Ebola Virus Glycoprotein Toxicity Is Mediated by a Dynamin-Dependent Protein-Trafficking Pathway

Vaccine Research Center, National Institute for Allergy and Infectious Disease, National Institutes of Health, Bethesda, Maryland 20814, USA.
Journal of Virology (Impact Factor: 4.44). 02/2005; 79(1):547-53. DOI: 10.1128/JVI.79.1.547-553.2005
Source: PubMed


Ebola virus infection causes a highly lethal hemorrhagic fever syndrome associated with profound immunosuppression through
its ability to induce widespread inflammation and cellular damage. Though GP, the viral envelope glycoprotein, mediates many
of these effects, the molecular events that underlie Ebola virus cytopathicity are poorly understood. Here, we define a cellular
mechanism responsible for Ebola virus GP cytotoxicity. GP selectively decreased the expression of cell surface molecules that
are essential for cell adhesion and immune function. GP dramatically reduced levels of αVβ3 without affecting the levels of
α2β1 or cadherin, leading to cell detachment and death. This effect was inhibited in vitro and in vivo by brefeldin A and
was dependent on dynamin, the GTPase. GP also decreased cell surface expression of major histocompatibility complex class
I molecules, which alters recognition by immune cells, and this effect was also dependent on the mucin domain previously implicated
in GP cytotoxicity. By altering the trafficking of select cellular proteins, Ebola virus GP inflicts cell damage and may facilitate
immune escape by the virus.

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    • "The present study further indicated that this GP function was common in filoviruses, including MARV. It is well documented that the MLR of EBOV GP plays a critical role in the morphological changes of GP-expressing cells, likely caused by steric shielding effects (Francica et al., 2009; Simmons et al., 2002; Sullivan et al., 2005; Takada et al., 2000; Yang et al., 2000). It was also reported that the MLR and sugar chains on the GP molecule were important for epitope shielding, suggesting that the MLR plays a crucial role in the steric shielding effect (Francica et al. 2010; Reynard et al., 2009). "
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    ABSTRACT: The viral envelope glycoprotein (GP) is thought to play important roles in the pathogenesis of filovirus infection. It is known that GP expressed on the cell surface forms a steric shield over host proteins such as major histocompatibility complex class I and integrin β1, which may result in the disorder of cell-to-cell contacts and/or inhibition of the immune response. However, it is not clarified whether this phenomenon contributes to the pathogenicity of filoviruses. In this study, we found that the steric shielding efficiency differed among filovirus strains and was correlated with the difference in their relative pathogenicities. While the highly glycosylated mucin-like region of GP was indispensable, the differential shielding efficiency did not necessarily depend on the primary structure of the mucin-like region, suggesting the importance of the overall properties (e.g., flexibility and stability) of the GP molecule for efficient shielding of host proteins.
    Preview · Article · Nov 2013 · Virology
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    • "Transient expression of GP in cultured mammalian cells causes cytopathic effects including cell rounding and detachment (Chan et al., 2000; Simmons et al., 2002). GP can down-regulate several cell surface molecules such as major histocompatibility complex class I (Sullivan et al., 2005) and integrin (Simmons et al., 2002). As Ebola GP is the protein that mediates infection and is the major antigen (Wilson et al., 2000), it is a critical target for vaccine development. "
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    ABSTRACT: Filoviruses (Ebola and Marburg viruses) cause severe and often fatal haemorrhagic fever in humans and non-human primates. The US Centers for Disease Control identifies Ebola and Marburg viruses as 'category A' pathogens (defined as posing a risk to national security as bioterrorism agents), which has lead to a search for vaccines that could prevent the disease. Because the use of such vaccines would be in the service of public health, the cost of production is an important component of their development. The use of plant biotechnology is one possible way to cost-effectively produce subunit vaccines. In this work, a geminiviral replicon system was used to produce an Ebola immune complex (EIC) in Nicotiana benthamiana. Ebola glycoprotein (GP1) was fused at the C-terminus of the heavy chain of humanized 6D8 IgG monoclonal antibody, which specifically binds to a linear epitope on GP1. Co-expression of the GP1-heavy chain fusion and the 6D8 light chain using a geminiviral vector in leaves of N. benthamiana produced assembled immunoglobulin, which was purified by ammonium sulphate precipitation and protein G affinity chromatography. Immune complex formation was confirmed by assays to show that the recombinant protein bound the complement factor C1q. Size measurements of purified recombinant protein by dynamic light scattering and size-exclusion chromatography also indicated complex formation. Subcutaneous immunization of BALB/C mice with purified EIC resulted in anti-Ebola virus antibody production at levels comparable to those obtained with a GP1 virus-like particle. These results show excellent potential for a plant-expressed EIC as a human vaccine.
    Full-text · Article · Feb 2011 · Plant Biotechnology Journal
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    • "As the majority of studies suggest a critical role of Ebola GP in causing cytotoxicity [3], [4], [8], [5], [23], [24], and GP interacts with VP40 and NP to form viral particles [13], [14], [15], we therefore investigated the cellular localization of GP, VP40 and NP when transiently expressed in HEK293T cells. Since Ebola GP induces cell rounding and detachment 24 hours after transfection [8], the cellular localization of Ebola GP was examined here 24 hours after transient transfection to try gain insight into the mechanism of GP cytotoxicity. "
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    ABSTRACT: The Filoviridae family comprises of Ebola and Marburg viruses, which are known to cause lethal hemorrhagic fever. However, there is no effective anti-viral therapy or licensed vaccines currently available for these human pathogens. The envelope glycoprotein (GP) of Ebola virus, which mediates entry into target cells, is cytotoxic and this effect maps to a highly glycosylated mucin-like region in the surface subunit of GP (GP1). However, the mechanism underlying this cytotoxic property of GP is unknown. To gain insight into the basis of this GP-induced cytotoxicity, HEK293T cells were transiently transfected with full-length and mucin-deleted (Δmucin) Ebola GP plasmids and GP localization was examined relative to the nucleus, endoplasmic reticulum (ER), Golgi, early and late endosomes using deconvolution fluorescent microscopy. Full-length Ebola GP was observed to accumulate in the ER. In contrast, GPΔmucin was uniformly expressed throughout the cell and did not localize in the ER. The Ebola major matrix protein VP40 was also co-expressed with GP to investigate its influence on GP localization. GP and VP40 co-expression did not alter GP localization to the ER. Also, when VP40 was co-expressed with the nucleoprotein (NP), it localized to the plasma membrane while NP accumulated in distinct cytoplasmic structures lined with vimentin. These latter structures are consistent with aggresomes and may serve as assembly sites for filoviral nucleocapsids. Collectively, these data suggest that full-length GP, but not GPΔmucin, accumulates in the ER in close proximity to the nuclear membrane, which may underscore its cytotoxic property.
    Full-text · Article · Jan 2011 · Virology Journal
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