Summary Project description 2nd funding period
The mega Dalton sized nuclear pore complexes NPCs are the major gateway across the nuclear envelope. We aim to decipher the molecular structure and dynamics of the pathogen-mediated transport mechanism through the NPC focusing on two different viral systems: the Human Immunodeficiency Virus (HIV) and the Hepatitis B virus (HBV). For large viral or subviral structures, it is currently not well understood what protein interactions with the NPC and motions inside the NPC can facilitate such processes. We use a bottom up approach, starting with defined components of the viral systems to study both generic and specific interactions with the NPC. To achieve molecular resolution we employ state of the art single molecule and super-resolution microscopy techniques to visualize protein flexibility and protein-protein interactions between the pathogens (HBV and HIV capsid structures) and the NPC with high spatial (nanometer) and temporal resolution. It has become clear that genetic code expansion (GCE) is well suited to introduce clickable functionalities into the proteins of interest. In the last funding period we have made substantial progress to minimally invasively engineer viral and NPC proteins with ultra-photostable small fluorescent dyes suitable for singlemolecule/super-resolution fluorescence observation (Fackler, Müller, Schultz) using this method. We will further develop GCE in the next funding period (with Müller, Grimm/Urban, Kräusslich, Lusic/Beck, Dao Thi, Johnsson) to satisfy the intricate demands of studying virus-NPC interactions with high resolution methods. Furthermore, we have been able to reveal how canonical nuclear transport can be both, fast and specific, and together with the newly engineered fluorescent viral capsids form HBV and HIV, we are now in the unique position to decipher the molecular steps of such large (subviral) structures passing the NPC. Our technologies allow visualizing similarities, differences and specifics of diverse pathogen-NPC interactions, enabling us to understand how selected viruses manage to hijack the nuclear transport machinery. Our study focuses on non-infectious model systems that benefit from their high genetic amenability and access to state of the art technology. Our results will nourish and complement the studies of more complex systems and greatly benefit from the possibility to collaborate with groups from virology (Müller, Urban/Grimm, Kräusslich). The goal is to strengthen the synergies between high-resolution model studies and the actual infectious and physiological systems to converge on a common picture of viral passage mechanism.