Project Summary
Ebola (EBOV) and Marburg (MARV) filoviruses cause severe hemorrhagic fever in humans with
up to 90% mortality rates. Their genome contains only seven genes including the viral matrix protein
VP40 which, when expressed in mammalian cells, is sufficient to produce virus-like particles (VLPs)
that are essentially indistinguishable from live virions. VP40 forms dimers, hexamers and octamers
mediated by different protein-protein (PPI) and protein-lipid (PLI) interactions that fulfill different and
essential roles in the viral lifecycle, making VP40 a “swiss army knife” of proteins. The fascinating
dynamic equilibria of VP40 and the availability of VLPs as a model system for direct observations
outside of a BSL4 laboratory make VP40 a unique system to rigorously study the biophysical basis for
viral budding as well as PPIs and PLIs in general. The significance of these studies is further increased
because VP40 is the most conserved protein upon virus passage through humans, but exploiting VP40
as a potential drug target is unlikely to succeed without understanding the physical basis for
oligomerization and function of VP40.
The Stahelin and Wiest laboratories, building on established collaborations with each other and
several other collaborators supplying specific expertise, will use computational, experimental and
structural biophysics methods to investigate the central hypothesis of this grant: that interdomain
interactions of VP40 are key regulators of VP40 structures during the viral life cycle. In two specific
aims, we will (i) Determine the biophysical mechanisms by which VP40 dimer, hexamer and octamers
form in silico, in vitro and in human cells and (ii) determine how mutations of VP40 that arise in humans
during the course of an outbreak as well as in animals during passage of virus contribute to VP40
conformational change and rearrangement into its separate oligomeric forms.
These questions will be studied using a tightly integrated approach using multiscale molecular
dynamics simulations on the µs timescale and free energy perturbation methods on the computational
side and hydrogen-deuterium exchange, cellular imaging of VLPs as well as more traditional
biophysical experiments such as ultracentrifugation and SPR to determine the binding constants of
wildtype VP40 from EBOV and MARV as well as pertinent mutants. This innovate and integrated
approach will not only provide careful validation of the results, but also provide detailed insights into the
PPIs and PLIs governing the oligomerization equilibria across many time- and lengths scale, thus
enabling a rigorous understanding of the biophysical principles for a biomedically very important
filovirus protein that will have a significant impact on understanding other PPIs and PLIs.