Study of the interaction between the influenza A virus polymerase and GTPase Rab11a

The influenza A virus (IAV) genome that consists of eight viral RNA (vRNA) segments is transcribed and replicated by the viral heterotrimeric RNA-dependent RNA polymerase (RdRP) complex, composed of polymerase acidic (PA), polymerase basic 1 (PB1), and PB2, subunits, in the cell nucleus. Replicated vRNA segments are assembled with RdRP and oligomeric nucleoprotein into viral ribonucleoprotein (vRNP) complexes which are exported from the nucleus and transported across the cytoplasm to be packed into progeny virions. The GTPase Rab11a associated with recycling endosomes is believed to contribute to this process by mediating the cytoplasmic transport of vRNPs. However, how vRNPs interact with Rab11a remains poorly understood. In this D.Phil. project, I have utilised a combination of biochemical and structural approaches to characterise the interaction between the IAV RdRP and Rab11a. Initially, using a GST pull-down assay, I showed that the interaction between the IAV RdRP and Rab11a is direct, and that the 627- domain of PB2 is required for this interaction. Furthermore, my study demonstrated for the first time that Rab11a has a differential specificity for vRNP but not cRNP. Analysis of the Rab11a interactome further revealed that IAV infection reduces Rab11a binding to Rab11-family interacting partners (Rab11-FIPs), thereby altering the stoichiometry of cytoplasmic motor proteins bound by Rab11a. Using mutant Rab11a in a GST pull-down assay, this study provides the first evidence that IAV RdRP binds to the same site on Rab11a as Rab11-FIPs. It was also investigated here whether Rab11a has a role in determining the host specificity of influenza viruses. However, this study could not identify any adaptive mutations in PB2 that may affect the interaction between RdRP and Rab11a. Moreover, the study also did not identify the exact site of Rab11a binding on the viral RdRP using cryo- electron microscopy. However, performing a small-angle X-ray scattering analysis I showed that C-terminal two thirds of PB2 (PB2-C) and Rab11a form a well-folded protein complex in solution, and confirmed in a controlled pull-down assay that the interaction between PB2-C and Rab11a is direct. Finally, I have also established a workflow to study the interaction between IAV vRNPs and Rab11a in the context of live infected cells using full field cryo-X-ray microscopy. The findings of my D.Phil. work expand our understanding of the cytoplasmic transport of the influenza virus vRNPs. Identifying the exact site of Rab11a binding on the viral RdRP could uncover a novel target site for the development of an influenza antiviral drug.