Characterization and Inhibition of Viral Nuclease Enzymes

• Author / Creator

  Todd, Brendan, Geoffrey John

• Viral infection accounts for significant morbidity, mortality, and economic loss worldwide. At no time in modern history has this been more apparent than it is now given the current global pandemic. This problem is compounded by the emergence of viral strains resistant to currently available therapeutics. Therefore, the identification and characterization of antiviral compounds with novel targets and mechanisms of action is urgently required. One such underexplored target that has recently shown promise is viral nuclease enzymes. These enzymes are required for the replication of several viruses including herpesviruses, influenza viruses, SARS-CoV-2, HIV, and others.
  In this thesis, I expressed and purified nuclease enzymes from three clinically relevant human viruses with the goal of developing biochemical systems to allow us to understand the mechanism of action of antiviral drugs. To this end, I expressed and purified the terminase complex of cytomegalovirus, the RNA dependent RNA polymerase heterotrimer of influenza B (FluB-ht), and the proofreading exonuclease complex of SARS-CoV-2. These enzymes were chosen as they had been shown to either be the target of or involved in resistance to recently approved FDA approved antiviral drugs. For example, terminase is the target of letermovir, FluB-ht is the target of baloxavir marboxil, and the SARS-CoV-2 proofreading exonuclease may be involved in resistance against nucleoside analogue inhibitors of SARS-CoV-2 such as remdesivir.
  First, I successfully expressed and purified the cytomegalovirus (CMV) terminase complex and developed assays to monitor its ATPase, nuclease, and DNA binding activities. As this complex is known to be the target of letermovir, it was our goal to elucidate the mechanism of action
  ii
  underlying this new FDA approved drug. The assays that I have developed will facilitate this process. Furthermore, by truncating our expression construct, I was able to show that a small protein lacking enzymatic activity, UL51, is necessary for terminase expression.
  Secondly, with regards to the influenza B polymerase heterotrimer (FluB-ht), I developed assays to monitor the endonuclease and transcriptional activities of the complex. These assays revealed that the active form of the FDA approved influenza endonuclease (PA) inhibitor baloxavir marboxil, baloxavir acid (BXA), is a slow tight binding competitive inhibitor. Furthermore, I was able to show that BXA is a selective inhibitor of the endonuclease as it displays a greater than 50-fold selectivity for the endonuclease activity.
  Finally, with respect to the SARS-CoV-2 exonuclease, I successfully purified the active complex and showed that this complex can excise incorporated nucleotide analogues with analogues that represent mismatched base-pairs being more readily excised. Furthermore, I was able to show that under the right circumstances, the excision of the FDA approved SARS-CoV-2 inhibitor remdesivir is possible. This revealed a possible avenue for the development of resistance against nucleoside analogue inhibitors of SARS-CoV-2 and produced an assay for the excision of nucleoside analogue inhibitors in vitro.

• Subjects / Keywords

  • Virology

• Graduation date

  Fall 2021

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-pnh3-pv31

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.