Ebola GP and cytotoxicity

Ebola GP and cytotoxicity - 2000 Nature America Inc.

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886 NATURE MEDICINE • VOLUME 6 • NUMBER 8 • AUGUST 2000 ARTICLES The pathogenicity and high mortality of Ebola virus infection has been noted in many independent outbreaks, 1–3 but the events that lead to the pathogenicity associated with severe disease are un- known. The terminal stages of infection are characterized by fever, hemorrhage and hypotensive shock, with prominent involvement of the reticuloendothelial and mononuclear phagocytic cell sys- tems 4–6 . Recent reports indicate that immune mechanisms may con- tribute to Ebola virus pathogenesis; inflammatory cytokine levels are higher in fatal cases than in infected survivors 7,8 and super- natants containing tumor necrosis factor a from Marburg-infected monocyte cultures increase permeability in human umbilical vein endothelial cell (HUVEC) cultures in vitro 6 . Although the virus ap- parently encodes proteins that induce cytotoxicity in these cells, the specific gene product(s) has not been characterized so far. Among the seven viral gene products, the glycoprotein (GP) is central to the virus life cycle. This protein is synthesized in two forms: one struc- tural protein that is incorporated into the virion and a second that is secreted (sGP), each with distinct biological functions. 9 Virion GP forms trimers, 10,11 binds preferentially to endothelium and has been used to ‘pseudotype’ a mouse retroviral vector that facilitates gene transfer preferentially to these cells. 9 During pseudotyping studies, there was a decrease in virus titer and altered cell morphology in retrovirus producer cells when the level of expression of Ebola GP 3 but not murine leukemia virus envelope glycoprotein, was in- creased. These findings indicated that the Ebola glycoprotein might directly contribute to cellular cytotoxic effects. We compared eukaryotic expression vectors encoding GP or sGP after transfection of 293 human embryonic kidney cells. GP, unlike sGP, substantially altered the phenotype of the cells. Cells rounded and detached within 24 hours after transfection (Fig. 1 a ), and cell death occurred 2–4 days later (data not shown). We mapped the re- gion of GP responsible for this effect using deletion mutations. Several mutant GP proteins retained functional activity, demon- strated by their ability to ‘pseudotype’ a mouse retroviral vector en- coding green fluorescent protein for gene delivery in HUVECs, but two mutants did not cause morphological changes or cellular toxic- ity. One of these GP proteins, GP( muc), was a mutant with an in- ternal deletion in a serine–threonine-rich, mucin-like region, which was expressed and functionally active at levels similar to those of wild-type GP (Fig. 1 b ). Fusion of sGP to a region downstream of the mucin domain (GP 2, ref. 12) also preserved the ability of this glyco- protein to ‘pseudotype,’ but eliminated its cytopathic effects (Fig. 1
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This note was uploaded on 07/12/2011 for the course BIO 620 taught by Professor Hardy during the Spring '11 term at University of Florida.

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Ebola GP and cytotoxicity - 2000 Nature America Inc.

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