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The Ebola Virus Matrix Protein VP40 Selectively Induces Vesiculation from Phosphatidylserine-enriched Membranes*

  • Smita P. Soni
    Affiliations
    Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, South Bend, Indiana 46617
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  • Robert V. Stahelin
    Correspondence
    To whom correspondence should be addressed: Indiana University School of Medicine-South Bend, 143 Raclin-Carmichael Hall, 1234 Notre Dame Ave., South Bend, IN 46617. Tel.: 574-631-5054; Fax: 574-631-7821
    Affiliations
    Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, South Bend, Indiana 46617

    Department of Chemistry and Biochemistry and the Eck Institute for Global Health, University of Notre Dame, Notre Dame, Indiana 46556
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grant AI081077, the Center for Rare and Neglected Diseases at the University of Notre Dame, and grants from the Indiana University School of Medicine-South Bend Imaging and Flow Cytometry Core Facility (to R. V. S.).
Open AccessPublished:October 14, 2014DOI:https://doi.org/10.1074/jbc.M114.586396
      Ebola virus is from the Filoviridae family of viruses and is one of the most virulent pathogens known with ∼60% clinical fatality. The Ebola virus negative sense RNA genome encodes seven proteins including viral matrix protein 40 (VP40), which is the most abundant protein found in the virions. Within infected cells VP40 localizes at the inner leaflet of the plasma membrane (PM), binds lipids, and regulates formation of new virus particles. Expression of VP40 in mammalian cells is sufficient to form virus-like particles that are nearly indistinguishable from the authentic virions. However, how VP40 interacts with the PM and forms virus-like particles is for the most part unknown. To investigate VP40 lipid specificity in a model of viral egress we employed giant unilamellar vesicles with different lipid compositions. The results demonstrate VP40 selectively induces vesiculation from membranes containing phosphatidylserine (PS) at concentrations of PS that are representative of the PM inner leaflet content. The formation of intraluminal vesicles was not significantly detected in the presence of other important PM lipids including cholesterol and polyvalent phosphoinositides, further demonstrating PS selectivity. Taken together, these studies suggest that PM phosphatidylserine may be an important component of Ebola virus budding and that VP40 may be able to mediate PM scission.

      Introduction

      Ebola virus is a filamentous, lipid-enveloped virus from the Filoviridae family and is one of most virulent pathogens that can infect humans. Ebola virus causes viral hemorrhagic fever, with a fatality rate of ∼60%. With no current FDA approved vaccines or drugs, Ebola virus may pose a serious threat (
      • Ksiazek T.G.
      Clinical virology of Ebola hemorrhagic fever (EHF): virus, virus antigen, and IgG and IgM antibody findings among EHF patients in Kikwit, Democratic Republic of the Congo, 1995.
      ,
      • Johnson K.M.
      • Lange J.V.
      • Webb P.A.
      • Murphy F.A.
      Isolation and partial characterisation of a new virus causing acute haemorrhagic fever in Zaire.
      ,
      • Feldmann H.
      Ebola: a growing threat?.
      ). Currently, an outbreak in Western Africa, the first large scale Ebola virus outbreak for this region; has caused more than 5800 infections and 2800 deaths as of September 2014 (
      • Farrar J.J.
      • Piot P.
      The Ebola emergency: immediate action, ongoing strategy.
      ). Thus, there is urgency toward developing a treatment. Recently, significant strides have been made in development of vaccines (
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      • Willet M.
      • Papaneri A.B.
      • Wirblich C.
      • Feldmann F.
      • Holbrook M.
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      • Feldmann H.
      • Schnell M.J.
      Antibody quality and protection from lethal Ebola virus challenge in nonhuman primates immunized with Rabies virus based bivalent vaccine.
      ), repositioning of previously approved FDA drugs (
      • Johansen L.M.
      • Brannan J.M.
      • Delos S.E.
      • Shoemaker C.J.
      • Stossel A.
      • Lear C.
      • Hoffstrom B.G.
      • DeWald L.E.
      • Schornberg K.L.
      • Scully C.
      • Lehár J.
      • Hensley L.E.
      • White J.M.
      • Olinger G.G.
      FDA-approved selective estrogen receptor modulators inhibit Ebola virus infection.
      ), or monoclonal antibodies (
      • Qiu X.
      • Wong G.
      • Audet J.
      • Bello A.
      • Fernando L.
      • Alimonti J.B.
      • Fausther-Bovendo H.
      • Wei H.
      • Aviles J.
      • Hiatt E.
      • Johnson A.
      • Morton J.
      • Swope K.
      • Bohorov O.
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      Reversion of advanced Ebola virus disease in nonhuman primates with ZMapp.
      ) that may be viable means of preventing or treating infections. The Ebola virus negative sense RNA genome encodes seven proteins including the transmembrane glycoprotein (
      • Lee J.E.
      • Fusco M.L.
      • Hessell A.J.
      • Oswald W.B.
      • Burton D.R.
      • Saphire E.O.
      Structure of the Ebola virus glycoprotein bound to an antibody from a human survivor.
      ), which mediates Ebola virus entry into the host cell using the Niemann-Pick Type C1 cholesterol transporter (
      • Miller E.H.
      • Obernosterer G.
      • Raaben M.
      • Herbert A.S.
      • Deffieu M.S.
      • Krishnan A.
      • Ndungo E.
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      • Pfeffer S.R.
      • Dye J.M.
      • Whelan S.P.
      • Brummelkamp T.R.
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      Ebola virus entry requires the host-programmed recognition of an intracellular receptor.
      ,
      • Côté M.
      • Misasi J.
      • Ren T.
      • Bruchez A.
      • Lee K.
      • Filone C.M.
      • Hensley L.
      • Li Q.
      • Ory D.
      • Chandran K.
      • Cunningham J.
      Small molecule inhibitors reveal Niemann-Pick C1 is essential for Ebola virus infection.
      ,
      • Carette J.E.
      • Raaben M.
      • Wong A.C.
      • Herbert A.S.
      • Obernosterer G.
      • Mulherkar N.
      • Kuehne A.I.
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      • Griffin A.M.
      • Ruthel G.
      • Dal Cin P.
      • Dye J.M.
      • Whelan S.P.
      • Chandran K.
      • Brummelkamp T.R.
      Ebola virus entry requires the cholesterol transporter Niemann-Pick C1.
      ). Although a number of studies have addressed the mechanism of cellular entry of the virus, less is known regarding how the virus replicates inside the host cell and forms an assembly site to egress from the inner leaflet of the host cell plasma membrane (PM).
      The abbreviations used are: PM
      plasma membrane
      BAR
      Bar/Amiphysin/Rvs
      ESCRT
      endosomal sorting complexes required for transport
      IRSp53
      insulin receptor tyrosine kinase substrate p53
      ITO
      indium tin oxide
      GUV
      giant unilamellar vesicle
      Lact C2
      lactadherin C2 domain
      LRPE
      1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl)
      MIM
      missing-in-metastasis
      PI(4,5)P2
      1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-4′,5′-bisphosphate)
      PIP3
      1,2-dioleoyl-sn-glycero-3-phospho-(1′-myo-inositol-3′,4′,5′-trisphosphate)
      POPC
      1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
      POPE
      1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine
      POPI
      1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoinositol
      POPS
      1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine
      VLPs
      virus-like particles
      VP40
      viral protein 40.
      The Ebola virus matrix protein VP40 is a peripheral membrane protein that regulates budding and egress from the host cell PM (
      • Jasenosky L.D.
      • Neumann G.
      • Lukashevich I.
      • Kawaoka Y.
      Ebola virus VP40-induced particle formation and association with the lipid bilayer.
      ,
      • McCarthy S.E.
      • Johnson R.F.
      • Zhang Y.A.
      • Sunyer J.O.
      • Harty R.N.
      Role for amino acids 212KLR214 of Ebola virus VP40 in assembly and budding.
      ,
      • Bornholdt Z.A.
      • Noda T.
      • Abelson D.M.
      • Halfmann P.
      • Wood M.R.
      • Kawaoka Y.
      • Saphire E.O.
      Structural rearrangement of ebola virus VP40 begets multiple functions in the virus life cycle.
      ). VP40, along with the minor matrix protein VP24, compose the layer that underlies the viral lipid envelope bridging the membrane embedded glycoprotein and the nucleocapsid (
      • Olejnik J.
      • Ryabchikova E.
      • Corley R.B.
      • Mühlberger E.
      Intracellular events and cell fate in filovirus infection.
      ,
      • Harty R.N.
      No exit: targeting the budding process to inhibit filovirus replication.
      ). VP40 can form VLPs when transfected into mammalian cells, which resemble authentic Ebola virions (
      • Jasenosky L.D.
      • Neumann G.
      • Lukashevich I.
      • Kawaoka Y.
      Ebola virus VP40-induced particle formation and association with the lipid bilayer.
      ,
      • Licata J.M.
      • Johnson R.F.
      • Han Z.
      • Harty R.N.
      Contribution of Ebola virus glycoprotein, nucleoprotein, and VP24 to budding of VP40 virus-like particles.
      ). However, how VP40 binds the PM and promotes scission with or without host protein hijacking is still not clear. VP40 is a dimer (
      • Bornholdt Z.A.
      • Noda T.
      • Abelson D.M.
      • Halfmann P.
      • Wood M.R.
      • Kawaoka Y.
      • Saphire E.O.
      Structural rearrangement of ebola virus VP40 begets multiple functions in the virus life cycle.
      ) that has an ∼43 amino acid N-terminal region of unknown structure, a N-terminal domain that mediates dimerization (
      • Bornholdt Z.A.
      • Noda T.
      • Abelson D.M.
      • Halfmann P.
      • Wood M.R.
      • Kawaoka Y.
      • Saphire E.O.
      Structural rearrangement of ebola virus VP40 begets multiple functions in the virus life cycle.
      ) and a C-terminal domain that binds membranes (
      • Jasenosky L.D.
      • Neumann G.
      • Lukashevich I.
      • Kawaoka Y.
      Ebola virus VP40-induced particle formation and association with the lipid bilayer.
      ,
      • Bornholdt Z.A.
      • Noda T.
      • Abelson D.M.
      • Halfmann P.
      • Wood M.R.
      • Kawaoka Y.
      • Saphire E.O.
      Structural rearrangement of ebola virus VP40 begets multiple functions in the virus life cycle.
      ,
      • Soni S.P.
      • Adu-Gyamfi E.
      • Yong S.S.
      • Jee C.S.
      • Stahelin R.V.
      The Ebola virus matrix protein deeply penetrates the plasma membrane: an important step in viral egress.
      ,
      • Adu-Gyamfi E.
      • Soni S.P.
      • Xue Y.
      • Digman M.A.
      • Gratton E.
      • Stahelin R.V.
      The Ebola virus matrix protein penetrates into the plasma membrane: a key step in viral protein 40 (VP40) oligomerization and viral egress.
      ,
      • Ruigrok R.W.
      • Schoehn G.
      • Dessen A.
      • Forest E.
      • Volchkov V.
      • Dolnik O.
      • Klenk H.D.
      • Weissenhorn W.
      Structural characterization and membrane binding properties of the matrix protein VP40 of Ebola virus.
      ,
      • Stahelin R.V.
      Membrane binding and bending in Ebola VP40 assembly and egress.
      ) and mediates oligomerization (
      • Bornholdt Z.A.
      • Noda T.
      • Abelson D.M.
      • Halfmann P.
      • Wood M.R.
      • Kawaoka Y.
      • Saphire E.O.
      Structural rearrangement of ebola virus VP40 begets multiple functions in the virus life cycle.
      ). The N terminus of VP40 has two late domains (
      • Martin-Serrano J.
      • Zang T.
      • Bieniasz P.D.
      HIV-1 and Ebola virus encode small peptide motifs that recruit Tsg101 to sites of particle assembly to facilitate egress.
      ,
      • Timmins J.
      • Schoehn G.
      • Ricard-Blum S.
      • Scianimanico S.
      • Vernet T.
      • Ruigrok R.W.
      • Weissenhorn W.
      Ebola virus matrix protein VP40 interaction with human cellular factors Tsg101 and Nedd4.
      ), a PTAP motif and a PPXY motif that are thought to mediate interactions with the cellular endosomal sorting complexes required for transport (ESCRT) machinery (
      • Henne W.M.
      • Buchkovich N.J.
      • Emr S.D.
      The ESCRT pathway.
      ). Presumably VP40 late domains regulate membrane scission through interactions with the ESCRT machinery, but studies overall on the VP40 late domains suggest there may be ESCRT-dependent and/or independent forms of Ebola virus egress in cell culture (
      • Neumann G.
      • Ebihara H.
      • Takada A.
      • Noda T.
      • Kobasa D.
      • Jasenosky L.D.
      • Watanabe S.
      • Kim J.H.
      • Feldmann H.
      • Kawaoka Y.
      Ebola virus VP40 late domains are not essential for viral replication in cell culture.
      ).
      Ebola virus VP40 has been shown to induce budding and scission from in vitro membranes, which is dependent upon insertion of a hydrophobic loop in its C-terminal domain (
      • Soni S.P.
      • Adu-Gyamfi E.
      • Yong S.S.
      • Jee C.S.
      • Stahelin R.V.
      The Ebola virus matrix protein deeply penetrates the plasma membrane: an important step in viral egress.
      ). VP40 penetrates into the hydrocarbon core of membranes that recapitulate the PM lipid composition (
      • Soni S.P.
      • Adu-Gyamfi E.
      • Yong S.S.
      • Jee C.S.
      • Stahelin R.V.
      The Ebola virus matrix protein deeply penetrates the plasma membrane: an important step in viral egress.
      ,
      • Adu-Gyamfi E.
      • Soni S.P.
      • Xue Y.
      • Digman M.A.
      • Gratton E.
      • Stahelin R.V.
      The Ebola virus matrix protein penetrates into the plasma membrane: a key step in viral protein 40 (VP40) oligomerization and viral egress.
      ). Single point mutations in a C-terminal domain hydrophobic patch significantly reduced both membrane penetration and viral egress (
      • Soni S.P.
      • Adu-Gyamfi E.
      • Yong S.S.
      • Jee C.S.
      • Stahelin R.V.
      The Ebola virus matrix protein deeply penetrates the plasma membrane: an important step in viral egress.
      ). Inhibition of membrane penetration in cell culture resulted in a reduction in PM association of VP40, reduced VP40 oligomerization, and VLP production (
      • Soni S.P.
      • Adu-Gyamfi E.
      • Yong S.S.
      • Jee C.S.
      • Stahelin R.V.
      The Ebola virus matrix protein deeply penetrates the plasma membrane: an important step in viral egress.
      ,
      • Adu-Gyamfi E.
      • Soni S.P.
      • Xue Y.
      • Digman M.A.
      • Gratton E.
      • Stahelin R.V.
      The Ebola virus matrix protein penetrates into the plasma membrane: a key step in viral protein 40 (VP40) oligomerization and viral egress.
      ). Penetration of VP40 more than halfway into one monolayer of the membrane bilayer along with VP40 oligomerization appears to be a driving force for regulating formation of VLPs (
      • Soni S.P.
      • Adu-Gyamfi E.
      • Yong S.S.
      • Jee C.S.
      • Stahelin R.V.
      The Ebola virus matrix protein deeply penetrates the plasma membrane: an important step in viral egress.
      ,
      • Adu-Gyamfi E.
      • Soni S.P.
      • Xue Y.
      • Digman M.A.
      • Gratton E.
      • Stahelin R.V.
      The Ebola virus matrix protein penetrates into the plasma membrane: a key step in viral protein 40 (VP40) oligomerization and viral egress.
      ).
      To better understand the process of VP40-mediated Ebola virus egress, it is crucial to understand how VP40 is able to interact with and bud from the PM to regulate formation of VLPs. Here we employ giant unilamellar vesicles (GUVs) as a model of viral budding to investigate the lipid selectivity of VP40-mediated budding. The GUVs have previously been utilized to understand membrane scission and to better understand the role of viral matrix proteins as well as peripheral proteins in membrane bending (
      • Soni S.P.
      • Adu-Gyamfi E.
      • Yong S.S.
      • Jee C.S.
      • Stahelin R.V.
      The Ebola virus matrix protein deeply penetrates the plasma membrane: an important step in viral egress.
      ,
      • Wollert T.
      • Wunder C.
      • Lippincott-Schwartz J.
      • Hurley J.H.
      Membrane scission by the ESCRT-III complex.
      ,
      • Solon J.
      • Gareil O.
      • Bassereau P.
      • Gaudin Y.
      Membrane deformations induced by the matrix protein of vesicular stomatitis virus in a minimal system.
      ,
      • Shnyrova A.V.
      • Ayllon J.
      • Mikhalyov I.I.
      • Villar E.
      • Zimmerberg J.
      • Frolov V.A.
      Vesicle formation by self-assembly of membrane-bound matrix proteins into a fluidlike budding domain.
      ,
      • Saarikangas J.
      • Zhao H.
      • Pykäläinen A.
      • Laurinmäki P.
      • Mattila P.K.
      • Kinnunen P.K.
      • Butcher S.J.
      • Lappalainen P.
      Molecular mechanisms of membrane deformation by I-BAR domain proteins.
      ). Here we varied the lipid compositions of GUVs to investigate the role of anionic lipids and cholesterol, which are enriched in the inner leaflet of the PM. VP40 selectively and rapidly induced budding and scission when PS was present at levels similar to the inner leaflet composition of the PM. Elucidating the effect of VP40 on GUVs composed of lipids that mimic biological membranes should help to reveal the mechanisms VP40 uses to remodel the host cell membrane.

      DISCUSSION

      The PM is an asymmetric bilayer where the outer leaflet is composed mainly of PC, sphingomyelin, and glycosphingolipids and the inner leaflet is enriched with PE, PS, PI, and PIPs (
      • van Meer G.
      • Voelker D.R.
      • Feigenson G.W.
      Membrane lipids: where they are and how they behave.
      ). Additionally, the PM is enriched in cholesterol although the distribution of cholesterol among the outer and inner leaflets is not as well understood. PS is the most abundant anionic lipid in the cytosolic leaflet at ∼15–20% (
      • Vance J.E.
      • Steenbergen R.
      Metabolism and functions of phosphatidylserine.
      ,
      • Vance J.E.
      • Tasseva G.
      Formation and function of phosphatidylserine and phosphatidylethanolamine in mammalian cells.
      ) and contributes to the recruitment of polycationic proteins as well as proteins containing PS binding domains (
      • Cho W.
      • Stahelin R.V.
      Membrane binding and subcellular targeting of C2 domains.
      ). PS has also been shown to interact with several viral matrix proteins (
      • Dick R.A.
      • Goh S.L.
      • Feigenson G.W.
      • Vogt V.M.
      HIV-1 Gag protein can sense the cholesterol and acyl chain environment in model membranes.
      ,
      • Zakowski J.J.
      • Petri Jr., W.A.
      • Wagner R.R.
      Role of matrix protein in assembling the membrane of vesicular stomatitis virus: reconstitution of matrix protein with negatively charged phospholipid vesicles.
      ) as well as VP40 (
      • Soni S.P.
      • Adu-Gyamfi E.
      • Yong S.S.
      • Jee C.S.
      • Stahelin R.V.
      The Ebola virus matrix protein deeply penetrates the plasma membrane: an important step in viral egress.
      ,
      • Adu-Gyamfi E.
      • Soni S.P.
      • Xue Y.
      • Digman M.A.
      • Gratton E.
      • Stahelin R.V.
      The Ebola virus matrix protein penetrates into the plasma membrane: a key step in viral protein 40 (VP40) oligomerization and viral egress.
      ,
      • Ruigrok R.W.
      • Schoehn G.
      • Dessen A.
      • Forest E.
      • Volchkov V.
      • Dolnik O.
      • Klenk H.D.
      • Weissenhorn W.
      Structural characterization and membrane binding properties of the matrix protein VP40 of Ebola virus.
      ,
      • Scianimanico S.
      • Schoehn G.
      • Timmins J.
      • Ruigrok R.H.
      • Klenk H.D.
      • Weissenhorn W.
      Membrane association induces a conformational change in the Ebola virus matrix protein.
      ). However, the lipid specificity of VP40 has not been explored in much detail (
      • Stahelin R.V.
      Membrane binding and bending in Ebola VP40 assembly and egress.
      ) and more rigorous analysis of VP40 lipid interactions is warranted.
      Here we demonstrate that VP40 selectively induces membrane budding and scission from membranes enriched with PS. VP40 also requires ∼10 mol % or more PS in the membrane to effectively associate with vesicles and induce vesiculation into GUVs compared with that of a PC background. This binding selectivity may further help explain VP40 localization and assembly at the PM inner leaflet where PS is enriched compared with other cytoplasmic sites (
      • Fairn G.D.
      • Schieber N.L.
      • Ariotti N.
      • Murphy S.
      • Kuerschner L.
      • Webb R.I.
      • Grinstein S.
      • Parton R.G.
      High-resolution mapping reveals topologically distinct cellular pools of phosphatidylserine.
      ). To demonstrate the selectivity of VP40 budding, other anionic lipids that are found at the PM inner leaflet were used including: PI, PI(4,5)P2, and PIP3. Budding of VP40 into these GUVs was nearly undetectable under all conditions employed. It should also be noted that even when approximately equimolar anionic charge was compared (e.g. 20% PI versus 20% PS, or 3% PIP3 versus 10% PS), VP40 was still highly selective for PS-dependent budding. Furthermore, cholesterol did not play a major role in the in vitro budding system as it did not enhance nor inhibit PS-dependent budding and scission.
      VP40 budding and scission effects were specific as the robust PS sensor Lact C2 did not induce detectable changes in GUVs at similar concentrations (e.g. 250 nm). At higher concentrations (5 μm Lact C2) positive membrane curvature changes were observed, which is consistent with membrane crowding effects (
      • Stachowiak J.C.
      • Schmid E.M.
      • Ryan C.J.
      • Ann H.S.
      • Sasaki D.Y.
      • Sherman M.B.
      • Geissler P.L.
      • Fletcher D.A.
      • Hayden C.C.
      Membrane bending by protein-protein crowding.
      ). Additionally, the I-BAR domains of IRSp53 and MIM, which induced negative membrane curvature changes but not scission in our GUV assays were consistent with the previous report (
      • Mattila P.K.
      • Pykäläinen A.
      • Saarikangas J.
      • Paavilainen V.O.
      • Vihinen H.
      • Jokitalo E.
      • Lappalainen P.
      Missing-in-metastasis and IRSp53 deform PI(4,5)P2-rich membranes by an inverse BAR domain-like mechanism.
      ). Taken together, VP40 selectively induces membrane vesiculation and scission in a PS-dependent manner. It should also be noted that in our study bacterially expressed VP40 was used. In human cells, VP40 has been shown to be Tyr phosphorylated (
      • García M.
      • Cooper A.
      • Shi W.
      • Bornmann W.
      • Carrion R.
      • Kalman D.
      • Nabel G.J.
      Productive replication of Ebola virus Is regulated by the c-Abl1 tyrosine kinase.
      ) and may also undergo other post-translational modifications that regulate the cellular membrane association and budding of viral particles.
      How does VP40 selectively induce budding and scission on PS-enriched membranes? Membrane bending and formation of a vesicle from a planar membrane requires a large degree of thermal energy (
      • Helfrich W.
      Elastic properties of lipid bilayers: theory and possible experiments.
      ). Tight protein packing on the membrane interface creates a highly dense surface where the energetic requirement to bend the membrane into a sphere is significantly lowered (
      • Shnyrova A.V.
      • Ayllon J.
      • Mikhalyov I.I.
      • Villar E.
      • Zimmerberg J.
      • Frolov V.A.
      Vesicle formation by self-assembly of membrane-bound matrix proteins into a fluidlike budding domain.
      ). Indeed, VP40 tightly packs on the membrane bilayer of VLPs (
      • Bharat T.A.
      • Noda T.
      • Riches J.D.
      • Kraehling V.
      • Kolesnikova L.
      • Becker S.
      • Kawaoka Y.
      • Briggs J.A.
      Structural dissection of Ebola virus and its assembly determinants using cryo-electron tomography.
      ), where VP40 dimers rearrange into hexamers that can concatenate into long filaments (
      • Bornholdt Z.A.
      • Noda T.
      • Abelson D.M.
      • Halfmann P.
      • Wood M.R.
      • Kawaoka Y.
      • Saphire E.O.
      Structural rearrangement of ebola virus VP40 begets multiple functions in the virus life cycle.
      ). VP40 has been shown to associate with PS containing liposomes (
      • Ruigrok R.W.
      • Schoehn G.
      • Dessen A.
      • Forest E.
      • Volchkov V.
      • Dolnik O.
      • Klenk H.D.
      • Weissenhorn W.
      Structural characterization and membrane binding properties of the matrix protein VP40 of Ebola virus.
      ), which can induce VP40 oligomerization (
      • Scianimanico S.
      • Schoehn G.
      • Timmins J.
      • Ruigrok R.H.
      • Klenk H.D.
      • Weissenhorn W.
      Membrane association induces a conformational change in the Ebola virus matrix protein.
      ) through structural rearrangement to a zig-zagging filament that may be able to interact both laterally and longitudinally with other VP40 filaments (
      • Bornholdt Z.A.
      • Noda T.
      • Abelson D.M.
      • Halfmann P.
      • Wood M.R.
      • Kawaoka Y.
      • Saphire E.O.
      Structural rearrangement of ebola virus VP40 begets multiple functions in the virus life cycle.
      ,
      • Adu-Gyamfi E.
      • Digman M.A.
      • Gratton E.
      • Stahelin R.V.
      Investigation of Ebola VP40 assembly and oligomerization in live cells using number and brightness analysis.
      ,
      • Adu-Gyamfi E.
      • Soni S.P.
      • Xue Y.
      • Digman M.A.
      • Gratton E.
      • Stahelin R.V.
      The Ebola virus matrix protein penetrates into the plasma membrane: a key step in viral protein 40 (VP40) oligomerization and viral egress.
      ). This structural rearrangement likely requires the membrane penetration of the C-terminal hydrophobic loop of VP40 (
      • Soni S.P.
      • Adu-Gyamfi E.
      • Yong S.S.
      • Jee C.S.
      • Stahelin R.V.
      The Ebola virus matrix protein deeply penetrates the plasma membrane: an important step in viral egress.
      ,
      • Adu-Gyamfi E.
      • Soni S.P.
      • Xue Y.
      • Digman M.A.
      • Gratton E.
      • Stahelin R.V.
      The Ebola virus matrix protein penetrates into the plasma membrane: a key step in viral protein 40 (VP40) oligomerization and viral egress.
      ). VP40 protein insertion in the PS containing membrane combined with the orientation and shape of the VP40 filament that forms might increase the curvature stress and provide sufficient thermal energy to bend membranes. Thus, as VP40 structures have not yet been solved in the presence of membrane, there may be distinct structures of membrane-bound VP40 that form increasing selectivity for PS-dependent scission.

      Acknowledgments

      We thank Erica Ollmann Saphire for helpful discussions.

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