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The Physicochemical Characterization of Proteins and RNA in Positive Strand RNA Viruses

Haddad, Christina
http://orcid.org/0000-0003-4082-2941
http://rave.ohiolink.edu/etdc/view?acc_num=case1680604529910535

Abstract Details

2023, Doctor of Philosophy, Case Western Reserve University, Chemistry.
Positive Strand RNA (PSR) viruses, such as coronaviruses and enteroviruses, cause serious health and economic threats worldwide, as seen with the COVID-19 pandemic. This has drawn attention to the importance of identifying new antivirals and molecular targets in RNA viruses. The multifunctionality of PSR genomes make them desirable targets for therapeutic intervention. Here, we present a class of antivirals that can inhibit SARS-CoV-2 replication in vitro by targeting conserved viral RNA structures at the 5’-end. Specifically, stem loops (SLs) 1, 4, 5a, and 6 of the viral 5’-region have shown a degree of binding with dimethyl amiloride molecules as determined by NMR structural analysis. These results open the door to potentially develop specific small molecules against SARS-CoV-2 and related coronaviruses. Upon investigating SL6, interesting structural dynamics features were observed at the budge region when exposed to different temperatures. From various Nuclear Magnetic Resonance (NMR) and single angle x-ray scattering (SAXS) experiments, experimental restrains were obtained in order to generate a 3D structure of SL6 using molecular dynamics simulations. In SARS-CoV-2, stem-loop 3, which contains the transcriptional regulatory sequence, was proven to bind to the host Unwinding Protein 1 (UP1) using electrophoretic mobility shift assay (EMSA), isothermal titration chromatography (ITC), and NMR, which possibly suggests that UP1 participates in the mechanism of transcription of sub-genomic RNA. In addition, another PSR virus, Enterovirus A71 (EV-A71), which is the etiological agent of the hand, foot, and mouth disease, has caused severe morbidity and high mortality rates in children for decades. Thus, understanding the mechanisms by which EV-A71 replicates within the cellular environment can bring to light efficient drug targets for viral inhibition. The 5’-untranslated region (5’-UTR) of the RNA genome is the control hub of viral replication and transcription in EV-A71. The multifunctional viral protein, 3C protease (3Cpro), is essential for viral protein processing and RNA synthesis, which occurs by binding to SLI of the 5’-UTR. Here, we investigate how RNA binding of SLID, a portion of SLI, can slightly affect the enzymatic activity of 3Cpro. We identify an overlooked dimerization surface on 3Cpro that is proximal to its active site and distal to its RNA binding domain. Our data show that RNA binding is allosterically coupled to 3Cpro dimerization. To that point, single, double, and triple point mutations in the 3Cpro dimerization domain attenuates viral growth and kinetics. Taken together, we present compelling data that demonstrates novel targeting surfaces on 3Cpro that can be pursued as novel antiviral targets.
Blanton S. Tolbert (Advisor)
Fu-Sen Liang (Committee Chair)
Thomas Gerken (Committee Member)
Robert Salomon (Committee Member)
Divita Mathur (Committee Member)
283 p.
Biochemistry; Biophysics; Chemistry; Virology
Positive Strand RNA Viruses, Enterovirus A71, SARS-CoV-2, 3C protease, RNA 5'-untranslated region, Biophysics, Structural Biology

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Citations

  • Haddad, C. (2023). The Physicochemical Characterization of Proteins and RNA in Positive Strand RNA Viruses [Doctoral dissertation, Case Western Reserve University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=case1680604529910535

    APA Style (7th edition)

  • Haddad, Christina. The Physicochemical Characterization of Proteins and RNA in Positive Strand RNA Viruses. 2023. Case Western Reserve University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=case1680604529910535.

    MLA Style (8th edition)

  • Haddad, Christina. "The Physicochemical Characterization of Proteins and RNA in Positive Strand RNA Viruses." Doctoral dissertation, Case Western Reserve University, 2023. http://rave.ohiolink.edu/etdc/view?acc_num=case1680604529910535

    Chicago Manual of Style (17th edition)

case1680604529910535
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© 2023, some rights reserved.Creative Commons License The Physicochemical Characterization of Proteins and RNA in Positive Strand RNA Viruses by Christina Haddad is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by Case Western Reserve University School of Graduate Studies and OhioLINK.