Hydrophobic region of Marburg Virus may indicate mechanism of protein trafficking to plasma membrane

Abstract

Ebola virus and Marburg virus has high fatality rate in humans and to date there are no FDA approved vaccines or therapeutics to treat it. Viral protein 40 (VP40) is the major virus matrix protein of these viruses that regulates assembly and egress of infectious Ebola and Marburg virus particles. It is well established that Ebola VP40 interacts with the inner leaflet of the plasma membrane using anionic phospholipids and penetrates into the hydrocarbon core of the plasma membrane. Marburg VP40 also interacts with anionic phospholipids at the inner leaflet of the plasma membrane but unlike Ebola VP40 its interaction is highly dependent on anionic charge density of the plasma membrane. Marburg VP40 protein did not have hydrophobic interactions with the hydrocarbon core of the plasma membrane despite it having a structurally similar hydrophobic loop region to Ebola VP40, which penetrate the plasma membrane. To investigate the function of this hydrophobic loop region of Marburg VP40, we created single double and triple amino acid mutants of this region specifically targeting L201, F281, L283, and F286 residues and observed the phenotype of the cells transfected with these mutants using a GFP tag. The single amino acid mutant L201A along with the double mutant (F281A/F286A) and triple mutant (F281A/L283A/F286A) resulted in localization of VP40 into intracellular accumulations that had vesicular structures, while all other mutants maintained the Wild Type phenotype by associating with the plasma membrane. When these mutants were co‐expressed with Early Endosome Antigen 1 (EEA1) protein it led to complete dissociation of these mutants from vesicle structures to become entirely cytosolic. EEA1 protein is early endosomal marker that interacts with phosphoinositide‐PI3P and Rab5 protein on early endosomes. Our observation suggests that these mutants are associated with early endosomes by likely interacting with PI3P and their mutant phenotype is likely to represent inability of these mutants to be trafficked to the plasma membrane. Marburg VP40 is known to use retrograde late endosomal pathway accumulating at multivesicular bodies (MVBs) before reaching the plasma membrane. Therefore, these mutants are likely to represent an early step of Marburg VP40 trafficking to plasma membrane and may provide valuable insight into Marburg VP40 membrane trafficking pathway.