Mutation of Ebola Virus Matrix Protein Cysteine Residues Increases Binding to Phosphatidylserine through Increased Flexibility of a Lipid Binding Loop

AbstractThe Ebola virus (EBOV) is a genetically simple negative sense RNA virus with only 7 genes yet it causes severe hemorrhagic fever in humans. The matrix protein VP40 of EBOV is the main driver of viral budding through binding to host plasma membrane lipids and formation of the filamentous, pleomorphic virus particles. To better understand this dynamic and complex process we have asked what the role of two highly conserved cysteine residues are in the C-terminal domain of VP40. Here we report that the mutation of Cys311to alanine increases VP40 membrane binding affinity for phosphatidylserine containing membranes. C311A has a significant increase in binding to PS compared to WT, has longer virus like particles, and displays evidence of increased budding. C314A also has an increase in PS binding compared to WT, however to a lesser extent. The double Cys mutant shares the phenotypes of the single mutants with increased binding to PS. Computational studies demonstrate these Cys residues, Cys311in particular, restrain a loop segment containing Lys residues that interact with the plasma membrane. Mutation of Cys311promotes membrane binding loop flexibility, alters internal VP40 H-bonding, and increases PS binding. To the best of our knowledge, this is the first evidence of mutations that increase VP40 affinity for biological membranes and the length of EBOV virus like particles. Together, our findings indicate these residues are important for membrane dynamics at the plasma membrane via the interaction with phosphatidylserine.

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Cysteine Mutations in the Ebolavirus Matrix Protein VP40 Promote Phosphatidylserine Binding by Increasing the Flexibility of a Lipid-Binding Loop

Viruses ◽

10.3390/v13071375 ◽

2021 ◽

Vol 13 (7) ◽

pp. 1375

Author(s):

Kristen A. Johnson ◽

Nisha Bhattarai ◽

Melissa R. Budicini ◽

Carolyn M. LaBonia ◽

Sarah Catherine B. Baker ◽

...

Keyword(s):

Plasma Membrane ◽

Matrix Protein ◽

Rna Virus ◽

Dominant Role ◽

Hemorrhagic Fever ◽

Lipid Binding ◽

Cysteine Residues ◽

Dynamic Interactions ◽

The Individual ◽

Binding Loop

Ebolavirus (EBOV) is a negative-sense RNA virus that causes severe hemorrhagic fever in humans. The matrix protein VP40 facilitates viral budding by binding to lipids in the host cell plasma membrane and driving the formation of filamentous, pleomorphic virus particles. The C-terminal domain of VP40 contains two highly-conserved cysteine residues at positions 311 and 314, but their role in the viral life cycle is unknown. We therefore investigated the properties of VP40 mutants in which the conserved cysteine residues were replaced with alanine. The C311A mutation significantly increased the affinity of VP40 for membranes containing phosphatidylserine (PS), resulting in the assembly of longer virus-like particles (VLPs) compared to wild-type VP40. The C314A mutation also increased the affinity of VP40 for membranes containing PS, albeit to a lesser degree than C311A. The double mutant behaved in a similar manner to the individual mutants. Computer modeling revealed that both cysteine residues restrain a loop segment containing lysine residues that interact with the plasma membrane, but Cys311 has the dominant role. Accordingly, the C311A mutation increases the flexibility of this membrane-binding loop, changes the profile of hydrogen bonding within VP40 and therefore binds to PS with greater affinity. This is the first evidence that mutations in VP40 can increase its affinity for biological membranes and modify the length of Ebola VLPs. The Cys311 and Cys314 residues therefore play an important role in dynamic interactions at the plasma membrane by modulating the ability of VP40 to bind PS.

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A Conserved Tryptophan in the Ebola Virus Matrix Protein C-Terminal Domain Is Required for Efficient Virus-Like Particle Formation

Pathogens ◽

10.3390/pathogens9050402 ◽

2020 ◽

Vol 9 (5) ◽

pp. 402 ◽

Cited By ~ 1

Author(s):

Kristen A. Johnson ◽

Rudramani Pokhrel ◽

Melissa R. Budicini ◽

Bernard S. Gerstman ◽

Prem P. Chapagain ◽

...

Keyword(s):

Plasma Membrane ◽

Matrix Protein ◽

Ebola Virus ◽

Membrane Localization ◽

Virus Like Particles ◽

Plasma Membrane Localization ◽

The Matrix ◽

Dynamics Simulations

The Ebola virus (EBOV) harbors seven genes, one of which is the matrix protein eVP40, a peripheral protein that is sufficient to induce the formation of virus-like particles from the host cell plasma membrane. eVP40 can form different structures to fulfil different functions during the viral life cycle, although the structural dynamics of eVP40 that warrant dimer, hexamer, and octamer formation are still poorly understood. eVP40 has two conserved Trp residues at positions 95 and 191. The role of Trp95 has been characterized in depth as it serves as an important residue in eVP40 oligomer formation. To gain insight into the functional role of Trp191 in eVP40, we prepared mutations of Trp191 (W191A or W191F) to determine the effects of mutation on eVP40 plasma membrane localization and budding as well as eVP40 oligomerization. These in vitro and cellular experiments were complemented by molecular dynamics simulations of the wild-type (WT) eVP40 structure versus that of W191A. Taken together, Trp is shown to be a critical amino acid at position 191 as mutation to Ala reduces the ability of VP40 to localize to the plasma membrane inner leaflet and form new virus-like particles. Further, mutation of Trp191 to Ala or Phe shifted the in vitro equilibrium to the octamer form by destabilizing Trp191 interactions with nearby residues. This study has shed new light on the importance of interdomain interactions in stability of the eVP40 structure and the critical nature of timing of eVP40 oligomerization for plasma membrane localization and viral budding.

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PI(4,5)P2 Binding Sites in the Ebola Virus Matrix Protein Modulate Assembly and Budding

10.1101/341248 ◽

2018 ◽

Author(s):

Kristen A. Johnson ◽

Melissa R. Budicini ◽

Sarah Urata ◽

Nisha Bhattarai ◽

Bernard S. Gerstman ◽

...

Keyword(s):

Plasma Membrane ◽

Matrix Protein ◽

Ebola Virus ◽

Treatment Options ◽

Treatment Strategies ◽

Hemorrhagic Fever ◽

Lipid Binding ◽

Cellular Assays ◽

Cell Plasma

AbstractEbola virus (EBOV) causes sever hemorrhagic fever in humans, can cause death in a large percentage of those infected, and still lacks FDA approved treatment options. In this study, we investigated how the essential EBOV protein, VP40, forms stable oligomers to mediate budding and assembly from the host cell plasma membrane. An array of in vitro and cellular assays identified and characterized two lysine rich regions that bind to PI(4,5)P2 and serve distinct functions through the lipid binding and assembly of the viral matrix layer. We found that when VP40 binds PI(4,5)P2, VP40 oligomers become extremely stable and long lived. Together, this work characterizes the molecular basis of PI(4,5)P2 binding by VP40, which stabilizes formation of VP40 oligomers necessary for viral assembly and budding. Quercetin, a natural product that lowers PI(4,5)P2 in the plasma membrane, inhibited budding of VP40 VLPs and may inform future treatment strategies against EBOV.

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The Ebola virus matrix protein VP40 hijacks the host plasma membrane to form virus envelope

The Journal of Lipid Research ◽

10.1194/jlr.ilr120000753 ◽

2020 ◽

Vol 61 (7) ◽

pp. 971-971

Author(s):

Souad Amiar ◽

Robert V. Stahelin

Keyword(s):

Plasma Membrane ◽

Matrix Protein ◽

Ebola Virus ◽

Virus Envelope ◽

Host Plasma Membrane

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Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus

Journal of Virology ◽

10.1128/jvi.01087-15 ◽

2015 ◽

Vol 89 (18) ◽

pp. 9440-9453 ◽

Cited By ~ 47

Author(s):

Emmanuel Adu-Gyamfi ◽

Kristen A. Johnson ◽

Mark E. Fraser ◽

Jordan L. Scott ◽

Smita P. Soni ◽

...

Keyword(s):

Plasma Membrane ◽

Host Cell ◽

Human Cell ◽

Mammalian Cells ◽

Matrix Protein ◽

Ebola Virus ◽

Cell Plasma Membrane ◽

Replication Process ◽

Cell Plasma

ABSTRACTLipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles.IMPORTANCEThe lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry.

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Ebola Virus VP35-VP40 Interaction Is Sufficient for Packaging 3E-5E Minigenome RNA into Virus-Like Particles

Journal of Virology ◽

10.1128/jvi.01857-05 ◽

2006 ◽

Vol 80 (11) ◽

pp. 5135-5144 ◽

Cited By ~ 44

Author(s):

Reed F. Johnson ◽

Sarah E. McCarthy ◽

Peter J. Godlewski ◽

Ronald N. Harty

Keyword(s):

Confocal Microscopy ◽

Reverse Transcriptase ◽

Matrix Protein ◽

Ebola Virus ◽

Genomic Rna ◽

Transfected Cells ◽

Virus Like Particles ◽

Rna Complexes ◽

Viral Genomic Rna ◽

Two Hybrid

ABSTRACT The packaging of viral genomic RNA into nucleocapsids and subsequently into virions is not completely understood. Phosphoprotein (P) and nucleoprotein (NP) interactions link NP-RNA complexes with P-L (polymerase) complexes to form viral nucleocapsids. The nucleocapsid then interacts with the viral matrix protein, leading to specific packaging of the nucleocapsid into the virion. A mammalian two-hybrid assay and confocal microscopy were used to demonstrate that Ebola virus VP35 and VP40 interact and colocalize in transfected cells. VP35 was packaged into budding virus-like particles (VLPs) as observed by protease protection assays. Moreover, VP40 and VP35 were sufficient for packaging an Ebola virus minignome RNA into VLPs. Results from immunoprecipitation-reverse transcriptase PCR experiments suggest that VP35 confers specificity of the nucleocapsid for viral genomic RNA by direct VP35-RNA interactions.

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The Minor Matrix Protein VP24 from Ebola Virus Lacks Direct Lipid-Binding Properties

Viruses ◽

10.3390/v12080869 ◽

2020 ◽

Vol 12 (8) ◽

pp. 869

Author(s):

Yuan Su ◽

Robert V. Stahelin

Keyword(s):

Protein Interactions ◽

Matrix Protein ◽

Ebola Virus ◽

Cellular Membrane ◽

Lipid Binding ◽

Confocal Imaging ◽

Host Cells ◽

Binding Ability ◽

Matrix Layer

Viral protein 24 (VP24) from Ebola virus (EBOV) was first recognized as a minor matrix protein that associates with cellular membranes. However, more recent studies shed light on its roles in inhibiting viral genome transcription and replication, facilitating nucleocapsid assembly and transport, and interfering with immune responses in host cells through downregulation of interferon (IFN)-activated genes. Thus, whether VP24 is a peripheral protein with lipid-binding ability for matrix layer recruitment has not been explored. Here, we examined the lipid-binding ability of VP24 with a number of lipid-binding assays. The results indicated that VP24 lacked the ability to associate with lipids tested regardless of VP24 posttranslational modifications. We further demonstrate that the presence of the EBOV major matrix protein VP40 did not promote VP24 membrane association in vitro or in cells. Further, no protein–protein interactions between VP24 and VP40 were detected by co-immunoprecipitation. Confocal imaging and cellular membrane fractionation analyses in human cells suggested VP24 did not specifically localize at the plasma membrane inner leaflet. Overall, we provide evidence that EBOV VP24 is not a lipid-binding protein and its presence in the viral matrix layer is likely not dependent on direct lipid interactions.

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Plasma membrane association facilitates conformational changes in the Marburg virus protein VP40 dimer

RSC Advances ◽

10.1039/c7ra02940c ◽

2017 ◽

Vol 7 (37) ◽

pp. 22741-22748 ◽

Cited By ~ 7

Author(s):

Nisha Bhattarai ◽

Jeevan B. GC ◽

Bernard S. Gerstman ◽

Robert V. Stahelin ◽

Prem P. Chapagain

Keyword(s):

Plasma Membrane ◽

Conformational Changes ◽

Ebola Virus ◽

Membrane Binding ◽

Marburg Virus ◽

Virus Protein ◽

Membrane Association ◽

Binding Interface

The membrane binding interface of the Marburg virus protein mVP40 dimer differs from that of the Ebola virus eVP40 dimer but membrane binding allows conformational changes in mVP40 that makes it structurally similar to the eVP40 dimer.

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Oligomerization and Ca2+/calmodulin control binding of the ER Ca2+-sensors STIM1 and STIM2 to plasma membrane lipids

Bioscience Reports ◽

10.1042/bsr20130089 ◽

2013 ◽

Vol 33 (5) ◽

Cited By ~ 30

Author(s):

Rajesh Bhardwaj ◽

Hans-Michael Müller ◽

Walter Nickel ◽

Matthias Seedorf

Keyword(s):

Plasma Membrane ◽

Membrane Lipids ◽

Lipid Binding ◽

Dependent Manner ◽

Activation Threshold ◽

Concentration Dependent Manner ◽

Rich Domain ◽

Α Helix ◽

Distinct Features ◽

Ca2 Channels

Ca2+ (calcium) homoeostasis and signalling rely on physical contacts between Ca2+ sensors in the ER (endoplasmic reticulum) and Ca2+ channels in the PM (plasma membrane). STIM1 (stromal interaction molecule 1) and STIM2 Ca2+ sensors oligomerize upon Ca2+ depletion in the ER lumen, contact phosphoinositides at the PM via their cytosolic lysine (K)-rich domains, and activate Ca2+ channels. Differential sensitivities of STIM1 and STIM2 towards ER luminal Ca2+ have been studied but responses towards elevated cytosolic Ca2+ concentration and the mechanism of lipid binding remain unclear. We found that tetramerization of the STIM1 K-rich domain is necessary for efficient binding to PI(4,5)P2-containing PM-like liposomes consistent with an oligomerization-driven STIM1 activation. In contrast, dimerization of STIM2 K-rich domain was sufficient for lipid binding. Furthermore, the K-rich domain of STIM2, but not of STIM1, forms an amphipathic α-helix. These distinct features of the STIM2 K-rich domain cause an increased affinity for PI(4,5)P2, consistent with the lower activation threshold of STIM2 and a function as regulator of basal Ca2+ levels. Concomitant with higher affinity for PM lipids, binding of CaM (calmodulin) inhibited the interaction of the STIM2 K-rich domain with liposomes in a Ca2+ and PI(4,5)P2 concentration-dependent manner. Therefore we suggest that elevated cytosolic Ca2+ concentration down-regulates STIM2-mediated ER–PM contacts via CaM binding.

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