Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion

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dc.contributor.authorde Voux, Alexen_ZA
dc.contributor.authorChan, Mei-Chien_ZA
dc.contributor.authorFolefoc, Asongna Ten_ZA
dc.contributor.authorMadziva, Michael Ten_ZA
dc.contributor.authorFlanagan, Colleen Aen_ZA
dc.date.accessioned2016-01-11T06:46:18Z
dc.date.available2016-01-11T06:46:18Z
dc.date.issued2013en_ZA
dc.description.abstractThe CCR5 chemokine receptor is a rhodopsin-like G protein-coupled receptor that mediates the effects of pro-inflammatory β-chemokines. CCR5 is also the major co-receptor for entry of human immunodeficiency virus (HIV) into human cells. G protein-coupled receptors exist in ensembles of active and inactive conformations. Active receptor conformations can be stabilized by mutations. Although binding of the HIV envelope protein to CCR5 stimulates cellular signaling, the CCR5 conformation that induces fusion of the viral membrane with cellular membranes is not known. We mutated conserved amino acids to generate constitutively active CCR5 receptors, which are stabilized in active conformations, and tested the ability of constitutively active CCR5 receptors to mediate HIV envelope-directed membrane fusion. Mutation of the Asp 3.49(125) and Arg 6.32(225) residues of CCR5 did not cause constitutive activity, but Lys or Pro substitutions for Thr 2.56(82) , in the TxP motif, caused high basal inositol phosphate signaling. Signaling did not increase in response to MIP-1β, suggesting that the Thr 2.56(82) mutants were fully stabilized in active conformations. The Thr 2.56(82) Lys mutation severely decreased cell surface CCR5 expression. Combining the Thr 2.56(82) Lys mutation with an Arg 6.32(225) Gln mutation partially reversed the decrease in expression. Mutants with Thr 2.56(82) Lys substitutions were poor mediators of HIV envelope-directed membrane fusion, but mutants with the Thr 2.65(82) Pro substitution exhibited full co-receptor function. Our results suggest that the Thr 2.65(82) Lys and Thr 2.65(82) Pro mutations stabilize distinct constitutively active CCR5 conformations. Lys in position 2.65(82) stabilizes activated receptor conformations that appear to be constitutively internalized and do not induce envelope-dependent membrane fusion, whereas Pro stabilizes activated conformations that are not constitutively internalized and fully mediate envelope-directed membrane fusion.en_ZA
dc.identifier.apacitationde Voux, A., Chan, M., Folefoc, A. T., Madziva, M. T., & Flanagan, C. A. (2013). Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion. <i>PLoS One</i>, http://hdl.handle.net/11427/16219en_ZA
dc.identifier.chicagocitationde Voux, Alex, Mei-Chi Chan, Asongna T Folefoc, Michael T Madziva, and Colleen A Flanagan "Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion." <i>PLoS One</i> (2013) http://hdl.handle.net/11427/16219en_ZA
dc.identifier.citationde Voux, A., Chan, M. C., Folefoc, A. T., Madziva, M. T., & Flanagan, C. A. (2013). Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion. PloS one, 8(1), e54532. doi:10.1371/journal.pone.0054532en_ZA
dc.identifier.ris TY - Journal Article AU - de Voux, Alex AU - Chan, Mei-Chi AU - Folefoc, Asongna T AU - Madziva, Michael T AU - Flanagan, Colleen A AB - The CCR5 chemokine receptor is a rhodopsin-like G protein-coupled receptor that mediates the effects of pro-inflammatory β-chemokines. CCR5 is also the major co-receptor for entry of human immunodeficiency virus (HIV) into human cells. G protein-coupled receptors exist in ensembles of active and inactive conformations. Active receptor conformations can be stabilized by mutations. Although binding of the HIV envelope protein to CCR5 stimulates cellular signaling, the CCR5 conformation that induces fusion of the viral membrane with cellular membranes is not known. We mutated conserved amino acids to generate constitutively active CCR5 receptors, which are stabilized in active conformations, and tested the ability of constitutively active CCR5 receptors to mediate HIV envelope-directed membrane fusion. Mutation of the Asp 3.49(125) and Arg 6.32(225) residues of CCR5 did not cause constitutive activity, but Lys or Pro substitutions for Thr 2.56(82) , in the TxP motif, caused high basal inositol phosphate signaling. Signaling did not increase in response to MIP-1β, suggesting that the Thr 2.56(82) mutants were fully stabilized in active conformations. The Thr 2.56(82) Lys mutation severely decreased cell surface CCR5 expression. Combining the Thr 2.56(82) Lys mutation with an Arg 6.32(225) Gln mutation partially reversed the decrease in expression. Mutants with Thr 2.56(82) Lys substitutions were poor mediators of HIV envelope-directed membrane fusion, but mutants with the Thr 2.65(82) Pro substitution exhibited full co-receptor function. Our results suggest that the Thr 2.65(82) Lys and Thr 2.65(82) Pro mutations stabilize distinct constitutively active CCR5 conformations. Lys in position 2.65(82) stabilizes activated receptor conformations that appear to be constitutively internalized and do not induce envelope-dependent membrane fusion, whereas Pro stabilizes activated conformations that are not constitutively internalized and fully mediate envelope-directed membrane fusion. DA - 2013 DB - OpenUCT DO - 10.1371/journal.pone.0054532 DP - University of Cape Town J1 - PLoS One LK - https://open.uct.ac.za PB - University of Cape Town PY - 2013 T1 - Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion TI - Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion UR - http://hdl.handle.net/11427/16219 ER - en_ZA
dc.identifier.urihttp://hdl.handle.net/11427/16219
dc.identifier.urihttp://dx.doi.org/10.1371/journal.pone.0054532
dc.identifier.vancouvercitationde Voux A, Chan M, Folefoc AT, Madziva MT, Flanagan CA. Constitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusion. PLoS One. 2013; http://hdl.handle.net/11427/16219.en_ZA
dc.language.isoengen_ZA
dc.publisherPublic Library of Scienceen_ZA
dc.publisher.departmentMRC/UCT Receptor Biology Research Groupen_ZA
dc.publisher.facultyFaculty of Health Sciencesen_ZA
dc.publisher.institutionUniversity of Cape Town
dc.rightsThis is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.en_ZA
dc.rights.holder© 2013 de Voux et alen_ZA
dc.rights.urihttp://creativecommons.org/licenses/by/4.0en_ZA
dc.sourcePLoS Oneen_ZA
dc.source.urihttp://journals.plos.org/plosoneen_ZA
dc.subject.otherMembrane fusionen_ZA
dc.subject.otherHIVen_ZA
dc.subject.otherCell fusionen_ZA
dc.subject.otherHEK 293 cellsen_ZA
dc.subject.otherSubstitution mutationen_ZA
dc.subject.otherChemokinesen_ZA
dc.subject.otherG protein coupled receptorsen_ZA
dc.subject.otherMembrane proteinsen_ZA
dc.titleConstitutively active CCR5 chemokine receptors differ in mediating HIV envelope-dependent fusionen_ZA
dc.typeJournal Articleen_ZA
uct.type.filetypeText
uct.type.filetypeImage
uct.type.publicationResearchen_ZA
uct.type.resourceArticleen_ZA
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