Structure-function studies of viral RNA-dependent RNA polymerases
Date
2018-09-18
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Abstract
Human norovirus (NV) and hepatitis C virus (HCV) are two major and related human pathogens belonging to the positive-strand RNA virus superfamily. NV belongs in the Caliciviridae family and is a major cause of gastroenteritis outbreaks throughout the world. Although there is substantial interest in the development of vaccines and antiviral therapeutics for norovirus infections, there are currently no effective antiviral treatments. Using a fluorescence-based primer extension assay, I have identified a series of structurally-related compounds that inhibit norovirus polymerase at micromolar concentrations. Previous work in our group has helped to structurally characterize human norovirus polymerase complexes with RNA, nucleoside triphosphates, and divalent metal ions. These structures have revealed the enzyme trapped indifferent conformations that are likely important for the catalytic cycle. Several enzyme-inhibitor complexes with alendronic acid and medronic acid inhibitors have been crystallized, and the structure determination of NV Pol complexes has been undertaken. This thesis presents new results correlating enzyme inhibition kinetics with crystallographic structure determination for several novel inhibitor complexes. In the second part of this thesis, work is described on characterizing interactions between RNA and HCV non-structural protein NS5B, the RNA-dependent RNA Polymerase (RDRP) central to HCV replication. HCV NS5B is highly-conserved and forms the core of the catalytic site of the replicase complex. I have determined the structure of a novel polymerase-RNA complex that shows the binding of a short RNA oligonucleotide to a region of the enzyme near a commonly-targeted site for the binding of allosteric inhibitors.
Description
Keywords
Macromolecular crystallography, Protein purification, Polymerase, Hepatitis C, Norovirus
Citation
Samanta, S. (2018). Structure-function studies of viral RNA-dependent RNA polymerases (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/33047