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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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The specificity of peptide inhibitors of Envelope-mediated membrane fusion is limited to the parental virus. HeLa cells expressing HTLV-1 (A) or BLV (B) envelope were used as effector cells and co-cultured with untransfected HeLa cells. Cells were incubated in the presence of the peptides Pcr-400, PBLV-391, Pcr-400 L/A a non-functional derivative of Pcr-400 [20], or the control HIV C helix mimetic peptide C34 [51]. (C) Syncytia formation between BLV infected FLK cells and non-infected HeLa cells. Syncytia were counted in 10 low-power light microscope fields. Data points show the mean ± SD of triplicate assays.
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Figure 3: The specificity of peptide inhibitors of Envelope-mediated membrane fusion is limited to the parental virus. HeLa cells expressing HTLV-1 (A) or BLV (B) envelope were used as effector cells and co-cultured with untransfected HeLa cells. Cells were incubated in the presence of the peptides Pcr-400, PBLV-391, Pcr-400 L/A a non-functional derivative of Pcr-400 [20], or the control HIV C helix mimetic peptide C34 [51]. (C) Syncytia formation between BLV infected FLK cells and non-infected HeLa cells. Syncytia were counted in 10 low-power light microscope fields. Data points show the mean ± SD of triplicate assays.

Mentions: Both HTLV-1 and BLV envelope induced widespread syncytium formation in cultures incubated in the absence of peptide inhibitors or in the presence of inactive control peptides (Figure 3A, B). However, in keeping with previous studies [20-22], HTLV envelope-mediated syncytium formation was robustly blocked in a dose-dependent manner by Pcr-400 with an IC50 of 0.28 ± 0.01 μM (Figure 3A). However, despite the marked conservation of amino acid sequence between the LHRs and coiled coils of HTLV-1 and BLV, Pcr-400 failed to inhibit membrane fusion induced by BLV envelope even at concentrations up to 15 μM (Figure 3B) and above (data not shown). Also, like the inactive control peptides, the BLV LHR-mimetic peptide at concentrations up to 20 μM (Figure 3A) and above (data not shown) failed to inhibit membrane fusion induced by HTLV-1 envelope. By contrast, the peptide PBLV-391 specifically antagonized BLV envelope-mediated membrane fusion (Figure 3B) with a calculated IC50 of 3.49 ± 0.03 μM; control peptides including C34 and Pcr-400 L/A did not interfere with BLV Env-induced membrane fusion (Figure 3B). In addition, PBLV-391 robustly antagonized membrane fusion induced by virally expressed envelope as shown by the inhibition of syncytium formation between chronically BLV infected FLK cells and target cells (Figure 3C); whereas, the HTLV-1 peptide inhibitor did not block BLV-induced membrane fusion. Thus, it appears that the inhibitory properties of the LHR-mimetic peptides are highly specific to the virus from which they were derived.


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)

The specificity of peptide inhibitors of Envelope-mediated membrane fusion is limited to the parental virus. HeLa cells expressing HTLV-1 (A) or BLV (B) envelope were used as effector cells and co-cultured with untransfected HeLa cells. Cells were incubated in the presence of the peptides Pcr-400, PBLV-391, Pcr-400 L/A a non-functional derivative of Pcr-400 [20], or the control HIV C helix mimetic peptide C34 [51]. (C) Syncytia formation between BLV infected FLK cells and non-infected HeLa cells. Syncytia were counted in 10 low-power light microscope fields. Data points show the mean ± SD of triplicate assays.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: The specificity of peptide inhibitors of Envelope-mediated membrane fusion is limited to the parental virus. HeLa cells expressing HTLV-1 (A) or BLV (B) envelope were used as effector cells and co-cultured with untransfected HeLa cells. Cells were incubated in the presence of the peptides Pcr-400, PBLV-391, Pcr-400 L/A a non-functional derivative of Pcr-400 [20], or the control HIV C helix mimetic peptide C34 [51]. (C) Syncytia formation between BLV infected FLK cells and non-infected HeLa cells. Syncytia were counted in 10 low-power light microscope fields. Data points show the mean ± SD of triplicate assays.
Mentions: Both HTLV-1 and BLV envelope induced widespread syncytium formation in cultures incubated in the absence of peptide inhibitors or in the presence of inactive control peptides (Figure 3A, B). However, in keeping with previous studies [20-22], HTLV envelope-mediated syncytium formation was robustly blocked in a dose-dependent manner by Pcr-400 with an IC50 of 0.28 ± 0.01 μM (Figure 3A). However, despite the marked conservation of amino acid sequence between the LHRs and coiled coils of HTLV-1 and BLV, Pcr-400 failed to inhibit membrane fusion induced by BLV envelope even at concentrations up to 15 μM (Figure 3B) and above (data not shown). Also, like the inactive control peptides, the BLV LHR-mimetic peptide at concentrations up to 20 μM (Figure 3A) and above (data not shown) failed to inhibit membrane fusion induced by HTLV-1 envelope. By contrast, the peptide PBLV-391 specifically antagonized BLV envelope-mediated membrane fusion (Figure 3B) with a calculated IC50 of 3.49 ± 0.03 μM; control peptides including C34 and Pcr-400 L/A did not interfere with BLV Env-induced membrane fusion (Figure 3B). In addition, PBLV-391 robustly antagonized membrane fusion induced by virally expressed envelope as shown by the inhibition of syncytium formation between chronically BLV infected FLK cells and target cells (Figure 3C); whereas, the HTLV-1 peptide inhibitor did not block BLV-induced membrane fusion. Thus, it appears that the inhibitory properties of the LHR-mimetic peptides are highly specific to the virus from which they were derived.

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