scholarly journals Ebola Virus Glycoprotein: Proteolytic Processing, Acylation, Cell Tropism, and Detection of Neutralizing Antibodies

2001 ◽  
Vol 75 (3) ◽  
pp. 1576-1580 ◽  
Author(s):  
Hiroshi Ito ◽  
Shinji Watanabe ◽  
Ayato Takada ◽  
Yoshihiro Kawaoka
Keyword(s):  
Endothelial Cells ◽  
Neutralizing Antibodies ◽  
Ebola Virus ◽  
Cell Types ◽  
Proteolytic Processing ◽  
Cell Tropism ◽  
Transmembrane Region ◽  
Virus Glycoprotein ◽  
Vesicular Stomatitis ◽  
Neutralizing Epitopes

ABSTRACT Using the vesicular stomatitis virus (VSV) pseudotype system, we studied the functional properties of the Ebola virus glycoprotein (GP). Amino acid substitutions at the GP cleavage site, which reduce glycoprotein cleavability and viral infectivity in some viruses, did not appreciably change the infectivity of VSV pseudotyped with GP. Likewise, removal of two acylated cysteine residues in the transmembrane region of GP showed no discernible effects on infectivity. Although most filoviruses are believed to target endothelial cells and hepatocytes preferentially, the GP-carrying VSV showed greater affinity for epithelial cells than for either of these cell types, indicating that Ebola virus GP does not necessarily have strong tropism toward endothelial cells and hepatocytes. Finally, when it was used to screen for neutralizing antibodies against Ebola virus GP, the VSV pseudotype system allowed us to detect strain-specific neutralizing activity that was inhibited by secretory GP (SGP). This finding provides evidence of shared neutralizing epitopes on GP and SGP molecules and indicates the potential of SGP to serve as a decoy for neutralizing antibodies.

scholarly journals Mucin-Like Domain of Ebola Virus Glycoprotein Enhances Selective Oncolytic Actions against Brain Tumors

2020 ◽  
Vol 94 (8) ◽  
Author(s):  
Xue Zhang ◽  
Tingting Zhang ◽  
John N. Davis ◽  
Andrea Marzi ◽  
Anthony M. Marchese ◽  
...  
Keyword(s):  
Brain Tumors ◽  
Ebola Virus ◽  
Fluorescent Protein ◽  
Brain Cells ◽  
Normal Brain ◽  
Immunodeficient Mice ◽  
Virus Glycoprotein ◽  
Vesicular Stomatitis ◽  
Immunocompetent Mice ◽  
Green Fluorescent

ABSTRACT Given that the Ebola virus (EBOV) infects a wide array of organs and cells yet displays a relative lack of neurotropism, we asked whether a chimeric vesicular stomatitis virus (VSV) expressing the EBOV glycoprotein (GP) might selectively target brain tumors. The mucin-like domain (MLD) of the EBOV GP may enhance virus immune system evasion. Here, we compared chimeric VSVs in which EBOV GP replaces the VSV glycoprotein, thereby reducing the neurotoxicity associated with wild-type VSV. A chimeric VSV expressing the full-length EBOV GP (VSV-EBOV) containing the MLD was substantially more effective and safer than a parallel construct with an EBOV GP lacking the MLD (VSV-EBOVΔMLD). One-step growth, reverse transcription-quantitative PCR, and Western blotting assessments showed that VSV-EBOVΔMLD produced substantially more progeny faster than VSV-EBOV. Using immunodeficient SCID mice, we focused on targeting human brain tumors with these VSV-EBOVs. Similar to the findings of our previous study in which we used an attenuated VSV-EBOV with no MLD that expressed green fluorescent protein (GFP) (VSV-EBOVΔMLD-GFP), VSV-EBOVΔMLD without GFP targeted glioma but yielded only a modest extension of survival. In contrast, VSV-EBOV containing the MLD showed substantially better targeting and elimination of brain tumors after intravenous delivery and increased the survival of brain tumor-bearing mice. Despite the apparent destruction of most tumor cells by VSV-EBOVΔMLD, the virus remained active within the SCID mouse brain and showed widespread infection of normal brain cells. In contrast, VSV-EBOV eliminated the tumors and showed relatively little infection of normal brain cells. Parallel experiments with direct intracranial virus infection generated similar results. Neither VSV-EBOV nor VSV-EBOVΔMLD showed substantive infection of the brains of normal immunocompetent mice. IMPORTANCE The Ebola virus glycoprotein contains a mucin-like domain which may play a role in immune evasion. Chimeric vesicular stomatitis viruses with the EBOV glycoprotein substituted for the VSV glycoprotein show greater safety and efficacy in targeting brain tumors in immunodeficient mice when the MLD was expressed within the EBOV glycoprotein than when EBOV lacked the mucin-like domain.

Abstract 180: The Myristolated Alanine-Rich C Kinase Substrate (MARCKS) Differentially Regulates Proliferation of Vascular Smooth Muscle Cells and Endothelial Cells through Affecting Protein Stability and Proteolytic Processing of the Kinase Interacting with Stathmin (KIS)

2016 ◽  
Vol 36 (suppl_1) ◽  
Author(s):  
Dan Yu ◽  
Charles Drucker ◽  
Rajabrata Sarkar ◽  
Dudley K Strickland ◽  
Thomas S Monahan
Keyword(s):  
Endothelial Cells ◽  
Smooth Muscle ◽  
Coronary Artery ◽  
Smooth Muscle Cells ◽  
Protein Stability ◽  
Muscle Cells ◽  
Cell Types ◽  
Proteolytic Processing ◽  
Functional Domain ◽  
Human Coronary Artery

Objective: Presently, the antiproliferative agents used in drug eluting stents and drug coated balloons inhibit both VSMC and endothelial cell (EC) proliferation, and thus these patients require dual antiplatelet therapy indefinitely. Identification of a VSMC-specific target to prevent proliferation represents a significant unmet clinical need. Previously we found that knockdown of MARCKS arrests VSMC proliferation through a p27 kip1 -dependent mechanism. Interestingly MARCKS knockdown increases EC proliferation. p27 kip1 is phosphorylated by KIS allowing it to exit the nucleus and be degraded. Here we seek to understand how MARCKS influences KIS protein expression in these two cell types. Approach and Results: We performed siRNA-mediated knock down of MARCKS in human coronary artery endothelial cells (CAECs) and human coronary artery smooth muscle cells (CASMCs). MARCKS knockdown did not affect KIS mRNA expression as determined with RT-PCR in either cell type. KIS protein stability was evaluated in the presence of cyclohexamide with Western blot. In CAECs, MARCKS knockdown increased KIS stability, however, in CASMCs, MARCKS knockdown significantly decreased KIS protein stability. In CASMCs, MARCKS knockdown significantly increased KIS ubiquitinization where as in CAECs, MARCKS knockdown decreased KIS ubiquitinization. Interestingly, the well-studied functional domain of MARCKS(ED domain) is not directly involved in KIS regulation. MARCKS mutants (S4G and S4D) rescued proliferation in VSMCs. MARCKS knockdown in vivo in the murine femoral wire injury model resulted in decreased medial bromodeoxyuridine (BrdU) integration and neointima formation. MARCKS knockdown enhanced endothelial barrier function recovery four days after injury as assessed by Evans Blue integration. Conclusions: MARCKS differentially regulates the protein stability and proteolytic processing of KIS in VSMCs and ECs. The differential interaction of MARCKS and KIS likely explains the observed difference in proliferation observed with MARCKS knockdown in these two cell types.

scholarly journals Ebola virus glycoprotein-mediated anoikis of primary human cardiac microvascular endothelial cells

Virology ◽  
2004 ◽  
Vol 321 (2) ◽  
pp. 181-188 ◽  
Author(s):  
Ratna B Ray ◽  
Arnab Basu ◽  
Robert Steele ◽  
Aster Beyene ◽  
Jane McHowat ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Ebola Virus ◽  
Microvascular Endothelial Cells ◽  
Virus Glycoprotein ◽  
Microvascular Endothelial ◽  
Cardiac Microvascular Endothelial Cells

scholarly journals Vesicular Stomatitis Virus Pseudotyped with Ebola Virus Glycoprotein Serves as a Protective, Noninfectious Vaccine against Ebola Virus Challenge in Mice

2017 ◽  
Vol 91 (17) ◽  
Author(s):  
Nicholas J. Lennemann ◽  
Andrew S. Herbert ◽  
Rachel Brouillette ◽  
Bethany Rhein ◽  
Russell A. Bakken ◽  
...  
Keyword(s):  
Single Dose ◽  
West Africa ◽  
Vesicular Stomatitis Virus ◽  
Ebola Virus ◽  
Dependent Manner ◽  
Wild Type ◽  
Vaccine Platform ◽  
Virus Glycoprotein ◽  
Conserved Regions ◽  
Vesicular Stomatitis

ABSTRACT The recent Ebola virus (EBOV) epidemic in West Africa demonstrates the potential for a significant public health burden caused by filoviral infections. No vaccine or antiviral is currently FDA approved. To expand the vaccine options potentially available, we assessed protection conferred by an EBOV vaccine composed of vesicular stomatitis virus pseudovirions that lack native G glycoprotein (VSVΔG) and bear EBOV glycoprotein (GP). These pseudovirions mediate a single round of infection. Both single-dose and prime/boost vaccination regimens protected mice against lethal challenge with mouse-adapted Ebola virus (ma-EBOV) in a dose-dependent manner. The prime/boost regimen provided significantly better protection than a single dose. As N-linked glycans are thought to shield conserved regions of the EBOV GP receptor-binding domain (RBD), thereby blocking epitopes within the RBD, we also tested whether VSVΔG bearing EBOV GPs that lack GP1 N-linked glycans provided effective immunity against challenge with ma-EBOV or a more distantly related virus, Sudan virus. Using a prime/boost strategy, high doses of GP/VSVΔG partially or fully denuded of N-linked glycans on GP1 protected mice against ma-EBOV challenge, but these mutants were no more effective than wild-type (WT) GP/VSVΔG and did not provide cross protection against Sudan virus. As reported for other EBOV vaccine platforms, the protection conferred correlated with the quantity of EBOV GP-specific Ig produced but not with the production of neutralizing antibodies. Our results show that EBOV GP/VSVΔG pseudovirions serve as a successful vaccination platform in a rodent model of Ebola virus disease and that GP1 N-glycan loss does not influence immunogenicity or vaccination success. IMPORTANCE The West African Ebola virus epidemic was the largest to date, with more than 28,000 people infected. No FDA-approved vaccines are yet available, but in a trial vaccination strategy in West Africa, recombinant, infectious VSV encoding the Ebola virus glycoprotein effectively prevented virus-associated disease. VSVΔG pseudovirion vaccines may prove as efficacious and have better safety, but they have not been tested to date. Thus, we tested the efficacy of VSVΔG pseudovirions bearing Ebola virus glycoprotein as a vaccine platform. We found that wild-type Ebola virus glycoprotein, in the context of this platform, provides robust protection of EBOV-challenged mice. Further, we found that removal of the heavy glycan shield surrounding conserved regions of the glycoprotein does not enhance vaccine efficacy.

scholarly journals Broadly neutralizing antibodies from human survivors target a conserved site in the Ebola virus glycoprotein HR2–MPER region

2018 ◽  
Vol 3 (6) ◽  
pp. 670-677 ◽  
Author(s):  
Andrew I. Flyak ◽  
Natalia Kuzmina ◽  
Charles D. Murin ◽  
Christopher Bryan ◽  
Edgar Davidson ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Ebola Virus ◽  
Broadly Neutralizing Antibodies ◽  
Virus Glycoprotein

scholarly journals Polymorphisms in Human Cytomegalovirus gO Exert Epistatic Influences on Cell-Free and Cell-To-Cell Spread, and Antibody Neutralization on gH Epitopes

2019 ◽  
Author(s):  
Le Zhang Day ◽  
Cora Stegmann ◽  
Eric P. Schultz ◽  
Jean-Marc Lanchy ◽  
Qin Yu ◽  
...  
Keyword(s):  
Human Cytomegalovirus ◽  
Viral Genome ◽  
Genetic Background ◽  
Neutralizing Antibodies ◽  
Important Determinant ◽  
Cell Types ◽  
Cell Tropism ◽  
Laboratory Studies ◽  
Antibody Neutralization ◽  
Cell Spread

ABSTRACTThe human cytomegalovirus (HCMV) glycoproteins H and L (gH/gL) can be bound by either gO, or the UL128-131 proteins to form complexes that facilitate entry and spread and the complexes formed are important targets of neutralizing antibodies. Strains of HCMV vary considerably in the levels of gH/gL/gO and gH/gL/UL128-131 and this can impact infectivity and cell tropism. In this report, we investigated how natural interstrain variation in the amino acid sequence of gO influences the biology of HCMV. Heterologous gO recombinants were constructed in which 6 of the 8 alleles or genotypes (GT) of gO were analyzed in the backgrounds of strain TR and Merlin (ME). The levels of gH/gL complexes were not affected, but there were impacts on entry, spread and neutralization by anti-gH antibodies. AD169 (AD) gO (GT1a) drastically reduced cell-free infectivity of both strains on fibroblasts and epithelial cells. PHgO(GT2a) increased cell-free infectivity of TR in both cell types, but spread in fibroblasts was impaired. In contrast, spread of ME in both cell types was enhanced by Towne (TN) gO (GT4), despite similar cell-free infectivity. TR expressing TNgO(GT4) was resistant to neutralization by anti-gH antibodies AP86 and 14-4b, whereas ADgO(GT1a) conferred resistance to 14-4b, but enhanced neutralization by AP86. Conversely, ME expressing ADgO(GT1a) was more resistant to 14-4b. These results suggest; 1) mechanistically distinct roles for gH/gL/gO in cell-free and cell-to-cell spread, 2) gO isoforms can differentially shield the virus from neutralizing antibodies, and 3) effects of gO polymorphisms are epistatically dependent on other variable loci.IMPORTANCEAdvances in HCMV population genetics have greatly outpaced understanding of the links between genetic diversity and phenotypic variation. Moreover, recombination between genotypes may shuffle variable loci into various combinations with unknown outcomes. UL74(gO) is an important determinant of HCMV infectivity, and one of the most diverse loci in the viral genome. By analyzing interstrain heterologous UL74(gO) recombinants, we show that gO diversity can have dramatic impacts on cell-free and cell-to-cell spread as well as on antibody neutralization and that the manifestation of these impacts can be subject to epistatic influences of the global genetic background. These results highlight the potential limitations of laboratory studies of HCMV biology that use single, isolated genotypes or strains.

Ebola Virus Glycoprotein Demonstrates Differential Cellular Localization in Infected Cell Types of Nonhuman Primates and Guinea Pigs

2001 ◽  
Vol 125 (5) ◽  
pp. 625-630
Author(s):  
Keith Steele ◽  
Bruce Crise ◽  
Ana Kuehne ◽  
Wayne Kell
Keyword(s):  
Infected Cell ◽  
Guinea Pigs ◽  
Cellular Localization ◽  
Nonhuman Primates ◽  
Ebola Virus ◽  
Cell Types ◽  
Virus Glycoprotein ◽  
293 Cells

Abstract Background.—In vitro studies have previously shown that Ebola virus glycoprotein (GP) is rapidly processed and largely released from infected cells, whereas other viral proteins, such as VP40, accumulate within cells. Objective.—To determine infected cell types in which Ebola virus GP and VP40, individually, localize in vivo. Methods.—Immunohistochemistry and in situ hybridization using GP- and VP40-specific antibodies and genetic probes were used to analyze archived tissues of experimentally infected nonhuman primates and guinea pigs and Vero E6 and 293 cells infected in vitro. Results.—The GP antigen was consistently present in hepatocytes, adrenal cortical cells, fibroblasts, fibroblastic reticular cells, ovarian thecal cells, and several types of epithelial cells, but was not detected in macrophages and blood monocytes of animals, nor in Vero cells and 293 cells. All GP-positive and GP-negative cell types analyzed contained VP40 antigen and both GP and VP40 RNAs. Conclusions.—Ebola virus GP appears to selectively accumulate in many cell types infected in vivo, but not in macrophages and monocytes. This finding suggests that many cell types may have a GP-processing pathway that differs from the pathway described by previous in vitro studies. Differential cellular localization of GP could be relevant to the pathogenesis of Ebola hemorrhagic fever.

scholarly journals Proteolytic Processing of the Ebola Virus Glycoprotein Is Not Critical for Ebola Virus Replication in Nonhuman Primates

2007 ◽  
Vol 81 (6) ◽  
pp. 2995-2998 ◽  
Author(s):  
Gabriele Neumann ◽  
Thomas W. Geisbert ◽  
Hideki Ebihara ◽  
Joan B. Geisbert ◽  
Kathleen M. Daddario-DiCaprio ◽  
...  
Keyword(s):  
Virus Replication ◽  
Nonhuman Primates ◽  
Ebola Virus ◽  
Proteolytic Processing ◽  
Surface Glycoprotein ◽  
Wild Type Virus ◽  
Virus Infectivity ◽  
Recognition Sequence ◽  
Furin Cleavage ◽  
Virus Glycoprotein

ABSTRACT Enveloped viruses often require cleavage of a surface glycoprotein by a cellular endoprotease such as furin for infectivity and virulence. Previously, we showed that Ebola virus glycoprotein does not require the furin cleavage motif for virus replication in cell culture. Here, we show that there are no appreciable differences in disease progression, hematology, serum biochemistry, virus titers, or lethality in nonhuman primates infected with an Ebola virus lacking the furin recognition sequence compared to those infected with wild-type virus. We conclude that glycoprotein cleavage by subtilisin-like endoproteases is not critical for Ebola virus infectivity and virulence in nonhuman primates.

scholarly journals Identification of Protective Epitopes on Ebola Virus Glycoprotein at the Single Amino Acid Level by Using Recombinant Vesicular Stomatitis Viruses

2003 ◽  
Vol 77 (2) ◽  
pp. 1069-1074 ◽  
Author(s):  
Ayato Takada ◽  
Heinz Feldmann ◽  
Ute Stroeher ◽  
Mike Bray ◽  
Shinji Watanabe ◽  
...  
Keyword(s):  
Amino Acid ◽  
Virus Infection ◽  
Ebola Virus ◽  
Hemorrhagic Fever ◽  
Passive Transfer ◽  
Single Amino Acid ◽  
Virus Glycoprotein ◽  
Vesicular Stomatitis ◽  
Encoding Gene ◽  
Ebola Virus Infection

ABSTRACT Ebola virus causes lethal hemorrhagic fever in humans, but currently there are no effective vaccines or antiviral compounds for this infectious disease. Passive transfer of monoclonal antibodies (MAbs) protects mice from lethal Ebola virus infection (J. A. Wilson, M. Hevey, R. Bakken, S. Guest, M. Bray, A. L. Schmaljohn, and M. K. Hart, Science 287:1664-1666, 2000). However, the epitopes responsible for neutralization have been only partially characterized because some of the MAbs do not recognize the short synthetic peptides used for epitope mapping. To identify the amino acids recognized by neutralizing and protective antibodies, we generated a recombinant vesicular stomatitis virus (VSV) containing the Ebola virus glycoprotein-encoding gene instead of the VSV G protein-encoding gene and used it to select escape variants by growing it in the presence of a MAb (133/3.16 or 226/8.1) that neutralizes the infectivity of the virus. All three variants selected by MAb 133/3.16 contained a single amino acid substitution at amino acid position 549 in the GP2 subunit. By contrast, MAb 226/8.1 selected three different variants containing substitutions at positions 134, 194, and 199 in the GP1 subunit, suggesting that this antibody recognized a conformational epitope. Passive transfer of each of these MAbs completely protected mice from a lethal Ebola virus infection. These data indicate that neutralizing antibody cocktails for passive prophylaxis and therapy of Ebola hemorrhagic fever can reduce the possibility of the emergence of antigenic variants in infected individuals.

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