PhD Thesis Presentation - A Novel Mechanism of Enhanced Transcription Activity and Fidelity for Influenza A Viral RNA-dependent RNA Polymerase

Influenza pandemics and seasonal IAV is a significant threat to avian, swine, and human populations. IAV is a negative-sense, segmented RNA virus that is a member of the family Orthomyxoviridae. The RNA-dependent RNA polymerase (RdRp) is the primary machinery for all viral transcription and genome replication, which has important consequences for evolution, transmission, pathogenicity, and host range restriction. The molecular mechanisms by which they control the rate and fidelity of NTP incorporation, however, remain unknown. To elucidate the mechanistic details of fidelity control and to identify potential anti-flu compounds targeting the IAV RdRp, I used a multi-level approach in this thesis to find clues from enzymology, structural biology, and virology aspects. I demonstrated that a positively charged residue, K235, in the RdRp PB1 subunit is critical for transcription activity and fidelity regulation. Contrary to popular belief, I found that a single Lysine-to-Arginine mutation on K235 can significantly improve both transcription fidelity and activity, indicating a high potential for vaccine development. Drug resistance to the neuraminidase enzyme found on the surface of IAV has grown to be a major global issue. Apart from the active sites of RdRp, the cap-binding domain and endonuclease domain of RdRp could be used as potential therapeutic targets for the development of new anti-flu drugs. By combining in silica docking and reporter assay, I successfully identified several natural compound scaffolds that inhibit the transcription activity of IAV RdRp with the assistance of collaborators. Additionally, I established a baculovirus-insect expression system for wildtype and mutant IAV RdRps in order to investigate the structural basis of the high-fidelity K235R mutant and the novel compounds' potential target sites. These findings, taken together, shed new light on the mechanisms underlying NTP incorporation and fidelity control, laying the groundwork for rational design of IAV vaccines and potential antiviral drugs.