INVESTIGATION OF THE NUCLEOTIDE SELECTION MECHANISM OF RNA-DEPENDENT RNA POLYMERASE

Open Access
Author:
Shi, Jingjing
Graduate Program:
Chemistry
Degree:
Master of Science
Document Type:
Master Thesis
Date of Defense:
November 07, 2018
Committee Members:
  • David D Boehr, Thesis Advisor
Keywords:
  • poliovirus
  • RdRp
  • fidelity
  • NMR
  • HSQC
  • norovirus
Abstract:
RNA viruses cause a number of acute and chronic diseases including the common cold, severe acute respiratory syndrome (SARS), and a more recent outbreak of Middle East respiratory syndrome (MERS). Vaccines developed against viruses have saved many lives. A traditional vaccine is a preparation of killed microorganisms, live attenuated organism, or living fully virulent organisms that is administered to produce or artificially increase immunity to a disease. However, safety concerns and efficacy are potential problems for the further development of new vaccines. One promising strategy to develop vaccines is by targeting the virally encoded RNA-dependent RNA polymerase (RdRp). The RdRp is conserved in most RNA viruses and these enzymes share a conserved structure and catalytical residues. It has been determined that RdRp error rate relates to viral attenuation. A too accurate RdRp loses adaptability to the host environment; RdRp with higher error rate are also detrimental because the potential of lethal mutagenesis. It is crucial to understand the nucleotide selection mechanism to further investigate the development of live, attenuated vaccines. Poliovirus (PV) RdRp can be used as a model to study the nucleotide selection mechanism. There have been great efforts in investigating the roles of different conserved structural motifs in poliovirus RdRp regarding RdRp error rate. This thesis will mainly focus on the motif D of RdRp and uses a combination of kinetic and NMR experiments. Motif D undergoes an “open” to “closed” conformational change during catalysis. Site-directed mutagenesis experiments suggest that the importance of motif D is not just the conformational change during catalysis but the rearrangement of the active site lysine residue. Other amino acid changes in motif D may contribute to the repositioning of the active site lysine residue and may be used to change the RdRp error rate. Besides motif D, long-range interactions also potentially contribute to the change of PV RdRp fidelity by the repositioning of the catalytic lysine residue. NMR is a great tool to study structural dynamic changes during catalysis. The 1H,13C-methyl resonance of Met354 in motif D has been used as a probe to indicate the structural change in motif D. Single amino acid substitutions (K228A and N370A) in motif D change the open-closed conformational equilibrium and by perturbing the motif D conformational state, the enzyme fidelity is altered. Experiments with other protein variants (K359H, K359H/I331F) involving the motif D lysine suggest a relationship between fidelity and replication speed. . Norovirus is also a single-stranded, positive-sense RNA virus and it also uses RdRp as the replicative enzyme. However, unlike poliovirus, there is no FDA approved norovirus vaccine. It is worthwhile to apply the knowledge from poliovirus to study antiviral strategies for norovirus. This thesis will introduce preliminary trials of applying tools developed from the poliovirus system to the study of norovirus RdRp.