PhD Defense by Victoria Thusgaard Ruhoff

Title: Reconstitution of viral budding
-A study on phase separation and spontaneous curvature in plasma membranes

Abstract: Every year, influenza cause global outbreaks and substantial research has been carried out to understand this infectious disease and develop e!ective treatments and prevention strategies. On a molecular level, the cell-to-cell transmission of influenza virus is critically dependent on formation of spherical and cylindrical protrusions from the cell surface. The degree of membrane bending observed in these progeny virion structures requires generation of significant forces, the origin of which are an active area of research. Influenza viruses bud from infected cells just after gathering a number of spike-proteins in a nanoscale budding region followed by outward bulging of the membrane. It has been suggested that this process could be driven or enhanced by crowding of spike proteins which are present at the budding region during initiation of the bud. The bulky spike proteins may collide stochastically (crowd) to create a lateral entropic pressure capable of inducing membrane bending, however the proteins must be highly concentrated to provide sufficient lateral crowding to generate such curvature. A long-standing hypothesis in the field is that membrane nanodomains are responsible for this protein clustering.

In this thesis, it was investigated whether membrane nanodomains can efficiently relocated and cluster virus proteins in a novel model system which allows generic testing of the membrane phase affinity of any membrane protein. It was found that all tested virus proteins exhibited strong phase affinity, but surprisingly for more fluid domains rather than for liquid ordered domains, which stands in contrast to previously observed behavior. The ability of virus proteins to generate membrane bending in cells and membrane vesicles was further assessed by development of a second novel assay which can directly measure the spontaneous curvature of a membrane containing integral membrane proteins. By measuring the potential of a membrane to bend inward versus outwards it is possible to directly measure the spontaneous curvature of a cell membrane and future work will reveal the effect of viral proteins on the tendency of a membrane to bend. Overall, it can be concluded that virus spike proteins are still likely candidates in assisting or even driving the budding process of progeny viruses and therefore a deeper understanding of how these proteins interact with membranes is of critical importance for understanding how viruses disseminate in our body.