Detecting lipid induced structural changes of Marburg Virus‐VP40 protein using Hydrogen‐Deuterium exchange mass spectroscopy (HDX‐MS)
Authors: Wijesinghe Kaveesha Jayani , Urata Sarah , Li Sheng , Stahelin Robert V. 
Published: November 2023

Marburg virus is a close relative of Ebola virus and belongs to the Filoviridae family and can cause severe hemorrhagic fever in humans which is often fatal. Filoviruses are lipid‐enveloped viruses where the lipid envelope is extracted from the host cell plasma membrane during the budding of nascent viral particles. Marburg virus matrix protein‐VP40 (mVP40) underlies the lipid envelope of the virus forming the viral matrix layer, which provides structural stability to viral particles. Inside an infected host cell mVP40 plays a crucial role by interacting with the cytoplasmic leaflet of the cell membrane and facilitating assembly of the viral matrix and budding of virions. Recent crystal structure determination of mVP40 revealed it to be a dimer in solution with a flat and extended “basic patch” which interacts with negatively charged phospholipids in the plasma membrane. The flat and extensive cationic lipid binding surface was in line with our previous findings, which showed mVP40 to be non‐specific in association with anionic lipids. mVP40 associates with the plasma membrane and in vitro membranes in a charge dependent manner. Even though oligomeric forms of mVP40 have not been crystalized yet, it is hypothesized that like its cousin Ebola VP40, Marburg VP40 forms hexamers that further assemble into linear filaments forming the viral matrix. In this study, we have used hydrogen deuterium exchange mass spectrometry (HDX‐MS) as an alternative tool to determine if membrane interaction of mVP40 results in structural changes that facilitate formation of oligomeric forms. We have compared the deuteration pattern of mVP40 protein in the presence and absence of anionic phospholipid membranes and identified regions of the protein showing significant changes in deuteration level in the presence of liposomes. These regions showed significant reduction in the deuteration levels in the presence of anionic phospholipids suggesting they have become less solvent accessible. Reduction of deuteration level observed in basic patch region of the protein is a result of direct interactions with phospholipid membranes. However, we also observed that an α‐helix at the C‐terminal domain and an antiparallel β‐sheet structure at the N‐terminal domain of the protein showed significant loss of solvent accessibility in the presence of liposomes. If the protein undergoes oligomerization and forms filamentous structures, the oligomerization sites would become solvent inaccessible. Therefore, these identified regions on the protein may be potential sites of oligomerization. Thus, we hypothesize that following initial interaction of mVP40 dimers with anionic phospholipids, mVP40 is also likely to assemble in an end‐to‐end manner using the CTD α4‐helix to form a linear hexamer. Further oligomerization of linear hexamers with another hexamer unit by interacting via the NTD of composing dimers will result in matrix assembly. Alternatively, the linear hexamer may be an intermediate that undergoes structural rearrangement similar to Ebola VP40 protein. The rearranged hexamer may then assemble into the matrix layer via NTD interactions.Support or Funding InformationThis research has been supported by the NIH (AI081077) to R.V.S, (1U19AI117905, R01GM020501, R01AI101436) to S.L, as well as CBBI fellowship support funded by the NIH to K.J.W (T32GM075762).