Molecular Architecture of Ebola Virus Assembly

Ebola belongs to the Filoviridae family of viruses, which are negative sense single stranded RNA filamentous viruses that cause severe hemorrhagic fever. Ebola hemorrhagic fever (EHF) is a major public health concern in Central and Eastern Africa and parts of Asia. During EHF outbreaks fatality has varied between 25-100 %. Since there are no prescribed vaccines or therapeutic treatment, Ebola virus remains a viable candidate for biological warfare. Further, most endemic regions of the virus have little resources and lack access to quality healthcare. Hence, a timely discovery of drug interventions is needed to stem the increasing outbreaks of the disease. The highly contagious nature of the virus makes it difficult for investigators to study the virus as a whole except in maximum containment facilities. Even under such strict precautionary environments Ebola virus is often inactivated with UV irradiation. The ability to study sections of the viral proteome and the use of reverse genetics offer the opportunity to study the virus assembly process. VP40, the most abundantly expressed protein of the virus assembles into virus like particles (VLPs) from the plasma membrane independent of other viral proteins. This suggests that VP40 possesses the essential information needed for virus assembly. The goal of this PhD dissertation was to investigate the mechanisms by which Ebola VP40 is targeted to the cell surface (plasma membrane) and remodels the host membrane in order to efficiently form virus particles and bud from the cell. Little is known regarding how Filoviridae proteins interact with host cell membranes in order to assemble and bud into new virions. A number of in vitro and cellular methods have been employed to understand factors important for membrane targeting of Ebola virus proteins. First, I have determined that VP40 binds and inserts into the plasma membrane bilayer using a hydrophobic loop in its C-terminal domain.. I also showed using surface plasmon resonance (SPR), liposome sedimentation assays and different pharmacological agents that alter membrane composition and charge that VP40 is recruited to the host cell surface by binding selectively to phosphatidylserine (PS). These results demonstrate that VP40 associates with and bends the plasma membrane in a phosphatidylserine dependent manner. Recent evidence has suggested that the oligomerization of VP40 represents a crucial step in viral particle synthesis yet, the underlying mechanism is not understood. To understand the mechanism of oligomerization of Ebola VP40 in cells, fluorescence fluctuation methods such as Number & Brightness methods (N and B), Raster Image Correlation Spectroscopy (RICS), and internal reflection fluorescent microscopy (TIRFM) were used to resolve the spatio-temporal organization of VP40 into virus like particles (VLPs). Results from this study indicate that VP40 molecules are recruited as monomeric protein from the cytosol to the cell surface. On the plasma membrane, it was observed that VP40 oligomerizes to form filamentous virus particles of different sizes and lengths. Lastly, I have attempted to define the role of actin in the assembly of Ebola VLPs through a number of in vivo fluorescent labeling methods. Raster image correlation spectroscopy (RICS) method was used to establish that actin co-ordinates the movement of Ebola VP40 protein. Using single particle tracking in live cells it was demonstrated that the Ebola VP40 VLPs utilize actin filopodia for assembly and egress. Most importantly, it was shown that inhibiting actin filopodia formation with pharmacological agents significantly reduced the egress of VLPs. Together, the results of this study present another paradigm to the search for new antivirals for filoviruses.