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Measuring the strength of interaction between the Ebola fusion peptide and lipid rafts: implications for membrane fusion and virus infection.

Freitas MS, Follmer C, Costa LT, Vilani C, Bianconi ML, Achete CA, Silva JL - PLoS ONE (2011)

Bottom Line: The depletion of cholesterol from living mammalian cells reduced the ability of EBO₁₆ to induce lipid mixing.Our study is the first to show a strong correlation between the initial step of Ebola virus infection and cholesterol, thus providing a rationale for Ebola virus proteins being co-localized with lipid-raft domains.Such features suggest these processes are excellent targets for therapeutic and vaccine approaches to viral diseases.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Instituto de Bioquímica Médica, Instituto Nacional de Ciências e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

ABSTRACT
The Ebola fusion peptide (EBO₁₆) is a hydrophobic domain that belongs to the GP2 membrane fusion protein of the Ebola virus. It adopts a helical structure in the presence of mimetic membranes that is stabilized by the presence of an aromatic-aromatic interaction established by Trp8 and Phe12. In spite of its infectious cycle becoming better understood recently, several steps still remain unclear, a lacuna that makes it difficult to develop strategies to block infection. In order to gain insight into the mechanism of membrane fusion, we probed the structure, function and energetics of EBO₁₆ and its mutant W8A, in the absence or presence of different lipid membranes, including isolated domain-resistant membranes (DRM), a good experimental model for lipid rafts. The depletion of cholesterol from living mammalian cells reduced the ability of EBO₁₆ to induce lipid mixing. On the other hand, EBO₁₆ was structurally sensitive to interaction with lipid rafts (DRMs), but the same was not observed for W8A mutant. In agreement with these data, W8A showed a poor ability to promote membrane aggregation in comparison to EBO₁₆. Single molecule AFM experiments showed a high affinity force pattern for the interaction of EBO₁₆ and DRM, which seems to be a complex energetic event as observed by the calorimetric profile. Our study is the first to show a strong correlation between the initial step of Ebola virus infection and cholesterol, thus providing a rationale for Ebola virus proteins being co-localized with lipid-raft domains. In all, the results show how small fusion peptide sequences have evolved to adopt highly specific and strong interactions with membrane domains. Such features suggest these processes are excellent targets for therapeutic and vaccine approaches to viral diseases.

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Related in: MedlinePlus

Effect of lipid vesicles in the FTIR spectra of the amide I band of wtBO16 (solid lines) and W8A (dashed lines) at 25°C.Peptides in 100% (A) and 50% (B) DMSO or in the presence of LUVs of different compositions: PC (C), PC∶PE∶PI∶Cho (D), PC∶PE∶SPM∶Cho (E), or in the presence of lipid rafts from Vero cells (F). The peptide concentration was 19.5 mM, and the LUV concentration was 44 mM. The ordinate represents absorption (in arbitrary units).
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pone-0015756-g003: Effect of lipid vesicles in the FTIR spectra of the amide I band of wtBO16 (solid lines) and W8A (dashed lines) at 25°C.Peptides in 100% (A) and 50% (B) DMSO or in the presence of LUVs of different compositions: PC (C), PC∶PE∶PI∶Cho (D), PC∶PE∶SPM∶Cho (E), or in the presence of lipid rafts from Vero cells (F). The peptide concentration was 19.5 mM, and the LUV concentration was 44 mM. The ordinate represents absorption (in arbitrary units).

Mentions: Secondary structures of wt and its mutant W8A in the presence of vesicles were examined using conventional FT-IR spectroscopy. Representative spectra of the amide I band for the peptides in the absence or in the presence of vesicles are shown in Fig. 3. The amide I band consists of the C = O stretching (76%), C-N stretching (14%) and C-C-N deformation (10%) modes and appears in the region from 1600 to 1700 cm−1. This band is highly sensitive to the secondary structure of proteins and serves as an indicator of α-helix, β-sheet, turn and random conformation. Both the wt and W8A peptides diluted in DMSO showed a similar profile, with broad spectra at a maximum around 1665 cm−1 (Fig. 3A). In general, peaks in between 1680 and 1660 are related to turn, suggesting an unfolded structure in the presence of a high amount of DMSO. However, in the presence of 50% DMSO, it was possible to observe a peak that arose at approximately 1625 cm−1, suggesting an increase of b-sheet structure for wtEBO16 that was not observed for W8A (Fig. 3B). The increase in β-sheet structure could be linked to peptide aggregation induced by the contact with water, since a flared spectrum, as observed for W8A, could be correlated to an increase of other structural components.


Measuring the strength of interaction between the Ebola fusion peptide and lipid rafts: implications for membrane fusion and virus infection.

Freitas MS, Follmer C, Costa LT, Vilani C, Bianconi ML, Achete CA, Silva JL - PLoS ONE (2011)

Effect of lipid vesicles in the FTIR spectra of the amide I band of wtBO16 (solid lines) and W8A (dashed lines) at 25°C.Peptides in 100% (A) and 50% (B) DMSO or in the presence of LUVs of different compositions: PC (C), PC∶PE∶PI∶Cho (D), PC∶PE∶SPM∶Cho (E), or in the presence of lipid rafts from Vero cells (F). The peptide concentration was 19.5 mM, and the LUV concentration was 44 mM. The ordinate represents absorption (in arbitrary units).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3020948&req=5

pone-0015756-g003: Effect of lipid vesicles in the FTIR spectra of the amide I band of wtBO16 (solid lines) and W8A (dashed lines) at 25°C.Peptides in 100% (A) and 50% (B) DMSO or in the presence of LUVs of different compositions: PC (C), PC∶PE∶PI∶Cho (D), PC∶PE∶SPM∶Cho (E), or in the presence of lipid rafts from Vero cells (F). The peptide concentration was 19.5 mM, and the LUV concentration was 44 mM. The ordinate represents absorption (in arbitrary units).
Mentions: Secondary structures of wt and its mutant W8A in the presence of vesicles were examined using conventional FT-IR spectroscopy. Representative spectra of the amide I band for the peptides in the absence or in the presence of vesicles are shown in Fig. 3. The amide I band consists of the C = O stretching (76%), C-N stretching (14%) and C-C-N deformation (10%) modes and appears in the region from 1600 to 1700 cm−1. This band is highly sensitive to the secondary structure of proteins and serves as an indicator of α-helix, β-sheet, turn and random conformation. Both the wt and W8A peptides diluted in DMSO showed a similar profile, with broad spectra at a maximum around 1665 cm−1 (Fig. 3A). In general, peaks in between 1680 and 1660 are related to turn, suggesting an unfolded structure in the presence of a high amount of DMSO. However, in the presence of 50% DMSO, it was possible to observe a peak that arose at approximately 1625 cm−1, suggesting an increase of b-sheet structure for wtEBO16 that was not observed for W8A (Fig. 3B). The increase in β-sheet structure could be linked to peptide aggregation induced by the contact with water, since a flared spectrum, as observed for W8A, could be correlated to an increase of other structural components.

Bottom Line: The depletion of cholesterol from living mammalian cells reduced the ability of EBO₁₆ to induce lipid mixing.Our study is the first to show a strong correlation between the initial step of Ebola virus infection and cholesterol, thus providing a rationale for Ebola virus proteins being co-localized with lipid-raft domains.Such features suggest these processes are excellent targets for therapeutic and vaccine approaches to viral diseases.

View Article: PubMed Central - PubMed

Affiliation: Centro Nacional de Ressonância Magnética Nuclear Jiri Jonas, Instituto de Bioquímica Médica, Instituto Nacional de Ciências e Tecnologia de Biologia Estrutural e Bioimagem, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil.

ABSTRACT
The Ebola fusion peptide (EBO₁₆) is a hydrophobic domain that belongs to the GP2 membrane fusion protein of the Ebola virus. It adopts a helical structure in the presence of mimetic membranes that is stabilized by the presence of an aromatic-aromatic interaction established by Trp8 and Phe12. In spite of its infectious cycle becoming better understood recently, several steps still remain unclear, a lacuna that makes it difficult to develop strategies to block infection. In order to gain insight into the mechanism of membrane fusion, we probed the structure, function and energetics of EBO₁₆ and its mutant W8A, in the absence or presence of different lipid membranes, including isolated domain-resistant membranes (DRM), a good experimental model for lipid rafts. The depletion of cholesterol from living mammalian cells reduced the ability of EBO₁₆ to induce lipid mixing. On the other hand, EBO₁₆ was structurally sensitive to interaction with lipid rafts (DRMs), but the same was not observed for W8A mutant. In agreement with these data, W8A showed a poor ability to promote membrane aggregation in comparison to EBO₁₆. Single molecule AFM experiments showed a high affinity force pattern for the interaction of EBO₁₆ and DRM, which seems to be a complex energetic event as observed by the calorimetric profile. Our study is the first to show a strong correlation between the initial step of Ebola virus infection and cholesterol, thus providing a rationale for Ebola virus proteins being co-localized with lipid-raft domains. In all, the results show how small fusion peptide sequences have evolved to adopt highly specific and strong interactions with membrane domains. Such features suggest these processes are excellent targets for therapeutic and vaccine approaches to viral diseases.

Show MeSH
Related in: MedlinePlus