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Structural and Mechanistic Insights into HIV Regulated Splicing

Morgan, Christopher Edward

Abstract Details

2018, Doctor of Philosophy, Case Western Reserve University, Chemistry.
Human Immunodeficiency virus type 1 (HIV-1) is an epidemic that affects about 37 million people globally. The disease is not yet curable, but is treated using highly active antiretroviral therapy (HAART), a drug cocktail that targets various proteins required for virulence and replication of the virus. HAART is a lifelong commitment that does not fully restore the immune system of the patient and, despite wide usage of the therapy, the HIV epidemic continues largely unperturbed. It is therefore important to better understand the viral lifecycle to elucidate new therapeutic targets. The 9 kb HIV genome produces more than 40 different mRNA transcripts through alternative splicing, a process that is tightly regulated by the hnRNP (silencing) and SR (enhancing) host protein families through interactions with various RNA splicing elements. Variations in the highly regulated alternative splicing process can cause perturbations in HIV replication, making this a novel therapeutic target. To that end, this thesis seeks to better understand HIV regulated splicing through structural and biophysical studies. Specifically, the research presented herein focuses on the structures of two HIV RNA elements, ESS3 of ssA7 and ESSV of ssA2, and how they function to silence splicing at a given site through interactions with the host protein hnRNP A1. First, the interaction between hnRNP A1 and ESS3 was characterized through structural, thermodynamic and kinetic techniques. Results showed that the hnRNP A1 – ESS3 complex forms at a 1 to 1 ratio and binding occurs site-specifically to a nucleobase pocket formed between RRM1 and the inter-RRM linker. Focus was then turned to successful development of a structural technique that combines NMR and SAXS to better define the structures of RNA in solution. Lastly, the solution structure of ESSV was solved using this hybrid NMR-SAXS approach, and its interactions with hnRNP A1 were characterized through Isothermal Titration Calorimetry, showing that hnRNP A1 binds to multiple sites on ESSV to silence splicing. Overall, this thesis sheds light on how RNA splicing is silenced at ssA7 and ssA2 through protein-RNA interactions and presents methods to better define and determine structures of RNA in solution.
Irene Lee, PhD (Committee Chair)
Michael Zagorski, PhD (Committee Member)
Paul Carey, PhD (Committee Member)
Eckhard Jankowsky, PhD (Committee Member)
Blanton Tolbert, PhD (Advisor)
295 p.

Recommended Citations

Citations

  • Morgan, C. E. (2018). Structural and Mechanistic Insights into HIV Regulated Splicing [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1523011774871958

    APA Style (7th edition)

  • Morgan, Christopher. Structural and Mechanistic Insights into HIV Regulated Splicing. 2018. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1523011774871958.

    MLA Style (8th edition)

  • Morgan, Christopher. "Structural and Mechanistic Insights into HIV Regulated Splicing." Doctoral dissertation, Case Western Reserve University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=case1523011774871958

    Chicago Manual of Style (17th edition)