Structure and interactions of endonuclease in the influenza a polymerase complex

Thesis

The influenza viral genome is contained in eight discrete segments which are transcribed and replicated separately by influenza viral polymerases (FluPol) before being assembled into nascent virions and trafficked out of the host cell (Zhu et al., 2023). FluPols undergo dramatic conformational changes and can perform either transcription or replication depending on cellular conditions and interaction with different host factors (Carrique et al., 2020). The endonuclease at the N-terminal end of the Polymerase Acidic (PA) subunit of FluPol is necessary for transcription; endonuclease performs “cap-snatching,” where host mRNA caps are cleaved and used to initiate transcription of viral mRNA (Dias et al., 2009). Interestingly, endonuclease remains structured in the cap-snatching incompetent conformations of FluPol and appears to interact with different subunits or external proteins depending on the FluPol conformation (Staller et al., 2024, Thierry et al., 2016; Vidic et al., 2016). Whether the interactions with different FluPol subunits regulate endonuclease activity is unknown. Based upon the hypothesis that regulation of promiscuous endonuclease activity could mediate conformational changes in the FluPol, I tested the interactions of endonuclease and three potential binding partners: viral nucleoprotein, a peptide from the Polymerase Basic 1 (PB1) subunit, and a peptide from the Polymerase Basic 2 (PB2) subunit. The same unstructured region of the endonuclease appeared to interact with viral nucleoprotein and the PB1 peptide. I introduced point mutations to knock out the interaction, confirming the residues implicated in binding, and performed activity assays to assess the enzymatic activity of wild type and mutant endonuclease with each potential binding partner, where the mutants displayed substantial loss of function. These results increase understanding of endonuclease interactions in the influenza polymerase and identify a single residue mutation which confers almost complete loss of function, offering insight into potential targets for novel antiviral therapies.

Authors: Rhyu, I

• DOI: 10.5287/ora-erpkyyyoo
• Type of award: MSc by Research
• Level of award: Masters
• Awarding institution: University of Oxford
• Language: English
• Keywords: flu; FluPol; endonuclease; influenza; endo; NMR; polymerase
• Subjects: Structural dynamics; Biochemistry; Structural biology