Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 May 21;52(20):3393-404.
doi: 10.1021/bi400040v. Epub 2013 May 7.

Conformational properties of peptides corresponding to the ebolavirus GP2 membrane-proximal external region in the presence of micelle-forming surfactants and lipids

Lauren K Regula  1 , Richard Harris, Fang Wang, Chelsea D Higgins, Jayne F Koellhoffer, Yue Zhao, Kartik Chandran, Jianmin Gao, Mark E Girvin, Jonathan R Lai
Affiliations

Conformational properties of peptides corresponding to the ebolavirus GP2 membrane-proximal external region in the presence of micelle-forming surfactants and lipids

Lauren K Regula et al. Biochemistry. .

Abstract

Ebola virus and Sudan virus are members of the family Filoviridae of nonsegmented negative-strand RNA viruses ("filoviruses") that cause severe hemorrhagic fever with fatality rates as high as 90%. Infection by filoviruses requires membrane fusion between the host and the virus; this process is facilitated by the two subunits of the envelope glycoprotein, GP1 (the surface subunit) and GP2 (the transmembrane subunit). The membrane-proximal external region (MPER) is a Trp-rich segment that immediately precedes the transmembrane domain of GP2. In the analogous glycoprotein for HIV-1 (gp41), the MPER is critical for membrane fusion and is the target of several neutralizing antibodies. However, the role of the MPER in filovirus GP2 and its importance in membrane fusion have not been established. Here, we characterize the conformational properties of peptides representing the GP MPER segments of Ebola virus and Sudan virus in the presence of micelle-forming surfactants and lipids, at pH 7 and 4.6. Circular dichroism spectroscopy and tryptophan fluorescence indicate that the GP2 MPER peptides bind to micelles of sodium dodecyl sulfate and dodecylphosphocholine (DPC). Nuclear magnetic resonance spectroscopy of the Sudan virus MPER peptide revealed that residues 644-651 interact directly with DPC, and that this interaction enhances the helical conformation of the peptide. The Sudan virus MPER peptide was found to moderately inhibit cell entry by a GP-pseudotyped vesicular stomatitis virus but did not induce leakage of a fluorescent molecule from a large unilammellar vesicle comprised of 1-palmitoyl-2-oleoylphosphatidylcholine or cause hemolysis. Taken together, this analysis suggests the filovirus GP2 MPER binds and inserts shallowly into lipid membranes.

PubMed Disclaimer

Conflict of interest statement

CONFLICT OF INTEREST STATEMENT

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Amino acid alignment of GP2 MPER regions from type members of the five species of genus Ebolavirus (BDBV, Bundbugyo virus; TAFV, Thai Forest virus). Residues that are identical in at least four of the viruses are highlighted in gray (hydrophobic) or boxed (polar). For comparison, the MPER segments of FIV and HIV-1 gp41 are included. Z-MPER and S-MPER peptides correspond the sequences shown here for EBOV and SUDV, respectively. N- and C-termini of peptides were blocked with acetyl and amide groups, respectively.
Figure 2
Figure 2
CD spectra of Z-MPER and S-MPER in various conditions. (A) Peptides with or without 60% TFE. (B – E) Z-MPER titrations with SDS (B and C) or DPC (D and E) pH 4.6 and 7.1. Data were acquired in SDS concentrations up to 10mM in 0.5mM increments, and in DPC concentrations up to 2mM in 0.1mM increments. (F – I) Similar analysis of S-MPER.
Figure 3
Figure 3
CD signal at 200nm and 222nm as a function of [P]:[L]. Relative CD signals were calculated by values at 200 nm and 222 nm for each concentration of SDS/DPC using the following equation: Relative CD Signal = 1−(θ−θminimum)/θmaximum. (A) S-MPER with SDS. (B) S-MPER with DPC. (C) Z-MPER with SDS. (D) Z-MPER with DPC.
Figure 4
Figure 4
Effects of micelle-forming detergents on tryptophan fluorescence. (A) Emission spectra of S-MPER alone and in the presence of 20mM SDS or 20mM DPC. (B and C) Effects of SDS (B) or DPC (C) on emission at 354nm. (D) Change in emission maximum (λem) upon detergent binding for S-MPER.
Figure 5
Figure 5
Nuclear magnetic resonance of S-MPER (A) Chemical shift deviations from random coil shifts for δCα-δCβ. (B) Secondary structure propensity (SSP) scores for S-MPER in H2O (black) and in 200mM DPC micelles (gray) calculated using 13Cα, 13Cβ and 1Hα chemical shifts. Positive values represent helical propensity. (C) Average amide chemical shift differences, {[(Δδ1H)2 + 0.2(Δδ15N)2]/2}1/2, for S-MPER in H2O vs. 200mM DPC micelles. The alternative chemical shift difference for the ambiguous assignment of the amide correlations of Q19/W20 is shown in gray. (D) Effect of a paramagnetic agent on S-MPER in 200mM DPC micelles in D2O for the correlation of the last protonated carbon of each amino acid in the aliphatic 1H,13C HSQC spectrum (except that the Cγ/Hγ and H/C3ε correlations were used for Pro and Trp). The ratio of integrated crosspeak intensities with and without 9mM 5-doxyl stearate is plotted along with the amino acid sequence. Residues where it was not possible to get reliable intensity ratios because of overlapping crosspeaks are marked with *. (E) α-Helical wheel representation of the C-terminal residues (–20) of S-MPER color coded by % intensity loss on addition of 9 mM 5-doxylstearate; >50% brown, 35–50% green, <35% blue.
Figure 6
Figure 6
Biological and membrane-perturbing assays with S-MPER. (A) Inhibition of VSV-GP entry by S-MPER. The potent entry inhibitor Tat-Ebo (ref. 41) was used as a positive control. (B) Control entry assay using VSV-G. (C) Leakage assays with 500 μM POPC LUVs. Experiments were performed twice independently with similar results, a representative data set is shown. (D) Hemolysis assays.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Kuhn JH, Bao Y, Bavari S, Becker S, Bradfute S, Brister JR, Bukreyev AA, Chandran K, Davey RA, Dolnik O, Dye JM, Enterlein S, Hensley LE, Honko AN, Jahrling PB, Johnson KM, Kobinger G, Leroy EM, Lever MS, Mühlberger E, Netesov SV, Olinger GG, Palacios G, Patterson JL, Paweska JT, Pitt L, Radoshitzky SR, Saphire EO, Smither SJ, Swanepoel R, Towner JS, van der Groen G, Volchkov VE, Wahl-Jensen V, Warren TK, Weidmann M, Nichol ST. Virus nomenclature below the species level: a standardized nomenclature for natural variants of viruses assigned to the family Filoviridae. Arch Virol. 2012;155:2083–2103. - PMC - PubMed
    1. Sanchez A, Kiley MP, Holloway BP, Auperin DD. Sequence analysis of the Ebola virus genome: organization, genetic elements, and comparison with the genome of Marburg virus. Virus Res. 1993;29:215–240. - PubMed
    1. Feldmann H, Geisbert TW. Ebola haemorrhagic fever. Lancet. 2011;377:849–862. - PMC - PubMed
    1. Sanchez A, Lukwiya M, Bausch D, Mahanty S, Sanchez AJ, Wagoner KD, Rollin PE. Analysis of human peripheral blood samples from fatal and nonfatal cases of Ebola (Sudan) hemorrhagic fever: cellular responses, virus load, and nitric oxide levels. J Virol. 2004;78:10370–10377. - PMC - PubMed
    1. Sullivan N, Yang ZY, Nabel GJ. Ebola virus pathogenesis: implications for vaccines and therapies. J Virol. 2003;77:9733–9737. - PMC - PubMed

Publication types

MeSH terms