Dissection of the pestiviral replicase and packaging complexes by proximity labelling and biorthogonal crosslinking approaches

The viral replicase assembly and the assembly of the pestiviral virion are both multi-step processes. Their underlying pathways are largely unknown, mainly due to technical difficulties in purifying the participating multiprotein complexes and carrying out structural studies. Both processes take place in close association with intracellular membranes and a yet unknown number of host factors. The pestiviral particle assembly is believed to occur within the ER, most likely in close proximity to the surface of lipid droplets similar to what has been reported for HCV. This project aims at elucidating the composition of multiprotein complexes important for pestiviral RNA replication and virion morphogenesis. Crucial for this aim is the ability (i) to identify viral or cellular proteins interacting with the pestiviral replicase and/or with the packaging complex and (ii) to identify amino acids directly involved in these protein interactions on the surface of these complexes and use this knowledge study the stepwise assembly process. As outlined in this application, we have established with proximity labeling and biorthogonal crosslinking two methods to identify viral/cellular replicase and packaging components for pestiviruses and dissect their role in replicase assembly in live cells. Especially the bioorthogonal dissection of replicase assembly is expected to generate a detailed landscape of protein-protein interactions required for this process and to identify hub elements important for the assembly of these complexes. Aim 1 will study the pestiviral replicase assembly in more detail. We identified viral and cellular proteins common to NS4B- and NS5A-specific proxisomes as potential components of the pestiviral replication complex. We will characterize the functional role(s) of the most promising cellular replicase components by generating and analyzing respective knockout cell lines for their ability to support RNA replication. Aim 2 will investigate the pestiviral Core proxisome to gain insights into pestiviral virion morphogenesis. With our newly established NCP7 Npro-V5-TuID-Core genome, we identified cellular proteins that are in close proximity to V5-TuID-Core during viral genome replication. We will analyze the most promising cellular components by generating respective knockout or knockdown cell lines for their ability to support RNA replication and virion morphogenesis. Together, this approach will inform us on the composition(s) of dedicated multi-protein complexes required for RNA replication and virion morphogenesis and the temporal and spatial regulation of their formation.