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HISTONE LYSINE METHYLTRANSFERASES SELECTIVELY RESTRICT HIV-1 IN CENTRAL MEMORY T-CELLS

Dobrowolski, Curtis Noel

Abstract Details

2018, Doctor of Philosophy, Case Western Reserve University, Molecular Virology.
Histone lysine methyltransferases (HKMTs) are key mediators of epigenetic silencing. Removal of these epigenetic blocks using inhibitors to EZH2, a key component to the polycomb repressive complex 2 (PRC2) or EHMT2, a methyltransferase induces HIV transcription from latent proviruses. We hypothesized that long-lived central memory cells will have the strongest epigenetic restrictions and therefore that these histone methyltransferase inhibitors will have a greater effect on central memory cells compared to other subsets. Memory CD4 T cells from three HIV positive CART treated donors where sorted into central memory (Tcm), effector memory (Tem) and transitional memory (Ttm) T cells using FACS. Each subset was pretreated with either GSK-343 (EZH2 inhibitor) or UNC-0638 (EHMT2 inhibitor) to prevent H3K27 or H3K9 methylation. After pretreatment, IL-15 was added to induce P-TEFb expression and HIV transcription. HIV transcripts were detected using EDITS (Envelope Detection by Induced Transcription-based Sequencing) by nested amplification of HIV Env splice junctions followed by deep sequencing using the Ion Torrent platform. Inhibition of EZH2 or G9a resulted in an increase of HIV transcription in memory CD4 T cells. When used in conjunction with an HDAC inhibitor (SAHA) or IL-15, proviral induction is synergistically potentiated. After memory T cells were sorted into Tcm, Tem and Ttm and treated with the EZH2 or EHMT2 inhibitor, there was a large induction of HIV transcription in the Tcm population. This became even more pronounced when used in conjunction with IL-15, and reached levels that were compare, or in some cases slightly higher, than induction by TCR stimulation using anti-CD3/CD28 beads. Inhibition of EZH2 or EHMT2 results in a significant increase of overall HIV transcription in memory T cells. The Tcm population, which are the most long lived of the memory T cell population, had enhanced strong epigenetic restrictions and responded much more strongly to histone methyltransferase inhibitors than the other memory T-cell subsets. The use of histone methyltransferase inhibitors in conjunction with other LRAs provide a new strategy for the reactivation for the deeply latent proviruses that accumulate in the central memory potential, as part of a “shock and kill” strategy.
John Tilton (Committee Chair)
Jonathan Karn (Advisor)
Jacek Skowronski (Committee Member)
Miguel Quinones-Mateu (Committee Member)
176 p.

Recommended Citations

Citations

  • Dobrowolski, C. N. (2018). HISTONE LYSINE METHYLTRANSFERASES SELECTIVELY RESTRICT HIV-1 IN CENTRAL MEMORY T-CELLS [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1522842870401743

    APA Style (7th edition)

  • Dobrowolski, Curtis. HISTONE LYSINE METHYLTRANSFERASES SELECTIVELY RESTRICT HIV-1 IN CENTRAL MEMORY T-CELLS. 2018. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1522842870401743.

    MLA Style (8th edition)

  • Dobrowolski, Curtis. "HISTONE LYSINE METHYLTRANSFERASES SELECTIVELY RESTRICT HIV-1 IN CENTRAL MEMORY T-CELLS." Doctoral dissertation, Case Western Reserve University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1522842870401743

    Chicago Manual of Style (17th edition)