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Investigating Phospholipid Binding Residues in the C Terminus of Ebola Virus Matrix Protein, VP40
Author(s) -
Budicini Melissa,
Johnson Kristen,
Stahelin Robert V.
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.630.1
Subject(s) - vp40 , ebola virus , viral matrix protein , random hexamer , microbiology and biotechnology , chemistry , ebolavirus , filoviridae , biology , biophysics , biochemistry , virus , virology , paramyxoviridae , viral disease
Ebola virus (EBOV) is a lipid‐enveloped virus that causes hemorrhagic fever and a fatality rate of 50–90%. Though EBOV only has 7 genes in its genome, one, VP40 is the main driver of viral egress. VP40 localizes to the plasma membrane where it forms virus like particles (VLPs). While the mechanism of the viral egress is not completely understood, previous studies have found that phospholipids phosphatidylserine (PS) and phosphatidylinositol 4,5‐bisphosphate (PIP 2 ) are required for plasma membrane localization, self‐oligomerization, VLP formation and VLP budding from the cell. VP40 transforms through self‐oligomerization from a dimer to a hexamer and eventually a longer filament. PS likely induces the dimer to hexamer transition while PIP 2 binding is required for stability of larger oligomers (n=12 or more). In an effort to understand the process of VP40 oligomerization, we have identified a likely PIP 2 binding pocket on the C terminus of VP40; the C terminus of VP40 is hypothesized to interact with the PM. This pocket shares structural similarity to the PIP 2 binding pocket of viral matrix protein HIV‐GAG from HIV‐1. Objective We are mutating potential binding residues and control residues to alanine on the C terminus of VP40 in order to understand the interactions between VP40, PIP 2 , and localization to the plasma membrane. Methods Site Directed Mutagenesis, Confocal Laser Scanning Microscopy (CLSM), Liposome Pelleting Assays (LPA), and Transmission Electron Microscopy (TEM). Results VP40 PM localization can be blocked by elimination of phospholipid substrates through various assays in live COS‐7 cells. We are now investigating how VP40 binds to phospholipids. We have made several mutations with site directed mutagenesis and expressed or purified each in live cells for CLSM, or for lipid binding assays (LPA, TEM). We have identified several amino acids in the proposed binding pocket that are required for PI(4,5)P 2 binding and oligomerization. Conclusions Phospholipid‐focused assays in live cells and in vitro assays reveal residues in the C‐terminal domain of VP40 that are required to bind to PI(4,5)P 2 . We aim to better understand this interaction and it's role in oligomerization through future assays. Support or Funding Information NIH AI081077