Phosphatidylinositol-Dependent Membrane Fusion Induced by a Putative Fusogenic Sequence of Ebola Virus |
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| Authors: | M. Bego?a Ruiz-Argüello Félix M. Go?i Francisca B. Pereira José L. Nieva |
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| Affiliation: | Grupo de Biomembranas (Unidad Asociada al CSIC), Departamento de Bioquímica, Universidad del País Vasco, 48080 Bilbao, Spain |
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| Abstract: | The membrane-interacting abilities of three sequences representing the putative fusogenic subdomain of the Ebola virus transmembrane protein have been investigated. In the presence of calcium, the sequence EBOGE (GAAIGLAWIPYFGPAAE) efficiently fused unilamellar vesicles composed of phosphatidylcholine, phosphatidylethanolamine, cholesterol, and phosphatidylinositol (molar ratio, 2:1:1:0.5), a mixture that roughly resembles the lipid composition of the hepatocyte plasma membrane. Analysis of the lipid dependence of the process demonstrated that the fusion activity of EBOGE was promoted by phosphatidylinositol but not by other acidic phospholipids. In comparison, EBOEA (EGAAIGLAWIPYFGPAA) and EBOEE (EGAAIGLAWIPYFGPAAE) sequences, which are similar to EBOGE except that they bear the negatively charged glutamate residue at the N terminus and at both the N and C termini, respectively, induced fusion to a lesser extent. As revealed by binding experiments, the glutamate residue at the N terminus severely impaired peptide-vesicle interaction. In addition, the fusion-competent EBOGE sequence did not associate significantly with vesicles lacking phosphatidylinositol. Tryptophan fluorescence quenching by vesicles containing brominated phospholipids indicated that the EBOGE peptide penetrated to the acyl chain level only when the membranes contained phosphatidylinositol. We conclude that binding and further penetration of the Ebola virus putative fusion peptide into membranes might be governed by the nature of the N-terminal residue and by the presence of phosphatidylinositol in the target membrane. Moreover, since insertion of such a peptide leads to membrane destabilization and fusion, the present data would be compatible with the involvement of this sequence in Ebola virus fusion.Ebola virus belongs to the Filoviridae family (23). This human pathogen occasionally causes epidemics of African hemorrhagic fever with a high rate of mortality (8, 23, 37). Little is known about the viral infectivity mechanism, and there is no specific treatment for Ebola virus hemorrhagic fever as yet. The most prominent pathology of Ebola virus infection includes necrosis of liver parenchyma as a direct consequence of virus replication (23). Ebola virus virions are composed of a helical nucleocapsid containing one linear, negative-sense, single-stranded RNA and surrounded by a lipidic envelope derived from the host cell plasma membrane (8, 23). The envelope contains solely one type of highly glycosylated protein (Ebola GP) arranged into oligomers, most probably trimers, which constitute the spikes that protrude from the virion surface (8, 30, 38, 39).The mode of entry of Ebola virus into target cells remains unknown. However it seems likely that the single surface protein Ebola GP is responsible for both receptor binding and membrane fusion during entry into the host cells. Homology analysis of its coding gene-derived sequence has identified several structural features that Ebola GP shares with other envelope fusion proteins derived from oncogenic retroviruses (12, 39). Just recently a detailed analysis has detected a high degree of structural homology between Ebola GP and the Rous sarcoma virus transmembrane protein (12). Several structural elements that might be involved in the ectodomain fusogenic function are shared by these viruses. In particular, there exists in both viruses an amino acid region bounded by cysteines that has at its center a sequence of approximately 16 uncharged and hydrophobic residues. Its location with respect to the viral membrane, the presence of a canonical fusion tripeptide (YFG in Ebola virus), and the fact that this sequence exhibits a high degree of identity among the Filoviridae members suggest that this region might constitute in Ebola virus the fusion peptide that is critical for virion-membrane fusion in the Retroviridae and other families (11, 40, 41).According to the most widely accepted mechanistic model proposed for the initial phase of the viral fusion process, activation of the viral spikes induces the exposure of previously buried hydrophobic fusion peptides in the vicinity of the target cell (5, 43). Further interaction of the viral fusion peptides with the cell membrane would depend mainly on the capacity for binding of these peptides to the membrane lipid components and could eventually trigger the process that brings about the actual merging of the viral and cell membranes via a currently unknown mechanism (41). This fact has justified the development of in vitro studies on the membrane-destabilizing effects of fusion peptides by using representative synthetic peptides of different viruses and model membranes (7, 15, 19, 29).The membrane environment into which the fusion peptide should partition obviously plays an important role in the process. Previous work from this laboratory has focused on the effect of the target membrane composition on viral fusion. Reports from this and other laboratories indicate the existence of conformational changes induced by lipidic components in the membrane-bound human immunodeficiency virus type 1 (HIV-1) fusion peptide (25, 28, 29), and we have identified a fusogenic conformation of the peptide represented by an extended β-type structure (25, 26, 28). The fusogenic interaction of the HIV-1 fusion peptide is, moreover, sensitive to factors that affect gp41 activity in vivo (27). Modulation of viral fusion by lipids has also been observed for complete virions and reconstituted systems fusing with model membranes (6, 24, 42). These observations indicate that enveloped viruses may optimize host interactions during the entry process, not only at the level of the selective binding to cell receptors but also at the level of the envelope fusion and subsequent capsid penetration.Our primary objective in this study was to confirm that the proposed fusogenic sequence for Ebola virus might interact with membranes, destabilize them, and eventually induce fusion. Because Ebola virus infects and replicates very efficiently in the liver, we initially employed as target membranes large unilamellar vesicles (LUV) made of a lipidic mixture that represents the hepatocyte plasma membrane composition (18). Our results demonstrate that this Ebola virus peptide interacts with phosphatidylinositol (PI)-containing membranes and induces vesicle fusion. Moreover, we show that the sequence lacking the negatively charged Glu residue at the N terminus interacts more efficiently with membranes. These data suggest that, similarly to the HIV-1 fusion peptide (26–28), the Ebola virus peptide segment under study may be important in viral fusion in vivo. |
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