Ebola and Marburg virus matrix layers are locally ordered assemblies of VP40 dimers

A key step in the life cycle of enveloped viruses is the budding of nascent virions from the host membrane. In filoviruses such as Ebola and Marburg virus, this process is achieved by the matrix protein VP40. When expressed alone, VP40 induces the budding of filamentous virus-like particles, suggesting that localization to the plasma membrane, oligomerization into a matrix layer, and the generation of membrane curvature are intrinsic properties of VP40. While a number of crystal structures of VP40 have been determined in various oligomerization states, there has been no direct information on the structure of assembled VP40 matrix layers within viruses or virus-like particles. Here, we present structures of Ebola and Marburg VP40 matrix layers in intact virus-like particles, as well as within intact Marburg viruses. We find that the matrix layers are formed from VP40 dimers which assemble into extended chains via C-terminal domain interactions. These chains stack into layers, forming a 2D lattice below the membrane surface. However, these 2D lattices are only locally ordered, forming a patchwork assembly across the membrane surfaces and suggesting that assembly may begin at multiple points. These observations define the structure and arrangement of the matrix protein layer that mediates the formation of filamentous filovirus particles.