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Defining the Structural Modulation of Cap-Independent Translation in Enterovirus 71

Dávila-Calderón, Jesse
http://orcid.org/0000-0002-9991-4536
http://rave.ohiolink.edu/etdc/view?acc_num=case1648818543714608

Abstract Details

2022, Doctor of Philosophy, Case Western Reserve University, Chemistry.
Enterovirus 71 (EV71), represents a persistent threat to global health and economies, as outbreaks are reported in the United States and globally each year. Infections are self-limited; however, prolonged infection in the immunocompromised can lead to severe neurological disorders and, eventually, death. As of the time of writing, there are no FDA-approved treatments against this pathogen. Thus, there is an immediate urgency to determine the mechanisms regulating host-virus interactions. EV71 utilizes a type I IRES element to initiate viral translation in a cap-independent pathway by recruiting multiple host proteins through a poorly understood mechanism. This thesis seeks to define the molecular and specificity determinants regulating the formation of IRES-protein complexes that modulate cap-independent translation. Herein, we studied the interactions of the negative translation modulator AUF1 with the conserved stem loop II (SLII) IRES domain. In chapter 2 we demonstrate that AUF1 and its isolated RRMs bind to the SLII bulge motif via a monophasic thermodynamic transition, where the bulk of the thermodynamic stability is conferred by the first RRM. Building on this knowledge, in chapter 3 we screened a library of RNA-targeting small molecules against the SLII IRES domain and found a potent inhibitor (DMA-135) of EV71 translation and replication. A combination of biophysical and functional studies revealed that DMA-135 functions by inducing a conformational change on SLII which stabilizes the formation of the repressive SLII:DMA-135:AUF1 complex. In chapter 4, we validated the proposed mechanism of action by generating (DMA-135)-EV71 resistant mutants, where the suppressor mutations mapped to SLII. Biophysical studies revealed that the suppressor mutations changed the local RNA structure around the SLII bulge which impaired DMA-135 and AUF1 binding. In chapter 5, we gathered the knowledge obtained in all previous chapters to delineate a pipeline for the identification and validation of RNA therapeutic targets and small molecules that function as inhibitors of RNA virus replication. In sum, the work presented in this thesis reveals that the integration of multiple biophysical, biochemical, and molecular biology approaches is necessary to develop a holistic understanding of cap-independent translation in EV71 and the identification of small molecule inhibitors that target viral RNA.
Blanton Tolbert (Advisor)
Fu-Sen Liang (Committee Chair)
Robert Salomon (Committee Member)
Thomas Gerken (Committee Member)
Shane Parker (Committee Member)
325 p.
Biophysics; Chemistry; Virology
Enterovirus 71; Cap-independent translation; RNA virus; biophysics; structural biology

Recommended Citations

Citations

  • Dávila-Calderón, J. (2022). Defining the Structural Modulation of Cap-Independent Translation in Enterovirus 71 [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1648818543714608

    APA Style (7th edition)

  • Dávila-Calderón, Jesse. Defining the Structural Modulation of Cap-Independent Translation in Enterovirus 71. 2022. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1648818543714608.

    MLA Style (8th edition)

  • Dávila-Calderón, Jesse. "Defining the Structural Modulation of Cap-Independent Translation in Enterovirus 71." Doctoral dissertation, Case Western Reserve University, 2022. http://rave.ohiolink.edu/etdc/view?acc_num=case1648818543714608

    Chicago Manual of Style (17th edition)

case1648818543714608
56
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This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.