Dynamics and nanoscale topology of the membrane-associated Rab11-vRNP complex in the context of influenza virus-infected cells – FluNanoTrack

Influenza A viruses (IAV) pose a serious threat to global public health. Development of improved influenza prevention and therapy, whether it be the universal vaccine or novel anti-influenza drugs, will depend strongly on fundamental science improving our understanding of the molecular mechanisms involved in viral replication and transmission. In FluNanoTrack, we focus on the macromolecular complex formed by the association of IAV ribonucleoproteins with the cellular GTPase Rab11.

The genomic RNAs of IAV are replicated in the nucleus of infected cells in the form of ribonucleoproteins (vRNPs) and transported to the plasma membrane. There is evidence that the cellular GTPase Rab11 binds vRNPs through a direct interaction with the viral PB2 protein (a component of vRNPs) and is essential for vRNP transport, however the mechanisms involved remain largely unknow. Based on our recent work, we hypothesize that the transport of Rab11-vRNP complexes is driven by their physical association with remodelled endoplasmic reticulum (ER) membranes and Rab11-dependent transport vesicles distinct from recycling endosomes. The overall goal of FluNanoTrack is to provide a detailed mechanistic description of the dynamics and nanoscale topology of Rab11-vRNP complexes in the cellular context. This will be achieved by combining the complementary expertise in molecular virology, biological imaging and high resolution microscopy of the three partners.

We will characterize the membrane-associated interactome and dynamics of Rab11-vRNP complexes in live IAV-infected cells. To this end, we will use i) a newly described split-APEX2 complementation assay for in vivo proximity labelling of host factors associated with a protein-protein complex of interest, followed by quantitative mass-spectrometry, and ii) the SplitFAST technology developed by Partner 2, a fluorescence complementation assay that allows for real-time visualization of the formation and dissociation of protein assemblies in live cells. Ad hoc virus-cell systems will be engineered using reverse genetics to produce PB2-tagged influenza viruses and the CRISPR-Cas9 technology to produce Rab11-tagged cell lines. A second objective is to develop innovative methods for imaging protein-protein interactions (PPI) with nanoscale resolution, using optical super-resolution microscopy or electron microscopy (EM). To our knowledge efficient methods are missing to date. Based on promising preliminary data, we aim to fill this gap by adapting and optimizing the SplitFAST technology. On one side the brightness and photostability of SplitFAST will be optimized for efficient use in Stimulated Emission Depletion (STED) microscopy. On the other side SplitFAST will be used with ROSgenic chromophores, in order to locally generate ROS upon PPI, and photoinduce the polymerization of osmiophilic polymer for EM detection. Finally, the resulting advanced SplitFAST complementation assays will be used to perform nanoscale imaging of Rab11-vRNP complexes in IAV-infected cells. We will unambiguously image Rab11-vRNP complexes and localize them relative to endomembrane and cytoskeleton markers using STED, and relative to the ultrastructural surroundings using EM.

The expected results of FluNanoTrack are new details on the association and dissociation of the Rab11-vRNP complex during the viral life cycle, extended knowledge about physically and functionally associated host proteins, and nanoscale images of Rab11-vRNP complexes that will inform on their topology at the membrane of cellular organelles and vRNP transport vesicles. Integration of the proteomic, live-imaging and high-resolution imaging datasets is expected to provide an unprecedented view of the molecular processes involved in the IAV multi-RNA genome transport. In addition, the proposed technical developments in STED microscopy and EM will have impact on other fields of studies well beyond the scope of the proposed project.