV to enter into a latent state are yet to be determined. The development of a suitable experimental system for studying HIV latency, which permits detailed biochemical analysis and drug screening, is a critical step in the effort to develop therapeutic strategies that will lead to viral eradication. The work outlined in this thesis reports that, when Jurkat T-cells are infected with lentiviral vectors that express the transactivator protein (Tat) in cis, HIV gene expression is gradually silenced. Silencing is enhanced when the lentiviral vectors carry an attenuated Tat gene. Following these observations we were able to develop a Jurkat cell-based model system of HIV latency. Detailed analysis of transcription initiation and elongation using chromatin immunoprecipitation (ChIP) assays confirms that Tat levels are restricted in latently infected cells but rise during proviral reactivation. ChIP assays using clones of latently infected cells demonstrate that the latent proviruses carry markers of repressive chromatin structure that were lost after TNF-alpha activation and replaced with a more transcriptionally permissive state. The progressive shutdown of HIV transcription suggests that epigenetic mechanisms targeting chromatin structures selectively restrict the HIV LTR.The information gained from the development and analysis of the Jurkat T-cell model has been applied to the development of a novel primary T-cell model. Primary CD4+ T-cells isolated from peripheral blood were activated and amplified by antibodies to the T-cell receptor (TCR) and then infected with lentiviral vectors carrying attenuated Tat. After sorting for the infected cells and re-amplification, the infected cells were allowed to spontaneously enter latency by long-term cultivation on feeder cell lines in the absence of TCR stimulation. Examination of the chromatin status of the latent provirus before and after TCR re-activation allowed for the corroboration of the reversal of heterochromatin structures to active chromatin structures seen in the Jurkat T-cell model. Thus, restrictive chromatin structures at the HIV LTR contribute to transcriptional silencing leading to latency in primary CD4+ T-cells.
Jonathan Karn, PhD (Advisor)
Lloyd Culp, PhD (Committee Chair)
Amiya Banerjee, PhD (Committee Member)
David McDonald, PhD (Committee Member)
203 p.
Virology
HIV; Latency; chromatin; epigenetics

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Citations

  • Pearson, R. (2009). Epigenetic Silencing of HIV Transcription Through Formation of Restrictive Chromatin Structures at the Viral LTR Drives the Progressive Entry of HIV into Latency [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1223040734

    APA Style (7th edition)

  • Pearson, Richard. Epigenetic Silencing of HIV Transcription Through Formation of Restrictive Chromatin Structures at the Viral LTR Drives the Progressive Entry of HIV into Latency. 2009. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1223040734.

    MLA Style (8th edition)

  • Pearson, Richard. "Epigenetic Silencing of HIV Transcription Through Formation of Restrictive Chromatin Structures at the Viral LTR Drives the Progressive Entry of HIV into Latency." Doctoral dissertation, Case Western Reserve University, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=case1223040734

    Chicago Manual of Style (17th edition)

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© 2008, all rights reserved.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.