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

2006 ◽  
Vol 80 (11) ◽  
pp. 5135-5144 ◽  
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.

scholarly journals Assembly of Severe Acute Respiratory Syndrome Coronavirus RNA Packaging Signal into Virus-Like Particles Is Nucleocapsid Dependent

2005 ◽  
Vol 79 (22) ◽  
pp. 13848-13855 ◽  
Author(s):  
Ping-Kun Hsieh ◽  
Shin C. Chang ◽  
Chu-Chun Huang ◽  
Ting-Ting Lee ◽  
Ching-Wen Hsiao ◽  
...  
Keyword(s):  
Severe Acute Respiratory Syndrome ◽  
Culture Medium ◽  
Binding Activity ◽  
Viral Rna ◽  
Structural Proteins ◽  
Genomic Rna ◽  
Packaging Signal ◽  
Virus Like Particles ◽  
N Protein ◽  
Viral Genomic Rna

ABSTRACT The severe acute respiratory syndrome coronavirus (SARS-CoV) was recently identified as the etiology of SARS. The virus particle consists of four structural proteins: spike (S), small envelope (E), membrane (M), and nucleocapsid (N). Recognition of a specific sequence, termed the packaging signal (PS), by a virus N protein is often the first step in the assembly of viral RNA, but the molecular mechanisms involved in the assembly of SARS-CoV RNA are not clear. In this study, Vero E6 cells were cotransfected with plasmids encoding the four structural proteins of SARS-CoV. This generated virus-like particles (VLPs) of SARS-CoV that can be partially purified on a discontinuous sucrose gradient from the culture medium. The VLPs bearing all four of the structural proteins have a density of about 1.132 g/cm3. Western blot analysis of the culture medium from transfection experiments revealed that both E and M expressed alone could be released in sedimentable particles and that E and M proteins are likely to form VLPs when they are coexpressed. To examine the assembly of the viral genomic RNA, a plasmid representing the GFP-PS580 cDNA fragment encompassing the viral genomic RNA from nucleotides 19715 to 20294 inserted into the 3′ noncoding region of the green fluorescent protein (GFP) gene was constructed and applied to the cotransfection experiments with the four structural proteins. The SARS-CoV VLPs thus produced were designated VLP(GFP-PS580). Expression of GFP was detected in Vero E6 cells infected with the VLP(GFP-PS580), indicating that GFP-PS580 RNA can be assembled into the VLPs. Nevertheless, when Vero E6 cells were infected with VLPs produced in the absence of the viral N protein, no green fluorescence was visualized. These results indicate that N protein has an essential role in the packaging of SARS-CoV RNA. A filter binding assay and competition analysis further demonstrated that the N-terminal and C-terminal regions of the SARS-CoV N protein each contain a binding activity specific to the viral RNA. Deletions that presumably disrupt the structure of the N-terminal domain diminished its RNA-binding activity. The GFP-PS-containing SARS-CoV VLPs are powerful tools for investigating the tissue tropism and pathogenesis of SARS-CoV.

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 Association of Human Immunodeficiency Virus Type 1 Vif with RNA and Its Role in Reverse Transcription

2000 ◽  
Vol 74 (19) ◽  
pp. 8938-8945 ◽  
Author(s):  
Markus Dettenhofer ◽  
Shan Cen ◽  
Bradley A. Carlson ◽  
Lawrence Kleiman ◽  
Xiao-Fang Yu
Keyword(s):  
Human Immunodeficiency Virus ◽  
Reverse Transcriptase ◽  
Human Immunodeficiency Virus Type ◽  
Virus Type ◽  
Reverse Transcription ◽  
Genomic Rna ◽  
Immunodeficiency Virus ◽  
Viral Genomic Rna ◽  
Hiv 1

ABSTRACT The vif gene of human immunodeficiency virus type 1 (HIV-1) is essential for viral replication, although the functional target of Vif remains elusive. HIV-1 vif mutant virions derived from nonpermissive H9 cells displayed no significant differences in the amount, ratio, or integrity of their protein composition relative to an isogenic wild-type virion. The amounts of the virion-associated viral genomic RNA and tRNA3 Lyswere additionally present at normal levels in vif mutant virions. We demonstrate that Vif associates with RNA in vitro as well as with viral genomic RNA in virus-infected cells. A functionally conserved lentivirus Vif motif was found in the double-stranded RNA binding domain of Xenopus laevis, Xlrbpa. The natural intravirion reverse transcriptase products were markedly reduced invif mutant virions. Moreover, purified vifmutant genomic RNA-primer tRNA complexes displayed severe defects in the initiation of reverse transcription with recombinant reverse transcriptase. These data point to a novel role for Vif in the regulation of efficient reverse transcription through modulation of the virion nucleic acid components.

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 Nedd4 Regulates Egress of Ebola Virus-Like Particles from Host Cells

2003 ◽  
Vol 77 (18) ◽  
pp. 9987-9992 ◽  
Author(s):  
Jiro Yasuda ◽  
Mitsuyoshi Nakao ◽  
Yoshihiro Kawaoka ◽  
Hisatoshi Shida
Keyword(s):  
Ubiquitin Ligase ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Dominant Negative ◽  
Host Cells ◽  
Virus Budding ◽  
Ww Domains ◽  
Virus Like Particles ◽  
Active Site Cysteine ◽  
Type D

ABSTRACT Ebola virus budding is mediated by two proline-rich motifs, PPxY and PTAP, within the viral matrix protein VP40. We have previously shown that a Nedd4-like protein BUL1, but not Nedd4, positively regulates budding of type D retrovirus Mason-Pfizer monkey virus (J. Yasuda, E. Hunter, M. Nakao, and H. Shida, EMBO Rep. 3:636-640, 2002). Here, we report that the cellular E3 ubiquitin ligase Nedd4 regulates budding of VP40-induced virus-like particles (VLPs) through interaction with the PPxY motif. Mutation of the active site cysteine (C894A), resulting in abrogation of ubiquitin ligase activity, impaired the function of Nedd4 on budding. In addition, the WW domains of Nedd4 are essential for binding to the viral PPxY motif, and a small fragment of Nedd4 containing only WW domains significantly inhibited Ebola VLP budding in a dominant-negative manner. Our findings suggest that the viruses containing PPxY as an L-domain motif specifically use E3 in the process of virus budding. We also examined the effects of overexpression of Tsg101 and its mutant. As expected, Tsg101 enhanced VP40-induced VLP release, and TsgΔC, which lacks its C-terminal half, inhibited VLP release. These results indicate that Nedd4, together with Tsg101, plays an important role in Ebola virus budding.

scholarly journals Recombinant Modified Vaccinia Virus Ankara Generating Ebola Virus-Like Particles

2017 ◽  
Vol 91 (11) ◽  
Author(s):  
Marc Schweneker ◽  
Andrea S. Laimbacher ◽  
Gert Zimmer ◽  
Susanne Wagner ◽  
Elisabeth M. Schraner ◽  
...  
Keyword(s):  
Vaccinia Virus ◽  
Viral Vectors ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Virus Disease ◽  
Hemorrhagic Fever ◽  
Human Cells ◽  
Transient Expression Assay ◽  
Virus Like Particles ◽  
Modified Vaccinia Virus Ankara

ABSTRACT There are currently no approved therapeutics or vaccines to treat or protect against the severe hemorrhagic fever and death caused by Ebola virus (EBOV). Ebola virus-like particles (EBOV VLPs) consisting of the matrix protein VP40, the glycoprotein (GP), and the nucleoprotein (NP) are highly immunogenic and protective in nonhuman primates against Ebola virus disease (EVD). We have constructed a modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) recombinant coexpressing VP40 and GP of EBOV Mayinga and the NP of Taï Forest virus (TAFV) (MVA-BN-EBOV-VLP) to launch noninfectious EBOV VLPs as a second vaccine modality in the MVA-BN-EBOV-VLP-vaccinated organism. Human cells infected with either MVA-BN-EBOV-VLP or MVA-BN-EBOV-GP showed comparable GP expression levels and transport of complex N-glycosylated GP to the cell surface. Human cells infected with MVA-BN-EBOV-VLP produced large amounts of EBOV VLPs that were decorated with GP spikes but excluded the poxviral membrane protein B5, thus resembling authentic EBOV particles. The heterologous TAFV NP enhanced EBOV VP40-driven VLP formation with efficiency similar to that of the homologous EBOV NP in a transient-expression assay, and both NPs were incorporated into EBOV VLPs. EBOV GP-specific CD8 T cell responses were comparable between MVA-BN-EBOV-VLP- and MVA-BN-EBOV-GP-immunized mice. The levels of EBOV GP-specific neutralizing and binding antibodies, as well as GP-specific IgG1/IgG2a ratios induced by the two constructs, in mice were also similar, raising the question whether the quality rather than the quantity of the GP-specific antibody response might be altered by an EBOV VLP-generating MVA recombinant. IMPORTANCE The recent outbreak of Ebola virus (EBOV), claiming more than 11,000 lives, has underscored the need to advance the development of safe and effective filovirus vaccines. Virus-like particles (VLPs), as well as recombinant viral vectors, have proved to be promising vaccine candidates. Modified vaccinia virus Ankara-Bavarian Nordic (MVA-BN) is a safe and immunogenic vaccine vector with a large capacity to accommodate multiple foreign genes. In this study, we combined the advantages of VLPs and the MVA platform by generating a recombinant MVA-BN-EBOV-VLP that would produce noninfectious EBOV VLPs in the vaccinated individual. Our results show that human cells infected with MVA-BN-EBOV-VLP indeed formed and released EBOV VLPs, thus producing a highly authentic immunogen. MVA-BN-EBOV-VLP efficiently induced EBOV-specific humoral and cellular immune responses in vaccinated mice. These results are the basis for future advancements, e.g., by including antigens from various filoviral species to develop multivalent VLP-producing MVA-based filovirus vaccines.

scholarly journals Sequence-specific interaction between HIV-1 matrix protein and viral genomic RNA revealed by in vitro genetic selection

RNA ◽  
2001 ◽  
Vol 7 (4) ◽  
pp. 576-584 ◽  
Author(s):  
PRAKASH PUROHIT ◽  
STEFAN DUPONT ◽  
MARIO STEVENSON ◽  
MICHAEL R. GREEN
Keyword(s):  
Matrix Protein ◽  
Specific Interaction ◽  
Genetic Selection ◽  
Genomic Rna ◽  
Viral Genomic Rna ◽  
Hiv 1

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

2004 ◽  
Vol 78 (14) ◽  
pp. 7344-7351 ◽  
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.

scholarly journals Interaction of Tsg101 with Marburg Virus VP40 Depends on the PPPY Motif, but Not the PT/SAP Motif as in the Case of Ebola Virus, and Tsg101 Plays a Critical Role in the Budding of Marburg Virus-Like Particles Induced by VP40, NP, and GP

2007 ◽  
Vol 81 (9) ◽  
pp. 4895-4899 ◽  
Author(s):  
Shuzo Urata ◽  
Takeshi Noda ◽  
Yoshihiro Kawaoka ◽  
Shigeru Morikawa ◽  
Hideyoshi Yokosawa ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Critical Role ◽  
Marburg Virus ◽  
Virus Like Particles

ABSTRACT Marburg virus (MARV) VP40 is a matrix protein that can be released from mammalian cells in the form of virus-like particles (VLPs) and contains the PPPY sequence, which is an L-domain motif. Here, we demonstrate that the PPPY motif is important for VP40-induced VLP budding and that VLP production is significantly enhanced by coexpression of NP and GP. We show that Tsg101 interacts with VP40 depending on the presence of the PPPY motif, but not the PT/SAP motif as in the case of Ebola virus, and plays an important role in VLP budding. These findings provide new insights into the mechanism of MARV budding.

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