Mapping of the VP40-Binding Regions of the Nucleoprotein of Ebola Virus

ABSTRACT Expression of Ebola virus nucleoprotein (NP) in mammalian cells leads to the formation of helical structures, which serve as a scaffold for the nucleocapsid. We recently found that NP binding with the matrix protein VP40 is important for nucleocapsid incorporation into virions (T. Noda, H. Ebihara, Y. Muramoto, K. Fujii, A. Takada, H. Sagara, J. H. Kim, H. Kida, H. Feldmann, and Y. Kawaoka, PLoS Pathog. 2:e99, 2006). To identify the region(s) on the NP molecule required for VP40 binding, we examined the interaction of a series of NP deletion mutants with VP40 biochemically and ultrastructurally. We found that both termini of NP (amino acids 2 to 150 and 601 to 739) are essential for its interaction with VP40 and for its incorporation into virus-like particles (VLPs). We also found that the C terminus of NP is important for nucleocapsid incorporation into virions. Of interest is that the formation of NP helices, which involves the N-terminal 450 amino acids of NP, is dispensable for NP incorporation into VLPs. These findings enhance our understanding of Ebola virus assembly and in so doing move us closer to the identification of targets for the development of antiviral compounds to combat Ebola virus infection.

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Association of Ebola Virus Matrix Protein VP40 with Microtubules

Journal of Virology ◽

10.1128/jvi.79.8.4709-4719.2005 ◽

2005 ◽

Vol 79 (8) ◽

pp. 4709-4719 ◽

Cited By ~ 54

Author(s):

Gordon Ruthel ◽

Gretchen L. Demmin ◽

George Kallstrom ◽

Melodi P. Javid ◽

Shirin S. Badie ◽

...

Keyword(s):

Amino Acids ◽

Mammalian Cells ◽

Matrix Protein ◽

Ebola Virus ◽

Life Cycles ◽

Binding Motif ◽

Physical Interaction ◽

Tubulin Polymerization ◽

In Cells

ABSTRACT Viruses exploit a variety of cellular components to complete their life cycles, and it has become increasingly clear that use of host cell microtubules is a vital part of the infection process for many viruses. A variety of viral proteins have been identified that interact with microtubules, either directly or via a microtubule-associated motor protein. Here, we report that Ebola virus associates with microtubules via the matrix protein VP40. When transfected into mammalian cells, a fraction of VP40 colocalized with microtubule bundles and VP40 coimmunoprecipitated with tubulin. The degree of colocalization and microtubule bundling in cells was markedly intensified by truncation of the C terminus to a length of 317 amino acids. Further truncation to 308 or fewer amino acids abolished the association with microtubules. Both the full-length and the 317-amino-acid truncation mutant stabilized microtubules against depolymerization with nocodazole. Direct physical interaction between purified VP40 and tubulin proteins was demonstrated in vitro. A region of moderate homology to the tubulin binding motif of the microtubule-associated protein MAP2 was identified in VP40. Deleting this region resulted in loss of microtubule stabilization against drug-induced depolymerization. The presence of VP40-associated microtubules in cells continuously treated with nocodazole suggested that VP40 promotes tubulin polymerization. Using an in vitro polymerization assay, we demonstrated that VP40 directly enhances tubulin polymerization without any cellular mediators. These results suggest that microtubules may play an important role in the Ebola virus life cycle and potentially provide a novel target for therapeutic intervention against this highly pathogenic virus.

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Biochemical and Functional Characterization of the Ebola Virus VP24 Protein: Implications for a Role in Virus Assembly and Budding

Journal of Virology ◽

10.1128/jvi.77.3.1793-1800.2003 ◽

2003 ◽

Vol 77 (3) ◽

pp. 1793-1800 ◽

Cited By ~ 89

Author(s):

Ziying Han ◽

Hani Boshra ◽

J. Oriol Sunyer ◽

Susan H. Zwiers ◽

Jason Paragas ◽

...

Keyword(s):

Lipid Membranes ◽

Mammalian Cells ◽

Matrix Protein ◽

Ebola Virus ◽

Virus Assembly ◽

Functional Characterization ◽

Minor Component ◽

Structural Features ◽

Structure And Function ◽

And Function

ABSTRACT The VP24 protein of Ebola virus is believed to be a secondary matrix protein and minor component of virions. In contrast, the VP40 protein of Ebola virus is the primary matrix protein and the most abundant virion component. The structure and function of VP40 have been well characterized; however, virtually nothing is known regarding the structure and function of VP24. Wild-type and mutant forms of VP24 were expressed in mammalian cells to gain a better understanding of the biochemical and functional nature of this viral protein. Results from these experiments demonstrated that (i) VP24 localizes to the plasma membrane and perinuclear region in both transfected and Ebola virus-infected cells, (ii) VP24 associates strongly with lipid membranes, (iii) VP24 does not contain N-linked sugars when expressed alone in mammalian cells, (iv) VP24 can oligomerize when expressed alone in mammalian cells, (v) progressive deletions at the N terminus of VP24 resulted in a decrease in oligomer formation and a concomitant increase in the formation of high-molecular-weight aggregates, and (vi) VP24 was present in trypsin-resistant virus like particles released into the media covering VP24-transfected cells. These data indicate that VP24 possesses structural features commonly associated with viral matrix proteins and that VP24 may have a role in virus assembly and budding.

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Contribution of Ebola Virus Glycoprotein, Nucleoprotein, and VP24 to Budding of VP40 Virus-Like Particles

Journal of Virology ◽

10.1128/jvi.78.14.7344-7351.2004 ◽

2004 ◽

Vol 78 (14) ◽

pp. 7344-7351 ◽

Cited By ~ 172

Author(s):

Jillian M. Licata ◽

Reed F. Johnson ◽

Ziying Han ◽

Ronald N. Harty

Keyword(s):

Amino Acids ◽

Matrix Protein ◽

Ebola Virus ◽

Virus Assembly ◽

Complete Understanding ◽

Virus Like Particles ◽

Virus Glycoprotein ◽

Membrane Bound ◽

Virus Proteins

ABSTRACT The VP40 matrix protein of Ebola virus buds from cells in the form of virus-like particles (VLPs) and plays a central role in virus assembly and budding. In this study, we utilized a functional budding assay and cotransfection experiments to examine the contributions of the glycoprotein (GP), nucleoprotein (NP), and VP24 of Ebola virus in facilitating release of VP40 VLPs. We demonstrate that VP24 alone does not affect VP40 VLP release, whereas NP and GP enhance release of VP40 VLPs, individually and to a greater degree in concert. We demonstrate further the following: (i) VP40 L domains are not required for GP-mediated enhancement of budding; (ii) the membrane-bound form of GP is necessary for enhancement of VP40 VLP release; (iii) NP appears to physically interact with VP40 as judged by detection of NP in VP40-containing VLPs; and (iv) the C-terminal 50 amino acids of NP may be important for interacting with and enhancing release of VP40 VLPs. These findings provide a more complete understanding of the role of VP40 and additional Ebola virus proteins during budding.

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Molecular docking enabled updated screening of the matrix protein VP40 from Ebola virus with millions of compounds in the MCULE database for potential inhibitors

Bioinformation ◽

10.6026/97320630015627 ◽

2019 ◽

Vol 15 (9) ◽

pp. 627-632

Author(s):

Hasanain Abdulhameed Odhar ◽

Keyword(s):

Molecular Docking ◽

Matrix Protein ◽

Ebola Virus ◽

The Matrix ◽

Potential Inhibitors

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Ebola Virus VP35 Antagonizes PKR Activity through Its C-Terminal Interferon Inhibitory Domain

Journal of Virology ◽

10.1128/jvi.00523-09 ◽

2009 ◽

Vol 83 (17) ◽

pp. 8993-8997 ◽

Cited By ~ 93

Author(s):

Michael Schümann ◽

Thorsten Gantke ◽

Elke Mühlberger

Keyword(s):

Amino Acids ◽

Ebola Virus ◽

Regulatory Factor ◽

Protein Kinase R ◽

Double Stranded Rna ◽

Basic Amino Acids ◽

Dsrna Binding ◽

Inhibitory Domain ◽

Ebola Virus Infection ◽

Terminal Cluster

ABSTRACT Ebola virus VP35 contains a C-terminal cluster of basic amino acids required for double-stranded RNA (dsRNA) binding and inhibition of interferon regulatory factor 3 (IRF3). VP35 also blocks protein kinase R (PKR) activation; however, the responsible domain has remained undefined. Here we show that the IRF inhibitory domain of VP35 mediates the inhibition of PKR and enhances the synthesis of coexpressed proteins. In contrast to dsRNA binding and IRF inhibition, alanine substitutions of at least two basic amino acids are required to abrogate PKR inhibition and enhanced protein expression. Moreover, we show that PKR activation is not only blocked but reversed by Ebola virus infection.

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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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Influenza Virus Matrix Protein Is the Major Driving Force in Virus Budding

Journal of Virology ◽

10.1128/jvi.74.24.11538-11547.2000 ◽

2000 ◽

Vol 74 (24) ◽

pp. 11538-11547 ◽

Cited By ~ 167

Author(s):

Paulino Gómez-Puertas ◽

Carmen Albo ◽

Esperanza Pérez-Pastrana ◽

Amparo Vivo ◽

Agustı́n Portela

Keyword(s):

Virus Particle ◽

Influenza A ◽

Matrix Protein ◽

Virus Assembly ◽

Microscopic Analysis ◽

Tubular Structures ◽

Electron Microscopic ◽

Particle Assembly ◽

M1 Protein ◽

The Matrix

ABSTRACT To get insights into the role played by each of the influenza A virus polypeptides in morphogenesis and virus particle assembly, the generation of virus-like particles (VLPs) has been examined in COS-1 cell cultures expressing, from recombinant plasmids, different combinations of the viral structural proteins. The presence of VLPs was examined biochemically, following centrifugation of the supernatants collected from transfected cells through sucrose cushions and immunoblotting, and by electron-microscopic analysis. It is demonstrated that the matrix (M1) protein is the only viral component which is essential for VLP formation and that the viral ribonucleoproteins are not required for virus particle formation. It is also shown that the M1 protein, when expressed alone, assembles into virus-like budding particles, which are released in the culture medium, and that the recombinant M1 protein accumulates intracellularly, forming tubular structures. All these results are discussed with regard to the roles played by the virus polypeptides during virus assembly.

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Role of Natural Killer Cells in Innate Protection against Lethal Ebola Virus Infection

Journal of Experimental Medicine ◽

10.1084/jem.20032141 ◽

2004 ◽

Vol 200 (2) ◽

pp. 169-179 ◽

Cited By ~ 97

Author(s):

Kelly L. Warfield ◽

Jeremy G. Perkins ◽

Dana L. Swenson ◽

Emily M. Deal ◽

Catharine M. Bosio ◽

...

Keyword(s):

Virus Infection ◽

Nk Cells ◽

Natural Killer ◽

Matrix Protein ◽

Ebola Virus ◽

Nk Cell ◽

Cytolytic Activity ◽

Virus Protein ◽

Lymphoid Tissues ◽

Ebola Virus Infection

Ebola virus is a highly lethal human pathogen and is rapidly driving many wild primate populations toward extinction. Several lines of evidence suggest that innate, nonspecific host factors are potentially critical for survival after Ebola virus infection. Here, we show that nonreplicating Ebola virus-like particles (VLPs), containing the glycoprotein (GP) and matrix protein virus protein (VP)40, administered 1–3 d before Ebola virus infection rapidly induced protective immunity. VLP injection enhanced the numbers of natural killer (NK) cells in lymphoid tissues. In contrast to live Ebola virus, VLP treatment of NK cells enhanced cytokine secretion and cytolytic activity against NK-sensitive targets. Unlike wild-type mice, treatment of NK-deficient or -depleted mice with VLPs had no protective effect against Ebola virus infection and NK cells treated with VLPs protected against Ebola virus infection when adoptively transferred to naive mice. The mechanism of NK cell–mediated protection clearly depended on perforin, but not interferon-γ secretion. Particles containing only VP40 were sufficient to induce NK cell responses and provide protection from infection in the absence of the viral GP. These findings revealed a decisive role for NK cells during lethal Ebola virus infection. This work should open new doors for better understanding of Ebola virus pathogenesis and direct the development of immunotherapeutics, which target the innate immune system, for treatment of Ebola virus infection.

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The Matrix Protein of Measles Virus Regulates Viral RNA Synthesis and Assembly by Interacting with the Nucleocapsid Protein

Journal of Virology ◽

10.1128/jvi.01056-09 ◽

2009 ◽

Vol 83 (20) ◽

pp. 10374-10383 ◽

Cited By ~ 98

Author(s):

Masaharu Iwasaki ◽

Makoto Takeda ◽

Yuta Shirogane ◽

Yuichiro Nakatsu ◽

Takanori Nakamura ◽

...

Keyword(s):

Plasma Membrane ◽

Mammalian Cells ◽

Measles Virus ◽

Rna Synthesis ◽

Matrix Protein ◽

Viral Rna ◽

M Protein ◽

Ribonucleoprotein Complex ◽

N Protein ◽

The Matrix

ABSTRACT The genome of measles virus (MV) is encapsidated by the nucleocapsid (N) protein and associates with RNA-dependent RNA polymerase to form the ribonucleoprotein complex. The matrix (M) protein is believed to play an important role in MV assembly by linking the ribonucleoprotein complex with envelope glycoproteins. Analyses using a yeast two-hybrid system and coimmunoprecipitation in mammalian cells revealed that the M protein interacts with the N protein and that two leucine residues at the carboxyl terminus of the N protein (L523 and L524) are critical for the interaction. In MV minigenome reporter gene assays, the M protein inhibited viral RNA synthesis only when it was able to interact with the N protein. The N protein colocalized with the M protein at the plasma membrane when the proteins were coexpressed in plasmid-transfected or MV-infected cells. In contrast, the N protein formed small dots in the perinuclear area when it was expressed without the M protein, or it was incapable of interacting with the M protein. Furthermore, a recombinant MV possessing a mutant N protein incapable of interacting with the M protein grew much less efficiently than the parental virus. Since the M protein has an intrinsic ability to associate with the plasma membrane, it may retain the ribonucleoprotein complex at the plasma membrane by binding to the N protein, thereby stopping viral RNA synthesis and promoting viral particle production. Consequently, our results indicate that the M protein regulates MV RNA synthesis and assembly via its interaction with the N protein.

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