Influenza A virus (IAV) contains a segmented RNA genome that is transcribed and replicated by the viral RNA polymerase in the cell nucleus. Replicated RNA segments are assembled with viral polymerase and oligomeric nucleoprotein into viral ribonucleoprotein (vRNP) complexes which are exported from the nucleus and transported across the cytoplasm to be packaged into progeny virions. Host 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 study, we utilised a combination of biochemical, proteomic, and biophysical approaches to characterise the interaction between the viral polymerase and Rab11a. Using pull-down assays we show that vRNPs but not cRNPs from infected cell lysates bind to Rab11a. We also show that the viral polymerase directly interacts with Rab11a and that the C-terminal two thirds of the PB2 polymerase subunit (PB2-C) comprising the cap-binding, mid-link, 627 and nuclear localization signal (NLS) domains mediate this interaction. Small-angle X-ray scattering (SAXS) experiments confirmed that PB2-C associates with Rab11a in solution forming a compact folded complex with a 1:1 stoichiometry. Furthermore, we demonstrate that the switch I region of Rab11a, that has been shown to be important for binding Rab11 family interacting proteins (Rab11-FIPs), is also important for PB2-C binding suggesting that IAV polymerase and Rab11-FIPs compete for the same binding site. Our findings expand our understanding of the interaction between the IAV polymerase and Rab11a in the cytoplasmic transport of vRNPs. Importance The influenza virus RNA genome segments are replicated in the cell nucleus and are assembled into viral ribonucleoprotein (vRNP) complexes with viral RNA polymerase and nucleoprotein (NP). Replicated vRNPs need to be exported from the nucleus and trafficked across the cytoplasm to the cell membrane where virion assembly takes place. The host GTPase Rab11a plays a role in vRNP trafficking. In this study we show that the viral polymerase directly interacts with Rab11a mediating the interaction between vRNPs and Rab11a. We map this interaction to the C-terminal domains of the PB2 polymerase subunit and the switch I region of Rab11a. Identifying the exact site of Rab11a binding on the viral polymerase could uncover a novel target site for the development of an influenza antiviral drug.
Journal of virology
Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, United Kingdom.