Biochemical Characterization and Inhibition of Influenza B Virus RNA-Dependent RNA Polymerase

• Author / Creator

  Raeisimakiani, Parisa

• The influenza virus can cause severe respiratory illness in humans and animals. There are four types of influenza viruses (A, B, C, and D); however, only type A and B cause severe diseases in humans. Influenza virus belongs to the family Orthomyxoviridae, which includes segmented negative-sense RNA viruses. The Influenza RNA-dependent RNA polymerase (RdRp) is responsible for both viral genome replication and transcription. RdRp functions are essential to the viral life cycle, suggesting that it is a logical target for antiviral drugs. The RdRp is a heterotrimeric protein consisting of a catalytically active polymerase subunit (PB1), an endonuclease subunit (PA), and a cap-binding subunit (PB2).

  In this study, we purified influenza B RdRp and studied inhibitors of PB1 and PA. Favipiravir is a drug that targets PB1. This compound is a purine analog that affects RNA synthesis. Recent in vitro selection experiments revealed resistance-conferring mutations in the RdRp complex. We developed biochemical assays to elucidate the underlying mechanisms associated with inhibition and resistance. We demonstrated that the mutant enzyme diminishes rates of incorporation of the inhibitor when compared with the wild type. We have also identified structurally distinct nucleotide analogue inhibitors of RdRp that are not affected by this mutation. Thus, the mechanism of resistance seems to be specific. Baloxavir targets the endonuclease subunit PA. The influenza RdRp complex possesses two active sites that contain two divalent metal ions: the polymerase active site in PB1 and the endonuclease active site in PA. Using the purified RdRp complex, we utilized RNA polymerization and endonuclease assays to test selected compounds for potential inhibitory effects. The data shows that Baloxavir is a selective endonuclease inhibitor, and RNA synthesis is solely affected at very high concentrations of the inhibitor. The collective data confirms that targeting the polymerase and endonuclease active sites is a feasible approach. Future studies will show whether novel therapies based on these types of drug combinations may improve clinical outcomes.

• Subjects / Keywords

  • RdRp

• Graduation date

  Spring 2021

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-067b-as73

• 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.