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Characterizing interactions between the cellular HIV-1 integration cofactor, LEDGF/p75 with chromatin and HIV-1 integrase

Eidahl, Jocelyn O
http://rave.ohiolink.edu/etdc/view?acc_num=osu1366147572

Abstract Details

2013, Doctor of Philosophy, Ohio State University, Pharmacy.
Continuous emergence of HIV-1 phenotypes resistant to currently available therapies requires the need for development of new drugs with novel targets to prevent HIV-1 replication. Integration is an essential step in the life cycle of HIV-1 allowing for the stable integration of the viral genome into its host genome. The viral enzyme integrase (IN) catalyzes 3’-viral cDNA processing and DNA strand transfer reactions leading to the integration of the viral cDNA into the host DNA. HIV-1 IN is a promising therapeutic target due to the lack of a cellular counterpart. The first approved HIV-1 IN inhibitor specifically targets the IN:viral cDNA complex causing the viral cDNA to displace from the active site preventing DNA strand transfer and integration. Emergence of HIV-1 strains resistant to this current IN inhibitor exist highlighting the need for new HIV-1 inhibitors with novel targets or mechanisms of inhibition. Interactions essential for HIV-1 integration are formed between IN and viral cDNA, IN and cellular lens epithelium-derived growth factor/p75 (LEDGF/p75), as well as cellular LEDGF/p75 and chromatin. All three interactions could present novel drug targets and were the focus of my studies throughout my dissertation research. By understanding these critical interactions between the virus and host, we have the potential to be exploit such interactions for development of novel classes of HIV-1 inhibitors. Cellular LEDGF/p75 has been extensively shown to play a critical role in HIV-1 integration by tethering HIV-1 IN to active genes in host chromatin enhancing viral cDNA integration. Identifying the component of chromatin recognized by LEDGF/p75 that allows this cellular protein to tether IN to active gene regions is being widely pursued in the field of HIV-1. Interactions between components of the N-terminal region of LEDGF/p75, the PWWP domain, and chromatin were studied at length. Extensive biophysical and structural studies were conducted to examine the binding affinity and mechanism of binding of the LEDGF/p75 PWWP domain to both DNA and protein components of mononucleosomes (MN). Nuclear Magnetic Resonance (NMR) spectroscopy was used to solve the previously unknown protein structure of the LEDGF/p75 PWWP domain. The binding interfaces between LEDGF/p75 PWWP domain and components of MNs were also examined. Binding interactions between a specific histone post-translational modification (PTM), H3K36me3, were characterized and significance for high affinity binding monitored. Determining the details of the interaction between the LEDGF/p75 PWWP domain and active gene regions in chromatin could aid in the development of a novel class of HIV-1 inhibitors. The specific cellular roles performed by LEDGF/p75 are relatively unknown. LEDGF/p75 is proposed to act as a chromatin tether in the cell by interacting with chromatin via the N-terminal region of the protein while simultaneously interacting with various proteins via a conserved C-terminal domain. A MS-based proteomic approach was designed to identify cellular proteins interacting with LEDGF/p75 with hope to uncover interproteome interactions between LEDGF/p75 and cellular proteins. Our goal was to identify cellular proteins interacting with LEDGF/p75, characterize their binding interaction, and the functional significance of their interaction. Inhibiting catalytic activity of HIV-1 IN through an allosteric mechanism was explored as a novel therapeutic target. Dynamic interplay between the tetramer of IN subunits is essential for the formation of the functionally active IN-viral cDNA complex. Binding to an allosteric site on HIV-1 IN that could restrict dynamic interplay between protein subunits and form a catalytically inactive IN complex was examined.
Mamuka Kvaratskhelia (Advisor)
213 p.
Pharmacy Sciences

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Citations

  • Eidahl, J. O. (2013). Characterizing interactions between the cellular HIV-1 integration cofactor, LEDGF/p75 with chromatin and HIV-1 integrase [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1366147572

    APA Style (7th edition)

  • Eidahl, Jocelyn. Characterizing interactions between the cellular HIV-1 integration cofactor, LEDGF/p75 with chromatin and HIV-1 integrase. 2013. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1366147572.

    MLA Style (8th edition)

  • Eidahl, Jocelyn. "Characterizing interactions between the cellular HIV-1 integration cofactor, LEDGF/p75 with chromatin and HIV-1 integrase." Doctoral dissertation, Ohio State University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=osu1366147572

    Chicago Manual of Style (17th edition)

osu1366147572
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© 2013, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.