Kinetic characterization of the inhibition, excision mechanisms, and fidelity of Hepatitis C Virus RNA-dependent RNA polymerase

dc.contributor.advisorJohnson, Kenneth A. (Kenneth Allen)
dc.contributor.committeeMemberRussell, Rick
dc.contributor.committeeMemberFast, Walter L
dc.contributor.committeeMemberDudley, Jaquelin P
dc.creatorVillalba, Brian
dc.date.accessioned2020-07-01T18:00:52Z
dc.date.available2020-07-01T18:00:52Z
dc.date.created2020-05
dc.date.issued2020-03-03
dc.date.submittedMay 2020
dc.date.updated2020-07-01T18:00:53Z
dc.description.abstractNS5B is the RNA-dependent RNA polymerase that catalyzes the replication of the Hepatitis C Virus genome. It is a major target for antiviral drugs including nucleoside analogs, such as the prodrugs Mericitabine and Sofosbuvir, which get metabolized to the chain terminators 2’-fluoro-2’-C-methylcytidine-5’-triphosphate and 2’fluoro-2’-C-methyluridine-5’-triphosphate, respectively. These analogs act as chain terminators after they are incorporated during RNA synthesis. Recently, work in our lab has shown that NS5B can efficiently remove chain-terminators by a nucleotide-mediated excision reaction that rescues RNA synthesis. In this study I use transient-state kinetics to probe the mechanism of inhibition for nucleoside analogs by directly measuring the rates of incorporation, pyrophosphorolysis, and ATP-mediated excision. I find that while CTP and CTP analogs are readily incorporated, they are efficiently excised. However, UTP is highly resistant to excision, and the 2’-C modifications of UTP serve to further inhibit excision. Furthermore, I use these same techniques to measure the in vitro fidelity of NS5B and uncover mechanisms for maintaining fidelity. The data demonstrate that NS5B exhibits a range of fidelity dependent on the nature of the mismatch. I also identified a slow-pyrophosphorolysis mechanism by NS5B used to further increase fidelity by decreasing k [subscript cat] /K [subscript m]. Together, this work offers insight into how current antiviral therapeutics escape excision, and can aid in the development of new antivirals by furthering our understanding of NS5B.
dc.description.departmentBiochemistry
dc.format.mimetypeapplication/pdf
dc.identifier.urihttps://hdl.handle.net/2152/81944
dc.identifier.urihttp://dx.doi.org/10.26153/tsw/8952
dc.language.isoen
dc.subjectHepatitis C Virus
dc.subjectRNA polymerase
dc.subjectEnzyme kinetics
dc.subjectNucleoside analogs
dc.titleKinetic characterization of the inhibition, excision mechanisms, and fidelity of Hepatitis C Virus RNA-dependent RNA polymerase
dc.typeThesis
dc.type.materialtext
thesis.degree.departmentBiochemistry
thesis.degree.disciplineBiochemistry
thesis.degree.grantorThe University of Texas at Austin
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy

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