The central structural feature of the membrane fusion protein subunit from the Ebola virus glycoprotein is a long triple-stranded coiled coil - PubMed (original) (raw)

The central structural feature of the membrane fusion protein subunit from the Ebola virus glycoprotein is a long triple-stranded coiled coil

W Weissenhorn et al. Proc Natl Acad Sci U S A. 1998.

Abstract

The ectodomain of the Ebola virus Gp2 glycoprotein was solubilized with a trimeric, isoleucine zipper derived from GCN4 (pIIGCN4) in place of the hydrophobic fusion peptide at the N terminus. This chimeric molecule forms a trimeric, highly alpha-helical, and very thermostable molecule, as determined by chemical crosslinking and circular dichroism. Electron microscopy indicates that Gp2 folds into a rod-like structure like influenza HA2 and HIV-1 gp41, providing further evidence that viral fusion proteins from diverse families such as Orthomyxoviridae (Influenza), Retroviridae (HIV-1), and Filoviridae (Ebola) share common structural features, and suggesting a common membrane fusion mechanism.

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Figures

Figure 1

Figure 1

(a) Primary structure of the Ebola virus Gp. The numbers indicate amino acid positions (including the signal peptide) for (i) potential carbohydrate sites (4), (ii) the cleavage of Gp into Gp1 and Gp2 occurring at position 501 (5, 6); and (iii) Gp2 anchoring in the membrane by amino acids 651–672. The sequence of the expressed construct pIIGp2(552–650) is shown with the a and d heptad positions of pIIGCN4 in-frame with the predicted a and d positions of the Ebola virus TM protein (14). The cysteines (601 and 608) involved in disulfide formation are indicated and those changed to Ser (556 and 609) are indicated by S. (b) Chemical crosslinking of pIIGp2(552–650). Crosslinked products were separated on 15% SDS/PAGE and bands are stained with Coomassie brilliant blue. Lane 1, not reduced; lane 2, reduced; lanes 3–6 with ethyleneglycol bis(-succinimidylsuccinate) crosslinking concentrations of 0.1, 0.5, 2.0, and 5.0 mM. Molecular weight standards are shown. Bands corresponding to monomer, dimer, and trimer are indicated.

Figure 2

Figure 2

(a) CD spectra of pIIGp2(552–650) recorded at 20°C and 95°C, and after one cycle of heating to 95°C and cooling down to 20°C (refolded). (b) Thermal denaturation curve of pIIGp2(552–650) recorded at 222 nm with a scan rate of 1°C per min. (c) Denaturation of pIIGp2(552–650) in guanidine hydrochloride. The CD signal was monitored at 222 nm.

Figure 3

Figure 3

Electron micrographs of pIIGp2(552–650). Diagrammatic representations of the molecules are shown. The average length of the rods is 13 nm.

Figure 4

Figure 4

Electron micrographs of influenza virus TBHA2 (proteolytic fragment of low pH-treated viral HA, HA2 residues 38–175; (20); influenza virus E.TBHA2 (_E. coli_-expressed HA2 residues 38–175; (21); HIV-1 Gp41 (residues 21–166, expressed in insect cells) (22); HIV-1 pIIGpPT (proteolytic fragment derived from E. coli; pIIGCN4 and gp41 residues 30–90 and 107- 167/+12; (23); and Ebola pIIGp2(552–650). Diagrammatic representations of the molecules are shown. Micrographs showing fields of particles are found in refs. –.

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