Abstract
Ebola virus (EBOV) causes viral hemorrhagic fever in humans and can have clinical fatality rates of ~60%. The EBOV genome consists of negative sense RNA that encodes seven proteins including viral protein 40 (VP40). VP40 is the major Ebola virus matrix protein and regulates assembly and egress of infectious Ebola virus particles. It is well established that VP40 assembles on the inner leaflet of the plasma membrane of human cells to regulate viral budding where VP40 can produce virus like particles (VLPs) without other Ebola virus proteins present. The mechanistic details, however, of VP40 lipid-interactions and protein-protein interactions that are important for viral release remain to be elucidated. Here, we mutated a loop region in the N-terminal domain of VP40 (Lys127, Thr129, and Asn130) and find that mutations (K127A, T129A, and N130A) in this loop region reduce plasma membrane localization of VP40. Additionally, using total internal reflection fluorescence microscopy and number and brightness analysis we demonstrate these mutations greatly reduce VP40 oligomerization. Lastly, VLP assays demonstrate these mutations significantly reduce VLP release from cells. Taken together, these studies identify an important loop region in VP40 that may be essential to viral egress.
Highlights
Ebola virus (EBOV) [1] and Marburg virus (MARV) [2] are viruses from the Filoviridae family, which are some of the most virulent pathogens that infect humans
Using a combination of cellular imaging, number and brightness analysis (N&B), and virus like particles (VLPs) release assays we find that a loop region in the N-terminal domain is important for viral protein 40 (VP40) plasma membrane (PM) localization, VP40 oligomerization, and VLP release
To further screen for regions of VP40 that are important for PM localization and viral egress we randomly introduced mutations into the VP40 N-terminal domain to screen for changes in PM localization
Summary
Ebola virus (EBOV) [1] and Marburg virus (MARV) [2] are viruses from the Filoviridae family, which are some of the most virulent pathogens that infect humans. VP40 has been shown to play an important role in viral transcription through formation of a RNA binding octameric ring [13,26], but the octameric ring has not been observed in infectious virions or VLPs. While a number of regions of VP40 that are key determinants of trafficking or release have been identified, much less mechanistic information is available on the molecular basis of VP40 interactions with human proteins or cellular membrane lipids. Mutations of cationic residues in the N-terminal domain outside of this loop region (K86A or K90A) did not appreciably alter PM localization of VP40 or VLP release This N-terminal domain loop region may play an important role in VP40-protein or VP40-membrane interactions with the host cell
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have