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Highly specific inhibition of leukaemia virus membrane fusion by interaction of peptide antagonists with a conserved region of the coiled coil of envelope.

Lamb D, Schüttelkopf AW, van Aalten DM, Brighty DW - Retrovirology (2008)

Bottom Line: Remarkably, despite this conservation, BLV envelope was profoundly resistant to inhibition by HTLV-1-derived LHR-mimetics.Notably, conserved leucine residues are critical to the inhibitory activity of the BLV LHR-based peptides.Homology modeling indicated that hydrophobic residues in the BLV LHR likely make direct contact with a pocket at the membrane-proximal end of the core coiled-coil and disruption of these interactions severely impaired the activity of the BLV inhibitor.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Biomedical Research Centre, College of Medicine, Ninewells Hospital, The University, Dundee, DD1 9SY, Scotland, UK. d.J.Lamb@dundee.ac.uk

ABSTRACT

Background: Human T-cell leukaemia virus (HTLV-1) and bovine leukaemia virus (BLV) entry into cells is mediated by envelope glycoprotein catalyzed membrane fusion and is achieved by folding of the transmembrane glycoprotein (TM) from a rod-like pre-hairpin intermediate to a trimer-of-hairpins. For HTLV-1 and for several virus groups this process is sensitive to inhibition by peptides that mimic the C-terminal alpha-helical region of the trimer-of-hairpins.

Results: We now show that amino acids that are conserved between BLV and HTLV-1 TM tend to map to the hydrophobic groove of the central triple-stranded coiled coil and to the leash and C-terminal alpha-helical region (LHR) of the trimer-of-hairpins. Remarkably, despite this conservation, BLV envelope was profoundly resistant to inhibition by HTLV-1-derived LHR-mimetics. Conversely, a BLV LHR-mimetic peptide antagonized BLV envelope-mediated membrane fusion but failed to inhibit HTLV-1-induced fusion. Notably, conserved leucine residues are critical to the inhibitory activity of the BLV LHR-based peptides. Homology modeling indicated that hydrophobic residues in the BLV LHR likely make direct contact with a pocket at the membrane-proximal end of the core coiled-coil and disruption of these interactions severely impaired the activity of the BLV inhibitor. Finally, the structural predictions assisted the design of a more potent antagonist of BLV membrane fusion.

Conclusion: A conserved region of the HTLV-1 and BLV coiled coil is a target for peptide inhibitors of envelope-mediated membrane fusion and HTLV-1 entry. Nevertheless, the LHR-based inhibitors are highly specific to the virus from which the peptide was derived. We provide a model structure for the BLV LHR and coiled coil, which will facilitate comparative analysis of leukaemia virus TM function and may provide information of value in the development of improved, therapeutically relevant, antagonists of HTLV-1 entry into cells.

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Substitution of a single arginine residue with alanine yields an improved inhibitor. The syncytium inhibition activity of the peptides Pcr-400, PBLV-391 and the derivative peptide PBLV-R403A was examined. The percentage syncytium inhibition following co-incubation of cells with the peptides is shown. Syncytia were counted in 10 low-power light microscope fields. Data points show the mean ± SD of triplicate assays. The asters show the data points for which the p values were calculated (see main text).
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Figure 6: Substitution of a single arginine residue with alanine yields an improved inhibitor. The syncytium inhibition activity of the peptides Pcr-400, PBLV-391 and the derivative peptide PBLV-R403A was examined. The percentage syncytium inhibition following co-incubation of cells with the peptides is shown. Syncytia were counted in 10 low-power light microscope fields. Data points show the mean ± SD of triplicate assays. The asters show the data points for which the p values were calculated (see main text).

Mentions: The accumulated experimental data correlate well with the structural model, implying that predications based on the BLV trimer-of-hairpins model are likely to be informative. The homology model of the BLV TM ectodomain (Figure 6) suggests that Arg403, a residue within the predicted α-helix of the LHR and mimicked by PBLV-391 peptide, may be electrostatically unfavourable for efficient binding of the C-terminal LHR into the groove of the core coiled-coil. We predicted that removing this unfavourable charge interaction would improve the binding of the peptide to the BLV coiled coil and thereby improve the inhibitory activity of the peptide. We therefore synthesized a peptide, PBLV-R403A, which incorporated an alanine residue in place of the arginine equivalent to Arg403 of Env (Table 1). As anticipated, substitution of the arginine residue resulted in a modest but highly consistent and significant (p < 0.0001, Student's t-test) improvement in peptide potency when compared to PBLV-391. The peptide PBLV-R403A is more than twice as potent as PBLV-391 in syncytium interference assays, with a calculated IC50 of 1.56 ± 0.05 μM compared to 3.49 μM ± 0.03 μM for PBLV-391 (Figure 6). The data show that a single amino-acid substitution in the predicted short α-helix of the LHR-mimetic peptide increases the ability of the peptide to block membrane fusion and provides further support for the utility of the model of the BLV TM core.


Highly specific inhibition of leukaemia virus membrane fusion by interaction of peptide antagonists with a conserved region of the coiled coil of envelope.

Lamb D, Schüttelkopf AW, van Aalten DM, Brighty DW - Retrovirology (2008)

Substitution of a single arginine residue with alanine yields an improved inhibitor. The syncytium inhibition activity of the peptides Pcr-400, PBLV-391 and the derivative peptide PBLV-R403A was examined. The percentage syncytium inhibition following co-incubation of cells with the peptides is shown. Syncytia were counted in 10 low-power light microscope fields. Data points show the mean ± SD of triplicate assays. The asters show the data points for which the p values were calculated (see main text).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2533354&req=5

Figure 6: Substitution of a single arginine residue with alanine yields an improved inhibitor. The syncytium inhibition activity of the peptides Pcr-400, PBLV-391 and the derivative peptide PBLV-R403A was examined. The percentage syncytium inhibition following co-incubation of cells with the peptides is shown. Syncytia were counted in 10 low-power light microscope fields. Data points show the mean ± SD of triplicate assays. The asters show the data points for which the p values were calculated (see main text).
Mentions: The accumulated experimental data correlate well with the structural model, implying that predications based on the BLV trimer-of-hairpins model are likely to be informative. The homology model of the BLV TM ectodomain (Figure 6) suggests that Arg403, a residue within the predicted α-helix of the LHR and mimicked by PBLV-391 peptide, may be electrostatically unfavourable for efficient binding of the C-terminal LHR into the groove of the core coiled-coil. We predicted that removing this unfavourable charge interaction would improve the binding of the peptide to the BLV coiled coil and thereby improve the inhibitory activity of the peptide. We therefore synthesized a peptide, PBLV-R403A, which incorporated an alanine residue in place of the arginine equivalent to Arg403 of Env (Table 1). As anticipated, substitution of the arginine residue resulted in a modest but highly consistent and significant (p < 0.0001, Student's t-test) improvement in peptide potency when compared to PBLV-391. The peptide PBLV-R403A is more than twice as potent as PBLV-391 in syncytium interference assays, with a calculated IC50 of 1.56 ± 0.05 μM compared to 3.49 μM ± 0.03 μM for PBLV-391 (Figure 6). The data show that a single amino-acid substitution in the predicted short α-helix of the LHR-mimetic peptide increases the ability of the peptide to block membrane fusion and provides further support for the utility of the model of the BLV TM core.

Bottom Line: Remarkably, despite this conservation, BLV envelope was profoundly resistant to inhibition by HTLV-1-derived LHR-mimetics.Notably, conserved leucine residues are critical to the inhibitory activity of the BLV LHR-based peptides.Homology modeling indicated that hydrophobic residues in the BLV LHR likely make direct contact with a pocket at the membrane-proximal end of the core coiled-coil and disruption of these interactions severely impaired the activity of the BLV inhibitor.

View Article: PubMed Central - HTML - PubMed

Affiliation: The Biomedical Research Centre, College of Medicine, Ninewells Hospital, The University, Dundee, DD1 9SY, Scotland, UK. d.J.Lamb@dundee.ac.uk

ABSTRACT

Background: Human T-cell leukaemia virus (HTLV-1) and bovine leukaemia virus (BLV) entry into cells is mediated by envelope glycoprotein catalyzed membrane fusion and is achieved by folding of the transmembrane glycoprotein (TM) from a rod-like pre-hairpin intermediate to a trimer-of-hairpins. For HTLV-1 and for several virus groups this process is sensitive to inhibition by peptides that mimic the C-terminal alpha-helical region of the trimer-of-hairpins.

Results: We now show that amino acids that are conserved between BLV and HTLV-1 TM tend to map to the hydrophobic groove of the central triple-stranded coiled coil and to the leash and C-terminal alpha-helical region (LHR) of the trimer-of-hairpins. Remarkably, despite this conservation, BLV envelope was profoundly resistant to inhibition by HTLV-1-derived LHR-mimetics. Conversely, a BLV LHR-mimetic peptide antagonized BLV envelope-mediated membrane fusion but failed to inhibit HTLV-1-induced fusion. Notably, conserved leucine residues are critical to the inhibitory activity of the BLV LHR-based peptides. Homology modeling indicated that hydrophobic residues in the BLV LHR likely make direct contact with a pocket at the membrane-proximal end of the core coiled-coil and disruption of these interactions severely impaired the activity of the BLV inhibitor. Finally, the structural predictions assisted the design of a more potent antagonist of BLV membrane fusion.

Conclusion: A conserved region of the HTLV-1 and BLV coiled coil is a target for peptide inhibitors of envelope-mediated membrane fusion and HTLV-1 entry. Nevertheless, the LHR-based inhibitors are highly specific to the virus from which the peptide was derived. We provide a model structure for the BLV LHR and coiled coil, which will facilitate comparative analysis of leukaemia virus TM function and may provide information of value in the development of improved, therapeutically relevant, antagonists of HTLV-1 entry into cells.

Show MeSH
Related in: MedlinePlus