Role of the mucin-like domain of the Ebola virus in modulating virus-glycosaminoglycan interactions

Ebola virus, one of the deadliest human pathogens, is a known candidate for severe outbreaks and has caused several thousand deaths in the more recent outbreaks alone. To fight against it, a detailed knowledge about its viral life cycle is fundamental to the development of efficient vaccines and drugs. In this project I suggest to investigate the role of the mucin-like domain (MLD) of the viral glycoprotein (GP) in modulating virus attachment, detachment and diffusion on glycosaminoglycans (GAGs), responsible for recruiting the virus at the cell surface. To do so, I will generate GP-containing pseudotyped viruses, mimicking the tropism of the pathogen. Specifically, I will compare the GP of the Zaire strain of EBOV, an MLD-deleted mutant and a natural mutant that occurred during the West Africa outbreak (2013-2016) that is reported to have an increased tropism for human cells. I will use advanced biophysical techniques to examine the interactions on a molecular level as well as on the cellular level. On a molecular level, I will study the binding strength of individual bonds formed between the GP and GAGs using force spectroscopy. In addition, I will investigate the attachment and detachment of virus particles from GAGs immobilized on a glass surface in a biomimetic fashion, using total internal fluorescence microscopy. Proceeding to a more physiological model using living cells, I plan to study the diffusion behavior at the cell surface of pseudotyped viruses carrying the various mutations in their MLD. Stepping up in complexity, in the last part of the project, I will investigate the role of the MLD in modulating the ability of the virus to cross the glycocalyx, the sugar coat of cells, by employing 3D tracking. Taken together this project will lead to a better understanding on how viral particle migrate on the cell surface and how the interactions function on a molecular level.