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

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

10.20944/preprints202007.0363.v1 ◽

2020 ◽

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

10.1101/286120 ◽

2018 ◽

Author(s):

Kristen A. Johnson ◽

Nisha Bhattarai ◽

Melissa R. Budicini ◽

Carolyn M. Shirey ◽

Sarah Catherine B. Baker ◽

...

Keyword(s):

Plasma Membrane ◽

Matrix Protein ◽

Ebola Virus ◽

Membrane Lipids ◽

Membrane Binding ◽

Lipid Binding ◽

Membrane Dynamics ◽

Cysteine Residues ◽

Virus Like Particles ◽

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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Investigation of the Lipid Binding Properties of the Marburg Virus Matrix Protein VP40

Journal of Virology ◽

10.1128/jvi.02607-15 ◽

2015 ◽

Vol 90 (6) ◽

pp. 3074-3085 ◽

Cited By ~ 15

Author(s):

Kaveesha J. Wijesinghe ◽

Robert V. Stahelin

Keyword(s):

Plasma Membrane ◽

Charge Density ◽

Host Cell ◽

Matrix Protein ◽

Lipid Binding ◽

Marburg Virus ◽

Binding Properties ◽

Content Type ◽

Anionic Charge

ABSTRACTMarburg virus (MARV), which belongs to the virus familyFiloviridae, causes hemorrhagic fever in humans and nonhuman primates that is often fatal. MARV is a lipid-enveloped virus that during the replication process extracts its lipid coat from the plasma membrane of the host cell it infects. MARV carries seven genes, one of which encodes its matrix protein VP40 (mVP40), which regulates the assembly and budding of the virions. Currently, little information is available on mVP40 lipid binding properties. Here, we have investigated thein vitroand cellular mechanisms by which mVP40 associates with lipid membranes. mVP40 associates with anionic membranes in a nonspecific manner that is dependent upon the anionic charge density of the membrane. These results are consistent with recent structural determination of mVP40, which elucidated an mVP40 dimer with a flat and extensive cationic lipid binding interface.IMPORTANCEMarburg virus (MARV) is a lipid-enveloped filamentous virus from the familyFiloviridae. MARV was discovered in 1967, and yet little is known about how its seven genes are used to assemble and form a new viral particle in the host cell it infects. The MARV matrix protein VP40 (mVP40) underlies the inner leaflet of the virus and regulates budding from the host cell plasma membrane.In vitroand cellular assays in this study investigated the mechanism by which mVP40 associates with lipids. The results demonstrate that mVP40 interactions with lipid vesicles or the inner leaflet of the plasma membrane are electrostatic but nonspecific in nature and are dependent on the anionic charge density of the membrane surface. Small molecules that can disrupt lipid trafficking or reduce the anionic charge of the plasma membrane interface may be useful in inhibiting assembly and budding of MARV.

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Equine-Origin Immunoglobulin Fragments Protect Nonhuman Primates from Ebola Virus Disease

Journal of Virology ◽

10.1128/jvi.01548-18 ◽

2018 ◽

Vol 93 (5) ◽

Cited By ~ 2

Author(s):

Hualei Wang ◽

Gary Wong ◽

Wenjun Zhu ◽

Shihua He ◽

Yongkun Zhao ◽

...

Keyword(s):

Large Scale ◽

Nonhuman Primates ◽

Ebola Virus ◽

Virus Disease ◽

Hemorrhagic Fever ◽

West African ◽

Clinical Testing ◽

The Past ◽

Over 40 Years

ABSTRACT Ebola virus (EBOV) infections result in aggressive hemorrhagic fever in humans, with fatality rates reaching 90% and with no licensed specific therapeutics to treat ill patients. Advances over the past 5 years have firmly established monoclonal antibody (MAb)-based products as the most promising therapeutics for treating EBOV infections, but production is costly and quantities are limited; therefore, MAbs are not the best candidates for mass use in the case of an epidemic. To address this need, we generated EBOV-specific polyclonal F(ab′)2 fragments from horses hyperimmunized with an EBOV vaccine. The F(ab′)2 was found to potently neutralize West African and Central African EBOV in vitro. Treatment of nonhuman primates (NHPs) with seven doses of 100 mg/kg F(ab′)2 beginning 3 or 5 days postinfection (dpi) resulted in a 100% survival rate. Notably, NHPs for which treatment was initiated at 5 dpi were already highly viremic, with observable signs of EBOV disease, which demonstrated that F(ab′)2 was still effective as a therapeutic agent even in symptomatic subjects. These results show that F(ab′)2 should be advanced for clinical testing in preparation for future EBOV outbreaks and epidemics. IMPORTANCE EBOV is one of the deadliest viruses to humans. It has been over 40 years since EBOV was first reported, but no cure is available. Research breakthroughs over the past 5 years have shown that MAbs constitute an effective therapy for EBOV infections. However, MAbs are expensive and difficult to produce in large amounts and therefore may only play a limited role during an epidemic. A cheaper alternative is required, especially since EBOV is endemic in several third world countries with limited medical resources. Here, we used a standard protocol to produce large amounts of antiserum F(ab′)2 fragments from horses vaccinated with an EBOV vaccine, and we tested the protectiveness in monkeys. We showed that F(ab′)2 was effective in 100% of monkeys even after the animals were visibly ill with EBOV disease. Thus, F(ab′)2 could be a very good option for large-scale treatments of patients and should be advanced to clinical testing.

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