Exploring Structural and Functional Properties of Marburg Virus Matrix Protein-VP40

Marburg virus causes deadly hemorrhagic fever in humans and non-human primates and is a close relative of Ebola virus. To date, there are no FDA approved vaccines or drugs that can be used to treat Ebola or Marburg virus infection, however, multiple therapeutic agents are in clinical trial showing promising results. The Marburg virus genome encodes seven proteins and the Matrix protein-VP40 (mVP40) is the most abundantly expressed protein in viral proteome. The VP40 protein has the capacity to interact with inner leaflet of the plasma membrane of the host cell and assemble into viral like particles (VLPs) independent of other viral proteins. Although a wealth of information is available on how Ebola virus matrix protein-VP40 (eVP40) interacts with the plasma membrane to assemble the viral matrix and form VLPs, information on mVP40 plasma membrane interactions were scarce.

The goal of this dissertation was to investigate membrane targeting properties of mVP40 and to understand the molecular basis of mVP40 lipid selectivity and self-assembly that regulates viral matrix formation and subsequent budding from the host cell. Using a number of cell-based assays and in vitro methods we uncovered that mVP40 interacts with the plasma membrane using electrostatic interactions and does not show binding specificity to a particular type of anionic phospholipid. mVP40 association with the plasma membrane was highly anionic charge dependent like that of established anionic charge sensors.

Next, we investigated if mVP40 membrane association induced structural changes in mVP40 using Hydrogen-Deuterium exchange Mass spectrometry (HDX-MS). We identified two potential oligomerization surfaces on mVP40 that facilitate mVP40 assembly into multimeric structures. Using site directed mutagenesis we further investigated these potential oligomerization surfaces and found that mutations to these interfaces independently did not induce any changes in plasma membrane localization of the mutant proteins, however, when both surfaces were mutated an altered phenotype was observed as the mutant protein showed increased cytosolic presence. Thus, this mutant may shed light on the molecular basis of mVP40 oligomerization. We also uncovered that unlike eVP40, mVP40 does not penetrate into the hydrocarbon region of the plasma membrane, however, multiple mutations to a mVP40 hydrophobic loop region resulted in altered cellular localization.Compared to the wildtype mVP40, mutants of this hydrophobic loop region showed altered interaction with host COP-ІІ transport machinery protein, sec24C by showing increased association indicating possible involvement of the CTD-hydrophobic loop region for intracellular trafficking. Thus, this study has elucidated structural and functional properties of mVP40 comprehensively to understand mVP40 as a potential therapeutic target such that Marburg virus infection can be treated through inhibiting mVP40 assembly at the plasma membrane.