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Viroporin potential of the lentivirus lytic peptide (LLP) domains of the HIV-1 gp41 protein.

Costin JM, Rausch JM, Garry RF, Wimley WC - Virol. J. (2007)

Bottom Line: Mechanisms by which HIV-1 mediates reductions in CD4+ cell levels in infected persons are being intensely investigated, and have broad implications for AIDS drug and vaccine development.These results suggest that the C-terminal domains of HIV-1 Env proteins may form an ion channel, or viroporin.Increased understanding of the function of LLP domains and their role in the viral replication cycle could allow for the development of novel HIV drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biotechnology Research Group, Department of Biology, Florida Gulf Coast University, 10501 FGCU Blvd. S., Fort Myers, FL 33965, USA. jcostin@fgcu.edu

ABSTRACT

Background: Mechanisms by which HIV-1 mediates reductions in CD4+ cell levels in infected persons are being intensely investigated, and have broad implications for AIDS drug and vaccine development. Virally induced changes in membrane ionic permeability induced by lytic viruses of many families contribute to cytopathogenesis. HIV-1 induces disturbances in plasma membrane ion transport. The carboxyl terminus of TM (gp41) contains potential amphipathic alpha-helical motifs identified through their structural similarities to naturally occurring cytolytic peptides. These sequences have been dubbed lentiviral lytic peptides (LLP) -1, -2, and -3.

Results: Peptides corresponding to the LLP domains (from a clade B virus) partition into lipid membranes, fold into alpha-helices and disrupt model membrane permeability. A peptide corresponding to the LLP-1 domain of a clade D HIV-1 virus, LLP-1D displayed similar activity to the LLP-1 domain of the clade B virus in all assays, despite a lack of amino acid sequence identity.

Conclusion: These results suggest that the C-terminal domains of HIV-1 Env proteins may form an ion channel, or viroporin. Increased understanding of the function of LLP domains and their role in the viral replication cycle could allow for the development of novel HIV drugs.

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Related in: MedlinePlus

LLP peptides disrupt lipid membranes. Tb3+/DPA assay for peptide induced membrane permeation. Disruption of 90%POPC:10%POPG large unilamellar vesicles (LUV's) containing entrapped terbium and external DPA by LLP peptides is indicated by green Tb/DPA fluorescence under UV illumination.
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Figure 4: LLP peptides disrupt lipid membranes. Tb3+/DPA assay for peptide induced membrane permeation. Disruption of 90%POPC:10%POPG large unilamellar vesicles (LUV's) containing entrapped terbium and external DPA by LLP peptides is indicated by green Tb/DPA fluorescence under UV illumination.

Mentions: The Tb3+/DPA assay, a high throughput leakage assay developed by Rausch and Wimley, 2001, was employed in order to determine each LLP peptide's ability to disturb lipid membrane integrity. This technique relies upon the greatly increased fluorescence emission that occurs when the lanthanide metal terbium interacts with the aromatic chelator dipicholinic acid (DPA). To this end, Tb3+ was entrapped in Large Unilamellar Vesicles composed of 10% POPG: 90% POPC diluted in a KPO4 buffer containing DPA. Only upon membrane disruption was terbium able to come into contact with DPA in the buffer generating a fluorescent complex. Various ratios of peptide:lipid were incubated together in a microwell plate and the resulting fluorescence emissions were monitored under UV irradiation. LLP -1, -2, and -1D peptides used in this study were not NBD labeled, but were N-terminally labeled with a tryptophan residue for quantification. The results are presented in Figure 4. LLP-1 and -2 disrupted LUV's at very low peptide:lipid ratios of approximately 1:1,300. Roughly 20 and 32 times as much peptide was required to induce leakage from vesicles with LLP-3 and LLP-1D respectively. Complete dissolution of membranes with Triton X-100 shows 100% leakage of vesicles versus virtually no leakage with distilled water. The known pore forming antibiotic alamethacin was used as an additional positive control and produced similar results in the assay as Triton X-100 (data not shown).


Viroporin potential of the lentivirus lytic peptide (LLP) domains of the HIV-1 gp41 protein.

Costin JM, Rausch JM, Garry RF, Wimley WC - Virol. J. (2007)

LLP peptides disrupt lipid membranes. Tb3+/DPA assay for peptide induced membrane permeation. Disruption of 90%POPC:10%POPG large unilamellar vesicles (LUV's) containing entrapped terbium and external DPA by LLP peptides is indicated by green Tb/DPA fluorescence under UV illumination.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2211469&req=5

Figure 4: LLP peptides disrupt lipid membranes. Tb3+/DPA assay for peptide induced membrane permeation. Disruption of 90%POPC:10%POPG large unilamellar vesicles (LUV's) containing entrapped terbium and external DPA by LLP peptides is indicated by green Tb/DPA fluorescence under UV illumination.
Mentions: The Tb3+/DPA assay, a high throughput leakage assay developed by Rausch and Wimley, 2001, was employed in order to determine each LLP peptide's ability to disturb lipid membrane integrity. This technique relies upon the greatly increased fluorescence emission that occurs when the lanthanide metal terbium interacts with the aromatic chelator dipicholinic acid (DPA). To this end, Tb3+ was entrapped in Large Unilamellar Vesicles composed of 10% POPG: 90% POPC diluted in a KPO4 buffer containing DPA. Only upon membrane disruption was terbium able to come into contact with DPA in the buffer generating a fluorescent complex. Various ratios of peptide:lipid were incubated together in a microwell plate and the resulting fluorescence emissions were monitored under UV irradiation. LLP -1, -2, and -1D peptides used in this study were not NBD labeled, but were N-terminally labeled with a tryptophan residue for quantification. The results are presented in Figure 4. LLP-1 and -2 disrupted LUV's at very low peptide:lipid ratios of approximately 1:1,300. Roughly 20 and 32 times as much peptide was required to induce leakage from vesicles with LLP-3 and LLP-1D respectively. Complete dissolution of membranes with Triton X-100 shows 100% leakage of vesicles versus virtually no leakage with distilled water. The known pore forming antibiotic alamethacin was used as an additional positive control and produced similar results in the assay as Triton X-100 (data not shown).

Bottom Line: Mechanisms by which HIV-1 mediates reductions in CD4+ cell levels in infected persons are being intensely investigated, and have broad implications for AIDS drug and vaccine development.These results suggest that the C-terminal domains of HIV-1 Env proteins may form an ion channel, or viroporin.Increased understanding of the function of LLP domains and their role in the viral replication cycle could allow for the development of novel HIV drugs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Biotechnology Research Group, Department of Biology, Florida Gulf Coast University, 10501 FGCU Blvd. S., Fort Myers, FL 33965, USA. jcostin@fgcu.edu

ABSTRACT

Background: Mechanisms by which HIV-1 mediates reductions in CD4+ cell levels in infected persons are being intensely investigated, and have broad implications for AIDS drug and vaccine development. Virally induced changes in membrane ionic permeability induced by lytic viruses of many families contribute to cytopathogenesis. HIV-1 induces disturbances in plasma membrane ion transport. The carboxyl terminus of TM (gp41) contains potential amphipathic alpha-helical motifs identified through their structural similarities to naturally occurring cytolytic peptides. These sequences have been dubbed lentiviral lytic peptides (LLP) -1, -2, and -3.

Results: Peptides corresponding to the LLP domains (from a clade B virus) partition into lipid membranes, fold into alpha-helices and disrupt model membrane permeability. A peptide corresponding to the LLP-1 domain of a clade D HIV-1 virus, LLP-1D displayed similar activity to the LLP-1 domain of the clade B virus in all assays, despite a lack of amino acid sequence identity.

Conclusion: These results suggest that the C-terminal domains of HIV-1 Env proteins may form an ion channel, or viroporin. Increased understanding of the function of LLP domains and their role in the viral replication cycle could allow for the development of novel HIV drugs.

Show MeSH
Related in: MedlinePlus