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

Pathogens ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 402 ◽  
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.

scholarly journals Mutation of Hydrophobic Residues in the C-Terminal Domain of the Marburg Virus Matrix Protein VP40 Disrupts Trafficking to the Plasma Membrane

Viruses ◽  
2020 ◽  
Vol 12 (4) ◽  
pp. 482 ◽  
Author(s):  
Kaveesha J. Wijesinghe ◽  
Luke McVeigh ◽  
Monica L. Husby ◽  
Nisha Bhattarai ◽  
Jia Ma ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Matrix Protein ◽  
Marburg Virus ◽  
Membrane Localization ◽  
Hydrophobic Residues ◽  
Plasma Membrane Localization ◽  
Terminal Domain ◽  
Loop Region

Marburg virus (MARV) is a lipid-enveloped negative sense single stranded RNA virus, which can cause a deadly hemorrhagic fever. MARV encodes seven proteins, including VP40 (mVP40), a matrix protein that interacts with the cytoplasmic leaflet of the host cell plasma membrane. VP40 traffics to the plasma membrane inner leaflet, where it assembles to facilitate the budding of viral particles. VP40 is a multifunctional protein that interacts with several host proteins and lipids to complete the viral replication cycle, but many of these host interactions remain unknown or are poorly characterized. In this study, we investigated the role of a hydrophobic loop region in the carboxy-terminal domain (CTD) of mVP40 that shares sequence similarity with the CTD of Ebola virus VP40 (eVP40). These conserved hydrophobic residues in eVP40 have been previously shown to be critical to plasma membrane localization and membrane insertion. An array of cellular experiments and confirmatory in vitro work strongly suggests proper orientation and hydrophobic residues (Phe281, Leu283, and Phe286) in the mVP40 CTD are critical to plasma membrane localization. In line with the different functions proposed for eVP40 and mVP40 CTD hydrophobic residues, molecular dynamics simulations demonstrate large flexibility of residues in the EBOV CTD whereas conserved mVP40 hydrophobic residues are more restricted in their flexibility. This study sheds further light on important amino acids and structural features in mVP40 required for its plasma membrane localization as well as differences in the functional role of CTD amino acids in eVP40 and mVP40.

scholarly journals Mutation of Hydrophobic Residues in the C-Terminal Domain of the Marburg Virus Matrix Protein VP40 Disrupts Trafficking to the Plasma Membrane

2020 ◽  
Author(s):  
Kaveesha J. Wijesinghe ◽  
Luke McVeigh ◽  
Monica L. Husby ◽  
Nisha Bhattarai ◽  
Jia Ma ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Matrix Protein ◽  
Marburg Virus ◽  
Membrane Localization ◽  
Hydrophobic Residues ◽  
Plasma Membrane Localization ◽  
Terminal Domain ◽  
Loop Region

Marburg virus (MARV) is a lipid-enveloped negative sense single stranded RNA virus, which can cause a deadly hemorrhagic fever. MARV encodes seven proteins, including VP40 (mVP40), a matrix protein that interacts with the cytoplasmic leaflet of the host cell plasma membrane. VP40 traffics to the plasma membrane inner leaflet, where it assembles to facilitate the budding of viral particles. VP40 is a multifunctional protein that interacts with several host proteins and lipids to complete the viral replication cycle, but many of these host-interactions remain unknown or are poorly characterized. In this study, we investigated the role of a hydrophobic loop region in the carboxy-terminal domain (CTD) of mVP40 that shares sequence similarity with the CTD of Ebola virus VP40 (eVP40). These conserved hydrophobic residues in eVP40 have been previously shown to be critical to plasma membrane localization and membrane insertion. An array of cellular experiments and confirmatory in vitro work strongly suggests proper orientation and hydrophobic residues (Phe281, Leu283, and Phe286) in the mVP40 CTD are critical to plasma membrane localization. In line with the different functions proposed for eVP40 and mVP40 CTD hydrophobic residues, molecular dynamics simulations demonstrate large flexibility of residues in the EBOV CTD whereas conserved mVP40 hydrophobic residues are more restricted in their flexibility. This study sheds further light on important amino acids and structural features in mVP40 required for its plasma membrane localization as well as differences in the functional role of CTD amino acids in eVP40 and mVP40.

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

2015 ◽  
Vol 89 (18) ◽  
pp. 9440-9453 ◽  
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.

scholarly journals Interdomain salt-bridges in the Ebola virus protein VP40 and their role in domain association and plasma membrane localization

2016 ◽  
Vol 25 (9) ◽  
pp. 1648-1658 ◽  
Author(s):  
Jeevan B. GC ◽  
Kristen A. Johnson ◽  
Monica L. Husby ◽  
Cary T. Frick ◽  
Bernard S. Gerstman ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ebola Virus ◽  
Membrane Localization ◽  
Virus Protein ◽  
Salt Bridges ◽  
Plasma Membrane Localization

scholarly journals Role of Ebola Virus Secreted Glycoproteins and Virus-Like Particles in Activation of Human Macrophages

2005 ◽  
Vol 79 (4) ◽  
pp. 2413-2419 ◽  
Author(s):  
Victoria Wahl-Jensen ◽  
Sabine K. Kurz ◽  
Paul R. Hazelton ◽  
Hans-Joachim Schnittler ◽  
Ute Ströher ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cell Activation ◽  
Ebola Virus ◽  
Target Cells ◽  
Primary Target ◽  
Necrosis Factor Alpha ◽  
Human Macrophages ◽  
Virus Like Particles

ABSTRACT Ebola virus, a member of the family Filoviridae, causes one of the most severe forms of viral hemorrhagic fever. In the terminal stages of disease, symptoms progress to hypotension, coagulation disorders, and hemorrhages, and there is prominent involvement of the mononuclear phagocytic and reticuloendothelial systems. Cells of the mononuclear phagocytic system are primary target cells and producers of inflammatory mediators. Ebola virus efficiently produces four soluble glycoproteins during infection: sGP, delta peptide (Δ-peptide), GP1, and GP1,2Δ. While the presence of these glycoproteins has been confirmed in blood (sGP) and in vitro systems, it is hypothesized that they are of biological relevance in pathogenesis, particularly target cell activation. To gain insight into their function, we expressed the four soluble glycoproteins in mammalian cells and purified and characterized them. The role of the transmembrane glycoprotein in the context of virus-like particles was also investigated. Primary human macrophages were treated with glycoproteins and virus-like particles and subsequently tested for activation by detection of several critical proinflammatory cytokines (tumor necrosis factor alpha, interleukin-6 [IL-6], and IL-1 beta) and the chemokine IL-8. The presentation of the glycoprotein was determined to be critical since virus-like particles, but not soluble glycoproteins, induced high levels of activation. We propose that the presentation of GP1,2 in the rigid form such as that observed on the surface of particles is critical for initiating a sufficient signal for the activation of primary target cells. The secreted glycoproteins do not appear to play any role in exogenous activation of these cells during Ebola virus infection.

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

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.

scholarly journals Role of Phosphatidylinositol 4,5-Bisphosphate in Regulating EHD2 Plasma Membrane Localization

PLoS ONE ◽  
2013 ◽  
Vol 8 (9) ◽  
pp. e74519 ◽  
Author(s):  
Laura C. Simone ◽  
Steve Caplan ◽  
Naava Naslavsky
Keyword(s):  
Plasma Membrane ◽  
Membrane Localization ◽  
Plasma Membrane Localization

Evidence for a role of caveolin-1 in neurokinin-1 receptor plasma-membrane localization, efficient signaling, and interaction with β-arrestin 2

2007 ◽  
Vol 330 (2) ◽  
pp. 231-245 ◽  
Author(s):  
Valentina Kubale ◽  
Zrinka Abramović ◽  
Azra Pogačnik ◽  
Anders Heding ◽  
Marjeta Šentjurc ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Localization ◽  
Neurokinin 1 Receptor ◽  
Caveolin 1 ◽  
Plasma Membrane Localization ◽  
Neurokinin 1
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