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Robert V. Stahelin - One of the best experts on this subject based on the ideXlab platform.
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lipid specific oligomerization of the marburg virus matrix Protein VP40 is regulated by two distinct interfaces for virion assembly
Journal of Biological Chemistry, 2021Co-Authors: Souad Amiar, Nisha Bhattarai, Bernard S Gerstman, Kaveesha J. Wijesinghe, Prem P. Chapagain, Monica L. Husby, S Angel, Robert V. StahelinAbstract:Abstract Marburg virus (MARV) is a lipid-enveloped virus harboring a negative sense RNA genome, which has caused sporadic outbreaks of viral hemorrhagic fever in Sub-Saharan Africa. MARV assembles and buds from the host cell plasma where MARV matrix Protein (mVP40) dimers associate with anionic lipids at the plasma membrane inner leaflet and undergo a dynamic and extensive self-oligomerization into the structural matrix layer. The MARV matrix layer confers the virion filamentous shape and stability but how host lipids modulate mVP40 oligomerization is mostly unknown. Using in vitro and cellular techniques, we present a mVP40 assembly model highlighting two distinct oligomerization interfaces: the (N-terminal domain (NTD) and C-terminal domain (CTD)) in mVP40. Cellular studies of NTD and CTD oligomerization interface mutants demonstrate the importance of each interface in matrix assembly. The assembly steps include Protein trafficking to the plasma membrane, homo-multimerization that induced Protein enrichment, plasma membrane fluidity changes and elongations at the plasma membrane. An ascorbate peroxidase derivative (APEX)-transmission electron microscopy method was employed to closely assess the ultrastructural localization and formation of viral particles for wild type mVP40 and NTD and CTD oligomerization interface mutants. Taken together, these studies present a mechanistic model of mVP40 oligomerization and assembly at the plasma membrane during virion assembly that requires interactions with phosphatidylserine for NTD-NTD interactions and phosphatidylinositol-4,5-bisphosphate for proper CTD-CTD interactions. These findings have broader implications in understanding budding of lipid-enveloped viruses from the host cell plasma membrane and potential strategies to target Protein-Protein or lipid-Protein interactions to inhibit virus budding.
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the minor matrix Protein vp24 from ebola virus lacks direct lipid binding properties
Viruses, 2020Co-Authors: Robert V. StahelinAbstract: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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Conformational Flexibility of the Protein-Protein Interfaces of the Ebola Virus VP40 Structural Matrix Filament.
The journal of physical chemistry. B, 2019Co-Authors: Elumalai Pavadai, Nisha Bhattarai, Prem P. Chapagain, Robert V. Stahelin, Prabin Baral, Bernard S GerstmanAbstract:The Ebola virus (EBOV) is a virulent pathogen that causes severe hemorrhagic fever with a high fatality rate in humans. The EBOV transformer Protein VP40 plays crucial roles in viral assembly and budding at the plasma membrane of infected cells. One of VP40’s roles is to form the long, flexible, pleomorphic filamentous structural matrix for the virus. Each filament contains three unique interfaces: monomer NTD–NTD to form a dimer, dimer-to-dimer NTD–NTD oligomerization to form a hexamer, and end-to-end hexamer CTD–CTD to build the filament. However, the atomic-level details of conformational flexibility of the VP40 filament are still elusive. In this study, we have performed explicit-solvent, all-atom molecular dynamic simulations to explore the conformational flexibility of the three different interface structures of the filament. Using dynamic network analysis and other calculational methods, we find that the CTD–CTD hexamer interface with weak interdomain amino acid communities is the most flexible, and the NTD–NTD oligomer interface with strong interdomain communities is the least flexible. Our study suggests that the high flexibility of the CTD–CTD interface may be essential for the supple bending of the Ebola filovirus, and such flexibility may present a target for molecular interventions to disrupt the Ebola virus functioning.
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pi 4 5 p2 binding sites in the ebola virus matrix Protein modulate assembly and budding
bioRxiv, 2018Co-Authors: Kristen A. Johnson, Nisha Bhattarai, Bernard S Gerstman, Prem P. Chapagain, Sarah Urata, Melissa R Budicini, Robert V. StahelinAbstract:Ebola 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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Graphene-VP40 interactions and potential disruption of the Ebola virus matrix filaments
Biochemical and biophysical research communications, 2017Co-Authors: Jeevan B., Nisha Bhattarai, Bernard S Gerstman, Robert V. Stahelin, Rudramani Pokhrel, Kristen A. Johnson, Prem P. ChapagainAbstract:Ebola virus infections cause hemorrhagic fever that often results in very high fatality rates. In addition to exploring vaccines, development of drugs is also essential for treating the disease and preventing the spread of the infection. The Ebola virus matrix Protein VP40 exists in various conformational and oligomeric forms and is a potential pharmacological target for disrupting the virus life-cycle. Here we explored graphene-VP40 interactions using molecular dynamics simulations and graphene pelleting assays. We found that graphene sheets associate strongly with VP40 at various interfaces. We also found that the graphene is able to disrupt the C-terminal domain (CTD-CTD) interface of VP40 hexamers. This VP40 hexamer-hexamer interface is crucial in forming the Ebola viral matrix and disruption of this interface may provide a method to use graphene or similar nanoparticle based solutions as a disinfectant that can significantly reduce the spread of the disease and prevent an Ebola epidemic.
Stephan Becker - One of the best experts on this subject based on the ideXlab platform.
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Ebola and Marburg virus matrix layers are locally ordered assemblies of VP40 dimers
2020Co-Authors: William Wan, Larissa Kolesnikova, Zachary A Bornholdt, Stephan Becker, Erica Ollmann Saphire, Alexander Koehler, Mairi Clarke, Michael J. Norris, John Ag BriggsAbstract:Abstract A key step in the life cycle of enveloped viruses is the budding of nascent virions from the host membrane. In filoviruses such as Ebola and Marburg virus, this process is achieved by the matrix Protein VP40. When expressed alone, VP40 induces the budding of filamentous virus-like particles, suggesting that localization to the plasma membrane, oligomerization into a matrix layer, and the generation of membrane curvature are intrinsic properties of VP40. While a number of crystal structures of VP40 have been determined in various oligomerization states, there has been no direct information on the structure of assembled VP40 matrix layers within viruses or virus-like particles. Here, we present structures of Ebola and Marburg VP40 matrix layers in intact virus-like particles, as well as within intact Marburg viruses. We find that the matrix layers are formed from VP40 dimers which assemble into extended chains via C-terminal domain interactions. These chains stack into layers, forming a 2D lattice below the membrane surface. However, these 2D lattices are only locally ordered, forming a patchwork assembly across the membrane surfaces and suggesting that assembly may begin at multiple points. These observations define the structure and arrangement of the matrix Protein layer that mediates the formation of filamentous filovirus particles.
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Analysis of the multifunctionality of Marburg virus VP40.
The Journal of general virology, 2018Co-Authors: Alexander Koehler, Larissa Kolesnikova, Sebastian Pfeiffer, Stephan BeckerAbstract:The Marburg virus (MARV) matrix Protein, VP40, is a multifunctional Protein that is essential for the assembly and release of viral particles, inhibition of the interferon response and viral transcription/replication. VP40 is assumed to be present as soluble monomers and membrane-bound higher-order oligomers. To investigate the functional relevance of oligomerization and lipid binding of VP40 we constructed mutants with impaired VP40–VP40 or VP40–lipid interactions and tested their capacity to bind the plasma membrane, to form virus-like particles (VLPs) and to inhibit viral RNA synthesis. All of the analysed VP40 mutants formed perinuclear aggregates and were defective in their delivery to the plasma membrane and in VLP production. The VP40 mutants that were competent for oligomerization but lacked VP40–lipid interactions formed fibril-like structures, influenced MARV inclusion body formation and inhibited viral transcription/replication more strongly than the VP40 wild-type. Altogether, mutations that interfere with VP40’s transition from monomer to higher-order oligomers and/or lipid interactions destroy the Protein’s multifunctionality.
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Ebola virus Proteins NP, VP35, and VP24 are essential and sufficient to mediate nucleocapsid transport
Proceedings of the National Academy of Sciences, 2018Co-Authors: Yuki Takamatsu, Larissa Kolesnikova, Stephan BeckerAbstract:The intracytoplasmic movement of nucleocapsids is a crucial step in the life cycle of enveloped viruses. Determination of the viral components necessary for viral nucleocapsid transport competency is complicated by the dynamic and complex nature of nucleocapsid assembly and the lack of appropriate model systems. Here, we established a live-cell imaging system based on the ectopic expression of fluorescent Ebola virus (EBOV) fusion Proteins, allowing the visualization and analysis of the movement of EBOV nucleocapsid-like structures with different Protein compositions. Only three of the five EBOV nucleocapsid Proteins—nucleoProtein, VP35, and VP24—were necessary and sufficient to form transport-competent nucleocapsid-like structures. The transport of these structures was found to be dependent on actin polymerization and to have dynamics that were undistinguishable from those of nucleocapsids in EBOV-infected cells. The intracytoplasmic movement of nucleocapsid-like structures was completely independent of the viral matrix Protein VP40 and the viral surface glycoProtein GP. However, VP40 greatly enhanced the efficiency of nucleocapsid recruitment into filopodia, the sites of EBOV budding.
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A Single Amino Acid Change in the Marburg Virus Matrix Protein VP40 Provides a Replicative Advantage in a Species-Specific Manner.
Journal of virology, 2015Co-Authors: Alexander Koehler, Gordian Schudt, Larissa Kolesnikova, Ulla Welzel, Astrid Herwig, Stephan BeckerAbstract:UNLABELLED Marburg virus (MARV) induces severe hemorrhagic fever in humans and nonhuman primates but only transient nonlethal disease in rodents. However, sequential passages of MARV in rodents boosts infection leading to lethal disease. Guinea pig-adapted MARV contains one mutation in the viral matrix Protein VP40 at position 184 (VP40D184N). The contribution of the D184N mutation to the efficacy of replication in a new host is unknown. In the present study, we demonstrated that recombinant MARV containing the D184N mutation in VP40 [rMARVVP40(D184N)] grew to higher titers than wild-type recombinant MARV (rMARVWT) in guinea pig cells. Moreover, rMARVVP40(D184N) displayed higher infectivity in guinea pig cells. Comparative analysis of VP40 functions indicated that neither the interferon (IFN)-antagonistic function nor the membrane binding capabilities of VP40 were affected by the D184N mutation. However, the production of VP40-induced virus-like particles (VLPs) and the recruitment of other viral Proteins to the budding site was improved by the D184N mutation in guinea pig cells, which resulted in the higher infectivity of VP40D184N-induced infectious VLPs (iVLPs) compared to that of VP40-induced iVLPs. In addition, the function of VP40 in suppressing viral RNA synthesis was influenced by the D184N mutation specifically in guinea pig cells, thus allowing greater rates of transcription and replication. Our results showed that the improved viral fitness of rMARVVP40(D184N) in guinea pig cells was due to the better viral assembly function of VP40D184N and its lower inhibitory effect on viral transcription and replication rather than modulation of the VP40-mediated suppression of IFN signaling. IMPORTANCE The increased virulence achieved by virus passaging in a new host was accompanied by mutations in the viral genome. Analyzing how these mutations affect the functions of viral Proteins and the ability of the virus to grow within new host cells helps in the understanding of the molecular mechanisms increasing virulence. Using a reverse genetics approach, we demonstrated that a single mutation in MARV VP40 detected in a guinea pig-adapted MARV provided a replicative advantage of rMARVVP40(D184N) in guinea pig cells. Our studies show that this replicative advantage of rMARV VP40D184N was based on the improved functions of VP40 in iVLP assembly and in the regulation of transcription and replication rather than on the ability of VP40 to combat the host innate immunity.
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131 Replication and assembly of filoviruses.
JAIDS Journal of Acquired Immune Deficiency Syndromes, 2014Co-Authors: Larissa Kolesnikova, Olga Dolnik, Gordian Schudt, Stephan BeckerAbstract:The family of Filoviridae comprises Marburg and Ebola virus which both cause severe life-threatening diseases characterized by high fever, rash, thrombocytopenia and hemorrhagic diathesis. The pathogenesis of the syndrome is not completely understood; probably the dynamic replication of filoviruses in the infected host leads to an uncoordinated immune response. Detailed understanding of the basic mechanisms of filoviral assembly and interaction with host cells is key to identify targets of antiviral intervention. The first sign of filovirus replication that can be detected microscopically in the infected cell is the formation of inclusions in the perinuclear region. Inclusions contain all filoviral nucleocapsid Proteins (NP, VP35, VP30, VP24, and L) but also the matrix Protein VP40 and a number of cellular Proteins. Viral nucleocapsids are formed within the inclusions by specific interactions among the viral Proteins. Mature nucleocapsids are transported across the cytoplasm to the plasma membrane with the help of the actin cytoskeleton. In the cell periphery nucleocapsids are associated with the matrix Protein and channeled into filopodia, the site of filoviral release. Nucleocapsids inside filopodia are cotransported together with the unconventional motor Protein myosin 10. Transport of nucleocapsids and release of viral particles is supported by the cellular ESCRT machinery.
Winfried Weissenhorn - One of the best experts on this subject based on the ideXlab platform.
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Conformational plasticity of the Ebola virus matrix Protein.
Protein Science, 2014Co-Authors: Jens Radzimanowski, Grégory Effantin, Winfried WeissenhornAbstract:Filoviruses are the causative agents of a severe and often fatal hemorrhagic fever with repeated outbreaks in Africa. They are negative sense single stranded enveloped viruses that can cross species barriers from its natural host bats to primates including humans. The small size of the genome poses limits to viral adaption, which may be partially overcome by conformational plasticity. Here we review the different conformational states of the Ebola virus (EBOV) matrix Protein VP40 that range from monomers, to dimers, hexamers, and RNA-bound octamers. This conformational plasticity that is required for the viral life cycle poses a unique opportunity for development of VP40 specific drugs. Furthermore, we compare the structure to homologous matrix Protein structures from Paramyxoviruses and Bornaviruses and we predict that they do not only share the fold but also the conformational flexibility of EBOV VP40.
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Conserved proline-rich region of Ebola virus matrix Protein VP40 is essential for plasma membrane targeting and virus-like particle release.
Journal of Infectious Diseases, 2011Co-Authors: Olivier Reynard, Winfried Weissenhorn, Audrey Page, Mathieu Mateo, Hervé Raoul, Kirill Nemirov, Viktor VolchkovAbstract:The matrix Protein VP40 is essential for Ebola virus (EBOV) and Marburg virus assembly and budding at the plasma membrane. In this study we have investigated the effect of single amino acid substitutions in a conserved proline-rich region of the EBOV VP40 located in the carboxy-terminal part of the Protein. We demonstrate that substitutions within this region result in an alteration of intracellular VP40 localization and also cause a reduction or a complete block of virus-like particle budding, a benchmark of VP40 function. Furthermore, some mutated VP40s revealed an enhanced binding with cellular Sec24C, a part of the coat Protein complex II (COPII) vesicular transport system. Analysis of the 3-dimensional structure of VP40 revealed the spatial proximity of the proline-rich region and an earlier identified site of interaction with Sec24C, thus allowing us to hypothesize that the altered intracellular localization of the VP40 mutants is a consequence of defects in their interaction with COPII-mediated vesicular transport.
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Filovirus assembly and budding.
Virology, 2006Co-Authors: Bettina Hartlieb, Winfried WeissenhornAbstract:Filoviruses belong to the order of negative-stranded non-segmented RNA viruses and are classified into two genera, Ebola and Marburg viruses. They have a characteristic filamentous shape, which is largely determined by the matrix Protein VP40. Although VP40 is the main driving force for assembly and budding from the host cell, the production of infectious virus involves an intricate interplay between all viral structural Proteins in addition to cellular factors, e.g., those that normally function in multi-vesicular body biogenesis. As a consequence, assembly and budding steps are defined to specific cellular compartments, and the recent progress in understanding how the different components are assembled into stable enveloped virus particles is reviewed.
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An all-atom model of the pore-like structure of hexameric VP40 from Ebola: structural insights into the monomer-hexamer transition.
Journal of structural biology, 2005Co-Authors: Tam Luong Nguyen, M. Javad Aman, Winfried Weissenhorn, G. Schoehn, Rekha G. Panchal, Ann R. Hermone, James C. Burnett, Connor F. Mcgrath, Dan W. Zaharevitz, Rick GussioAbstract:Abstract : The matrix Protein VP40 is an indispensable component of viral assembly and budding by the Ebola virus. VP40 is a monomer in solution, but can fold into hexameric and octameric states, two oligomeric conformations that play central roles in the Ebola viral life cycle. While the X-ray structures of monomeric and octameric VP40 have been determined, the structure of hexameric VP40 has only been solved by three-dimensional electron microscopy (EM) to a resolution of approximately 30A. In this paper, we present the refinement of the EM reconstruction of truncated hexameric VP40 to approximately 20A and the construction of an all-atom model (residues 44-212) using the EM model at approximately 20A and the X-ray structure of monomeric VP40 as templates. The hexamer model suggests that the monomer-hexamer transition involves a conformational change in the N-terminal domain that is not evident during octamerization and therefore, may provide the basis for elucidating the biological function of VP40.
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structural studies on the ebola virus matrix Protein VP40 indicate that matrix Proteins of enveloped rna viruses are analogues but not homologues
Fems Microbiology Letters, 2004Co-Authors: Joanna Timmins, Rob W.h. Ruigrok, Winfried WeissenhornAbstract:Matrix Proteins are the driving force of assembly of enveloped viruses. Their main function is to interact with and polymerize at cellular membranes and link other viral components to the matrix–membrane complex resulting in individual particle shapes and ensuring the integrity of the viral particle. Although matrix Proteins of different virus families show functional analogy, they share no sequence or structural homology. Their diversity is also evident in that they use a variety of late domain motifs to commit the cellular vacuolar Protein sorting machinery to virus budding. Here, we discuss the structural and functional aspects of the filovirus matrix Protein VP40 and compare them to other known matrix Protein structures from vesicular stomatitis virus, influenza virus and retroviral matrix Proteins.
Prem P. Chapagain - One of the best experts on this subject based on the ideXlab platform.
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lipid specific oligomerization of the marburg virus matrix Protein VP40 is regulated by two distinct interfaces for virion assembly
Journal of Biological Chemistry, 2021Co-Authors: Souad Amiar, Nisha Bhattarai, Bernard S Gerstman, Kaveesha J. Wijesinghe, Prem P. Chapagain, Monica L. Husby, S Angel, Robert V. StahelinAbstract:Abstract Marburg virus (MARV) is a lipid-enveloped virus harboring a negative sense RNA genome, which has caused sporadic outbreaks of viral hemorrhagic fever in Sub-Saharan Africa. MARV assembles and buds from the host cell plasma where MARV matrix Protein (mVP40) dimers associate with anionic lipids at the plasma membrane inner leaflet and undergo a dynamic and extensive self-oligomerization into the structural matrix layer. The MARV matrix layer confers the virion filamentous shape and stability but how host lipids modulate mVP40 oligomerization is mostly unknown. Using in vitro and cellular techniques, we present a mVP40 assembly model highlighting two distinct oligomerization interfaces: the (N-terminal domain (NTD) and C-terminal domain (CTD)) in mVP40. Cellular studies of NTD and CTD oligomerization interface mutants demonstrate the importance of each interface in matrix assembly. The assembly steps include Protein trafficking to the plasma membrane, homo-multimerization that induced Protein enrichment, plasma membrane fluidity changes and elongations at the plasma membrane. An ascorbate peroxidase derivative (APEX)-transmission electron microscopy method was employed to closely assess the ultrastructural localization and formation of viral particles for wild type mVP40 and NTD and CTD oligomerization interface mutants. Taken together, these studies present a mechanistic model of mVP40 oligomerization and assembly at the plasma membrane during virion assembly that requires interactions with phosphatidylserine for NTD-NTD interactions and phosphatidylinositol-4,5-bisphosphate for proper CTD-CTD interactions. These findings have broader implications in understanding budding of lipid-enveloped viruses from the host cell plasma membrane and potential strategies to target Protein-Protein or lipid-Protein interactions to inhibit virus budding.
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lipid specific Protein oligomerization is regulated by two interfaces in marburg virus matrix Protein VP40
bioRxiv, 2020Co-Authors: Souad Amiar, Nisha Bhattarai, Bernard S Gerstman, Kaveesha J. Wijesinghe, Prem P. Chapagain, Monica L. Husby, S AngelAbstract:Marburg virus major matrix Protein (mVP40) dimers associate with anionic lipids at the plasma membrane and undergo a dynamic and extensive self-oligomerization into the structural matrix layer which confers the virion shape and stability. Using a myriad of in vitro and cellular techniques, we present a mVP40 assembly model highlighting two distinct oligomerization interfaces (N-terminal domain (NTD) and C-terminal domain (CTD)) in mVP40. Cellular studies of NTD and CTD oligomerization interface mutants demonstrated the importance of each interface in the mVP40 matrix assembly through Protein trafficking to the plasma membrane and homo-multimerization that induced Protein enrichment, plasma membrane fluidity changes and elongations at the plasma membrane. A novel APEX-TEM method was employed to closely assess the ultrastructural localization of and formation of viral particles for wild type and mutants. Taken together, these studies present a mechanistic model of mVP40 oligomerization and assembly at the plasma membrane during virion assembly.
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Conformational Flexibility of the Protein-Protein Interfaces of the Ebola Virus VP40 Structural Matrix Filament.
The journal of physical chemistry. B, 2019Co-Authors: Elumalai Pavadai, Nisha Bhattarai, Prem P. Chapagain, Robert V. Stahelin, Prabin Baral, Bernard S GerstmanAbstract:The Ebola virus (EBOV) is a virulent pathogen that causes severe hemorrhagic fever with a high fatality rate in humans. The EBOV transformer Protein VP40 plays crucial roles in viral assembly and budding at the plasma membrane of infected cells. One of VP40’s roles is to form the long, flexible, pleomorphic filamentous structural matrix for the virus. Each filament contains three unique interfaces: monomer NTD–NTD to form a dimer, dimer-to-dimer NTD–NTD oligomerization to form a hexamer, and end-to-end hexamer CTD–CTD to build the filament. However, the atomic-level details of conformational flexibility of the VP40 filament are still elusive. In this study, we have performed explicit-solvent, all-atom molecular dynamic simulations to explore the conformational flexibility of the three different interface structures of the filament. Using dynamic network analysis and other calculational methods, we find that the CTD–CTD hexamer interface with weak interdomain amino acid communities is the most flexible, and the NTD–NTD oligomer interface with strong interdomain communities is the least flexible. Our study suggests that the high flexibility of the CTD–CTD interface may be essential for the supple bending of the Ebola filovirus, and such flexibility may present a target for molecular interventions to disrupt the Ebola virus functioning.
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A cylindrical assembly model and dynamics of the Ebola virus VP40 structural matrix.
Scientific reports, 2018Co-Authors: Elumalai Pavadai, Bernard S Gerstman, Prem P. ChapagainAbstract:The Ebola filovirus causes severe hemorrhagic fever with a high fatality rate in humans. The primary structural matrix Protein VP40 displays transformer-Protein characteristics and exists in different conformational and oligomeric states. VP40 plays crucial roles in viral assembly and budding at the plasma membrane of the infected cells and is capable of forming virus-like particles without the need for other Ebola Proteins. However, no experimental three-dimensional structure for any filovirus VP40 cylindrical assembly matrix is currently available. Here, we use a Protein-Protein docking approach to develop cylindrical assembly models for an Ebola virion and also for a smaller structural matrix that does not contain genetic material. These models match well with the 2D averages of cryo-electron tomograms of the authentic virion. We also used all-atom molecular dynamics simulations to investigate the stability and dynamics of the cylindrical models and the interactions between the side-by-side hexamers to determine the amino acid residues that are especially important for stabilizing the hexamers in the cylindrical ring configuration matrix assembly. Our models provide helpful information to better understand the assembly processes of filoviruses and such structural studies may also lead to the design and development of antiviral drugs.
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pi 4 5 p2 binding sites in the ebola virus matrix Protein modulate assembly and budding
bioRxiv, 2018Co-Authors: Kristen A. Johnson, Nisha Bhattarai, Bernard S Gerstman, Prem P. Chapagain, Sarah Urata, Melissa R Budicini, Robert V. StahelinAbstract:Ebola 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.
Sina Bavari - One of the best experts on this subject based on the ideXlab platform.
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Glucopyranosyl lipid adjuvant enhances immune response to Ebola virus-like particle vaccine in mice
Vaccine, 2019Co-Authors: Melek M. E. Sunay, Karen A. Martins, Jesse T. Steffens, Melissa K Gregory, Sean A. Vantongeren, Neal Van Hoeven, Preston G. Garnes, Sina BavariAbstract:The identification of adjuvants that promote lasting antigen-specific immunity and augment vaccine efficacy are integral to the development of new Protein-based vaccines. The Ebola virus-like particle (VLP) vaccine expressing Ebola virus glycoProtein (GP) and matrix Protein (VP40) was used in this study to evaluate the ability of TLR4 agonist glucopyranosyl lipid adjuvant (GLA) formulated in a stable emulsion (SE) to enhance immunogenicity and promote durable protection against mouse-adapted Ebola virus (ma-EBOV). Antibody responses and Ebola-specific T cell responses were evaluated post vaccination. Survival analysis after lethal ma-EBOV challenge was performed 4 weeks and 22 weeks following final vaccination. GLA-SE enhanced EBOV-specific immunity and resulted in long-term protection against challenge with ma-EBOV infection in a mouse model. Specifically, GLA-SE elicited Th1-skewed antibodies and promoted the generation of EBOV GP-specific polyfunctional T cells. These results provide further support for the utility of TLR4 activating GLA-SE-adjuvanted vaccines.
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Development of a liquid chromatography high resolution mass spectrometry method for the quantitation of viral envelope glycoProtein in Ebola virus-like particle vaccine preparations
Clinical proteomics, 2016Co-Authors: Lisa H. Cazares, Tara Kenny, Michael D. Ward, Ernst E. Brueggemann, Paul S. Demond, Christopher R. Mahone, Karen A. Martins, Jonathan E. Nuss, Trevor Glaros, Sina BavariAbstract:Background Ebola virus like particles (EBOV VLPs, eVLPs), are produced by expressing the viral transmembrane glycoProtein (GP) and structural matrix Protein VP40 in mammalian cells. When expressed, these Proteins self-assemble and bud from ‘host’ cells displaying morphology similar to infectious virions. Several studies have shown that rodents and non-human primates vaccinated with eVLPs are protected from lethal EBOV challenge. The mucin-like domain of envelope glycoProtein GP1 serves as the major target for a productive humoral immune response. Therefore GP1 concentration is a critical quality attribute of EBOV vaccines and accurate measurement of the amount of GP1 present in eVLP lots is crucial to understanding variability in vaccine efficacy.
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virus like particle vaccination protects nonhuman primates from lethal aerosol exposure with marburgvirus vlp vaccination protects macaques against aerosol challenges
Viruses, 2016Co-Authors: John M Dye, Kelly L Warfield, Jay Wells, Robert Unfer, Sergey Shulenin, Donald K Nichols, Mohammad Javad Aman, Sina BavariAbstract:Marburg virus (MARV) was the first filovirus to be identified following an outbreak of viral hemorrhagic fever disease in Marburg, Germany in 1967. Due to several factors inherent to filoviruses, they are considered a potential bioweapon that could be disseminated via an aerosol route. Previous studies demonstrated that MARV virus-like particles (VLPs) containing the glycoProtein (GP), matrix Protein VP40 and nucleoProtein (NP) generated using a baculovirus/insect cell expression system could protect macaques from subcutaneous (SQ) challenge with multiple species of marburgviruses. In the current study, the protective efficacy of the MARV VLPs in conjunction with two different adjuvants: QS-21, a saponin derivative, and poly I:C against homologous aerosol challenge was assessed in cynomolgus macaques. Antibody responses against the GP antigen were equivalent in all groups receiving MARV VLPs irrespective of the adjuvant; adjuvant only-vaccinated macaques did not demonstrate appreciable antibody responses. All macaques were subsequently challenged with lethal doses of MARV via aerosol or SQ as a positive control. All MARV VLP-vaccinated macaques survived either aerosol or SQ challenge while animals administered adjuvant only exhibited clinical signs and lesions consistent with MARV disease and were euthanized after meeting the predetermined criteria. Therefore, MARV VLPs induce IgG antibodies recognizing MARV GP and VP40 and protect cynomolgus macaques from an otherwise lethal aerosol exposure with MARV.
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Assembly of Ebola virus matrix Protein VP40 is regulated by latch-like properties of N and C terminal tails.
PloS one, 2012Co-Authors: Leslie P. Silva, Sina Bavari, Michael Vanzile, Javad Aman, David C. SchriemerAbstract:The matrix Protein VP40 coordinates numerous functions in the viral life cycle of the Ebola virus. These range from the regulation of viral transcription to morphogenesis, packaging and budding of mature virions. Similar to the matrix Proteins of other nonsegmented, negative-strand RNA viruses, VP40 proceeds through intermediate states of assembly (e.g. octamers) but it remains unclear how these intermediates are coordinated with the various stages of the life cycle. In this study, we investigate the molecular basis of synchronization as governed by VP40. Hydrogen/deuterium exchange mass spectrometry was used to follow induced structural and conformational changes in VP40. Together with computational modeling, we demonstrate that both extreme N and C terminal tail regions stabilize the monomeric state through a direct association. The tails appear to function as a latch, released upon a specific molecular trigger such as RNA ligation. We propose that triggered release of the tails permits the coordination of late-stage events in the viral life cycle, at the inner membrane of the host cell. Specifically, N-tail release exposes the L-domain motifs PTAP/PPEY to the transport and budding complexes, whereas triggered C-tail release could improve association with the site of budding.
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involvement of vacuolar Protein sorting pathway in ebola virus release independent of tsg101 interaction
The Journal of Infectious Diseases, 2007Co-Authors: Lynn S Silvestri, Timothy Nelle, Dana L Swenson, George Kallstrom, Gordon Ruthel, Sina Bavari, Kelly L Warfield, Patrick L Iversen, Javad M AmanAbstract:Budding of Ebola virus (EBOV) particles from the plasma membrane of infected cells requires viral and host Proteins. EBOV virus matrix Protein VP40 recruits TSG101, an ESCRT-1 (host cell endosomal sorting complex required for transport-1) complex Protein in the vacuolar Protein sorting (vps) pathway, to the plasma membrane during budding. Involvement of other vps Proteins in EBOV budding has not been established. Therefore, we used VP40 deletion analysis, virus-like particle-release assays, and confocal microscopy to investigate the potential role of ESCRT-1 Proteins VPS4, VPS28, and VPS37B in EBOV budding. We found that VP40 could redirect each Protein from endosomes to the cell surface independently of TSG101 interaction. A lack of VPS4 adenosine triphosphatase activity reduced budding by up to 80%. Inhibition of VPS4 gene expression by use of phosphorodiamidite morpholino antisense oligonucleotides protected mice from lethal EBOV infection. These data show that EBOV can use vps Proteins independently of TSG101 for budding and reveal VPS4 as a potential target for filovirus therapeutics.
