Live‐Cell Imaging of Ebola Virus Matrix Protein VP40

Abstract

Intro: The Ebola Virus (EBOV), which causes severe hemorrhagic fever with a high mortality rate, acquires its lipid coat and exits the host cell by budding from the plasma membrane (PM). EBOV uses only 7 genes in its replication life cycle of which Viral Matrix Protein 40 (VP40) plays a critical role in assembly and budding as well as RNA transcription. VP40 alone can assemble into Virus-Like Particles (VLPs) and bud from cells like authentic EBOV. Live-cell imaging alongside in vitro studies is shedding light on the mechanisms by which VP40 fills its multiple roles in the EBOV life cycle. Objective: We aim to further elucidate the relationship between VP40 structure and function in the EBOV life cycle by using live-cell imaging experiments. Methods Confocal Microscopy, Total Internal Reflection Fluorescence Microscopy Results Wild-Type VP40 localizes to the PM and forms VLPs. Mutations in the N-terminal dimerization interface of VP40 that also predispose octameric ring formation abolish PM localization and VLPs but cause distinct intracellular structures to appear. A mutation in the C-terminal oligomerization interface abolishes VLPs but does not eliminate PM localization of certain punctate structures. A single mutation in a conserved cationic patch of 6 lysine residues abolishes PM localization. Conclusions Dimerization and oligomerization as well as interaction with the PM via a cationic patch appear to play crucial roles in the trafficking of VP40 to the PM and assembly of VLPs. Additionally, rearrangement of VP40 into octameric ring structures shows distinct intracellular localization. Financial Support: NIH AI081077