The Effects of Point Mutations on the Dimerization Domain of Ebola Virus Protein VP40

Abstract

The Ebola Virus (EBOV) is a deadly virus first identified in 1976 from the Ebola River region of Zaire (presently the Democratic Republic of Congo). EBOV is known for causing viral hemorrhagic fever and it can cause a high mortality rate in infected patients. VP40 is the matrix protein of EBOV (eVP40), which has an N‐terminal domain responsible for dimerization and oligomerization of the protein to form filaments of dimers at the plasma membrane inner leaflet of the host cell. The eVP40 dimerization and filament formation is necessary for viral budding from the host cell membrane. This project aims to investigate which amino acid residues in this N‐terminal dimerization domain are necessary for the protein to be able to oligomerize at the plasma membrane inner leaflet. First, computational screens were performed to determine which residues may play a role in dimerization, and then mutant plasmids with changes at these residues harboring a green fluorescent protein tag were created. Based on the computational screen, we hypothesized amino acid residues Arg52, Ile54, Ala55, Asp56, Asp57, and His61 would be essential for dimerization of eVP40. Mutants of these residues were then transfected into Cos‐7 cells and imaged using confocal microscopy and compared to WT eVP40. Cellular images were analyzed using ImageJ to determine the amount of fluorescently‐tagged protein present at the plasma membrane. Confocal imaging confirmed our central hypothesis and the computational data as these mutations had a significant loss of eVP40 plasma membrane localization. The next steps in this project will be to perform virus‐like particle (VLP) collection to further analyze the effects of point mutations on the dimerization domain as it relates to VLP formation budding out of the host cell.