GIB-1aren Env gainazal proteinaren palmitoilazioak duen eragina kolesterolaren elkarrekintzan: GIB-1aren Env fusio proteinaren ingurune lipidikoaren azterketa.

[Euskaraz] Giza Immunoeskasiaren Birusaren (GIBaren) infekzioa mundu osoan zehar zabaldutako Hartutako Immuno Eskasiaren Sindromearen (HIESaren) kausa da. GIBaren mintzean jatorri birikoko proteina bakarra adierazten da: gainazal glikoproteina (Env). Env bi azpiunitatez osatutako trimero gisa txertatzen da mintzean: mintz kanpoko gp120 proteina eta gp41 transmintz proteina. GIBaren gp41 proteinak, Lentivirus generoko gainontzeko birus gehienek ez bezala, domeinu zitoplasmatiko (DZ) luzea du; gainera, GIBaren andui ezberdinetan sekuentzia sendoki kontserbatzen da. Honek, DZak GIBaren bizi-zikloan funtzio gakoa izan dezakeela iradokitzen du. Gure taldean burututako ikerketen emaitzek, Env proteinak kolesterolarekin elkar eragiteko gp41aren DZa nahitaezko eragilea dela frogatu dute, baita elkarrekintza hori Env proteina guztien taldekatzea (clustering-a) emateko beharrezkoa dela ere; gainera, Env azaleko glikoproteinen multzokatze hau ezinbestekoa da birusak infekzio gaitasuna eskuratu dezan. Palmitoilazioak proteinak, Env barne, kolesterolean aberatsak diren domeinuetara, baltsa lipidikoetara alegia, bideratzen dituenaren ebidentzia dago. Hala ere, ikerketa horiek detergenteekiko mintz erresistenteen (DRM, Detergent Resistant Membrane) metodoetan oinarrituta daude eta teknika honen mugak direla eta, ez dute zuzenki frogatzen baltsa lipidikoetarako asoziazioa. Izan ere, DRM erauzketa baldintzek lipido-proteina asoziazio berriak sortaraz ditzakete. Hala, lan honen helburua, gp41 proteinaren DZko palmitoilazioak Env-kolesterol elkarrekintzan daukan papera in vivo aztertzea da, klik kimika metodoaren eta lipido fotoaktibagarrien erabilerarekin. Esperimentuak gauzatzeko GIB-1NL4-3 C762S mutantea erabili da, zeinak 762. aminoazido posizioan zisteina serina ordezkapena duen eta beraz, palmitoilazioa galtzen duen. Ikerkuntza lan honek DZaren palmitoilazioa Env-kolesterol elkarrekintzaren erantzule ez dela frogatzen du, interakzioa DZaren eta mintzeko kolesterolaren artean zuzenean ematen delarik.[English] The Human Immunodeficiency Virus (HIV) infection is the cause of the worldwide spread Acquired Immune Deficiency Syndrome (AIDS). HIV expresses a single viral glycoprotein in the viral membrane: envelope (Env). This glycoprotein is formed by a trimer formed by two subunits, a surface gp120 protein and a transmembrane gp41 protein. In HIV, the cytoplasmic domain (CTD) sequence of the gp41 protein is uncommonly long among other lentiviruses and highly conserved within HIV serotypes, suggesting possible essential function in HIV´s life cycle. Preliminary results from the group show that the CTD is responsible for protein-cholesterol interaction and that such interaction is necessary for Env clustering within viral membrane. Moreover, clustering of Env has been proved to be a prerequisite for viral infection. Palmitoylation is thought to be one of the responsible factors for targeting membrane proteins, including Env, to cholesterol rich domains, commonly called, lipid rafts. However, studies were conducted by using DRM (Detergent Resistant Membrane) method which presents limitations in the establishment of a reliable direct association between proteins and lipid rafts. Since DRM extraction conditions can form new lipid-protein assemblies, the objective of this study was to clarify the role of palmitoylation in the interaction of the gp41 CTD-cholesterol in vivo by new techniques, such as, Click chemistry and radioactive photoactivable lipids. Experiments were performed with the HIV-1NL4-3 C762S mutant, which presents the substitution of the cysteine residue to serine in the 762nd position of the CTD, and therefore, it lacks of palmytoilation. The results of this scientific study demonstrate that the interaction of Env protein to cholesterol through the CTD is not due to palmitoylation, but direct domain-viral membrane interaction.[ES] La infección por el virus de la inmunodeficiencia humana (VIH) es la causa de la propagación mundial del síndrome de inmunodeficiencia adquirida (SIDA). El VIH expresa una sola glicoproteína viral en la membrana viral: envoltura (Env). Esta glicoproteína está formada por un trímero formado por dos subunidades, una proteína gp120 de superficie y una proteína gp41 transmembrana. En el VIH, la secuencia del dominio citoplasmático (CTD) de la proteína gp41 es poco común entre otros lentivirus y está muy conservada en los serotipos del VIH, lo que sugiere una posible función esencial en el ciclo de vida del VIH. Los resultados preliminares del grupo muestran que el CTD es responsable de la interacción proteína-colesterol y que dicha interacción es necesaria para la agrupación de Env dentro de la membrana viral. Además, se ha demostrado que la agrupación de Env es un requisito previo para la infección viral. Se cree que la palmitoilación es uno de los factores responsables de dirigir las proteínas de membrana, incluida Env, a dominios ricos en colesterol, comúnmente llamados balsas lipídicas. Sin embargo, los estudios se realizaron utilizando el método DRM (Detergent Resistant Membrane) que presenta limitaciones en el establecimiento de una asociación directa confiable entre proteínas y balsas lipídicas. Dado que las condiciones de extracción de DRM pueden formar nuevos ensamblajes lípido-proteína, el objetivo de este estudio fue aclarar el papel de la palmitoilación en la interacción de la gp41 CTD-colesterol in vivo mediante nuevas técnicas, como la química Click y los lípidos fotoactivables radiactivos. Se realizaron experimentos con el mutante HIV-1NL4-3 C762S, que presenta la sustitución del residuo cisteína por serina en la posición 762 del CTD, y por tanto, carece de palmitoilación. Los resultados de este estudio científico demuestran que la interacción de la proteína Env con el colesterol a través del CTD no se debe a la palmitoilación, sino a la interacción dominio directo-membrana viral

GIB-1aren Env gainazal proteinaren palmitoilazioak duen eragina kolesterolaren elkarrekintzan: GIB-1aren Env fusio proteinaren ingurune lipidikoaren azterketa.

[Euskaraz] Giza Immunoeskasiaren Birusaren (GIBaren) infekzioa mundu osoan zehar zabaldutako Hartutako Immuno Eskasiaren Sindromearen (HIESaren) kausa da. GIBaren mintzean jatorri birikoko proteina bakarra adierazten da: gainazal glikoproteina (Env). Env bi azpiunitatez osatutako trimero gisa txertatzen da mintzean: mintz kanpoko gp120 proteina eta gp41 transmintz proteina. GIBaren gp41 proteinak, Lentivirus generoko gainontzeko birus gehienek ez bezala, domeinu zitoplasmatiko (DZ) luzea du; gainera, GIBaren andui ezberdinetan sekuentzia sendoki kontserbatzen da. Honek, DZak GIBaren bizi-zikloan funtzio gakoa izan dezakeela iradokitzen du. Gure taldean burututako ikerketen emaitzek, Env proteinak kolesterolarekin elkar eragiteko gp41aren DZa nahitaezko eragilea dela frogatu dute, baita elkarrekintza hori Env proteina guztien taldekatzea (clustering-a) emateko beharrezkoa dela ere; gainera, Env azaleko glikoproteinen multzokatze hau ezinbestekoa da birusak infekzio gaitasuna eskuratu dezan. Palmitoilazioak proteinak, Env barne, kolesterolean aberatsak diren domeinuetara, baltsa lipidikoetara alegia, bideratzen dituenaren ebidentzia dago. Hala ere, ikerketa horiek detergenteekiko mintz erresistenteen (DRM, Detergent Resistant Membrane) metodoetan oinarrituta daude eta teknika honen mugak direla eta, ez dute zuzenki frogatzen baltsa lipidikoetarako asoziazioa. Izan ere, DRM erauzketa baldintzek lipido-proteina asoziazio berriak sortaraz ditzakete. Hala, lan honen helburua, gp41 proteinaren DZko palmitoilazioak Env-kolesterol elkarrekintzan daukan papera in vivo aztertzea da, klik kimika metodoaren eta lipido fotoaktibagarrien erabilerarekin. Esperimentuak gauzatzeko GIB-1NL4-3 C762S mutantea erabili da, zeinak 762. aminoazido posizioan zisteina serina ordezkapena duen eta beraz, palmitoilazioa galtzen duen. Ikerkuntza lan honek DZaren palmitoilazioa Env-kolesterol elkarrekintzaren erantzule ez dela frogatzen du, interakzioa DZaren eta mintzeko kolesterolaren artean zuzenean ematen delarik.[English] The Human Immunodeficiency Virus (HIV) infection is the cause of the worldwide spread Acquired Immune Deficiency Syndrome (AIDS). HIV expresses a single viral glycoprotein in the viral membrane: envelope (Env). This glycoprotein is formed by a trimer formed by two subunits, a surface gp120 protein and a transmembrane gp41 protein. In HIV, the cytoplasmic domain (CTD) sequence of the gp41 protein is uncommonly long among other lentiviruses and highly conserved within HIV serotypes, suggesting possible essential function in HIV´s life cycle. Preliminary results from the group show that the CTD is responsible for protein-cholesterol interaction and that such interaction is necessary for Env clustering within viral membrane. Moreover, clustering of Env has been proved to be a prerequisite for viral infection. Palmitoylation is thought to be one of the responsible factors for targeting membrane proteins, including Env, to cholesterol rich domains, commonly called, lipid rafts. However, studies were conducted by using DRM (Detergent Resistant Membrane) method which presents limitations in the establishment of a reliable direct association between proteins and lipid rafts. Since DRM extraction conditions can form new lipid-protein assemblies, the objective of this study was to clarify the role of palmitoylation in the interaction of the gp41 CTD-cholesterol in vivo by new techniques, such as, Click chemistry and radioactive photoactivable lipids. Experiments were performed with the HIV-1NL4-3 C762S mutant, which presents the substitution of the cysteine residue to serine in the 762nd position of the CTD, and therefore, it lacks of palmytoilation. The results of this scientific study demonstrate that the interaction of Env protein to cholesterol through the CTD is not due to palmitoylation, but direct domain-viral membrane interaction.[ES] La infección por el virus de la inmunodeficiencia humana (VIH) es la causa de la propagación mundial del síndrome de inmunodeficiencia adquirida (SIDA). El VIH expresa una sola glicoproteína viral en la membrana viral: envoltura (Env). Esta glicoproteína está formada por un trímero formado por dos subunidades, una proteína gp120 de superficie y una proteína gp41 transmembrana. En el VIH, la secuencia del dominio citoplasmático (CTD) de la proteína gp41 es poco común entre otros lentivirus y está muy conservada en los serotipos del VIH, lo que sugiere una posible función esencial en el ciclo de vida del VIH. Los resultados preliminares del grupo muestran que el CTD es responsable de la interacción proteína-colesterol y que dicha interacción es necesaria para la agrupación de Env dentro de la membrana viral. Además, se ha demostrado que la agrupación de Env es un requisito previo para la infección viral. Se cree que la palmitoilación es uno de los factores responsables de dirigir las proteínas de membrana, incluida Env, a dominios ricos en colesterol, comúnmente llamados balsas lipídicas. Sin embargo, los estudios se realizaron utilizando el método DRM (Detergent Resistant Membrane) que presenta limitaciones en el establecimiento de una asociación directa confiable entre proteínas y balsas lipídicas. Dado que las condiciones de extracción de DRM pueden formar nuevos ensamblajes lípido-proteína, el objetivo de este estudio fue aclarar el papel de la palmitoilación en la interacción de la gp41 CTD-colesterol in vivo mediante nuevas técnicas, como la química Click y los lípidos fotoactivables radiactivos. Se realizaron experimentos con el mutante HIV-1NL4-3 C762S, que presenta la sustitución del residuo cisteína por serina en la posición 762 del CTD, y por tanto, carece de palmitoilación. Los resultados de este estudio científico demuestran que la interacción de la proteína Env con el colesterol a través del CTD no se debe a la palmitoilación, sino a la interacción dominio directo-membrana viral

Cholesterol in the Viral Membrane is a Molecular Switch Governing HIV-1 Env Clustering

HIV-1 entry requires the redistribution of envelope glycoproteins (Env) into a cluster and the presence of cholesterol (chol) in the viral membrane. However, the molecular mechanisms underlying the specific role of chol in infectivity and the driving force behind Env clustering remain unknown. Here, gp41 is demonstrated to directly interact with chol in the viral membrane via residues 751-854 in the cytoplasmic tail (CT751-854). Super-resolution stimulated emission depletion (STED) nanoscopy analysis of Env distribution further demonstrates that both truncation of gp41 CT751-854 and depletion of chol leads to dispersion of Env clusters in the viral membrane and inhibition of virus entry. This work reveals a direct interaction of gp41 CT with chol and indicates that this interaction is an important orchestrator of Env clustering.The authors are grateful to Barbara Müller, N. Landau, and Tom Hope for providing the plasmids pCHIV and pCAGGS NL4-3 Env, pMM310, and peGFP-Vpr, respectively. Proteomic analysis was performed by the SGIKER service of the University of the Basque Country. The authors would like to thank Advanced Light Microscopy Unit at the Centre for Genomic Regulation (CRG), Barcelona, Spain for the access to Leica STED microscope. The following reagents were obtained through the NIH AIDS Reagent Program (Division of NIAID, NIH): Anti-HIV-1 gp41 Hybridome (Chessie 8) (Cat# 526) from Dr. George Lewis; Antiviral bicyclam JM-2987 (hydrobromide salt of AMD-3100) from NIAID, DAIDS (cat# 8128). This project was supported by the Basque Government (grant number IT1264-19 to M.L. and F.-X.C.) and the Spanish Ministry of Science, Innovation, and Universities (BFU-2015-68981-P). This work was supported in part by the Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government. J.A.N.-G. was supported by a FI predoctoral fellowship from the Basque Government and currently by Fundación Biofísica Bizkaia. A.A. was supported by Fundación Biofísica Bizkaia. S.O. was supported by an IKASIKER fellowship from the Basque Government. J.C. was supported by European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No. 793830. H.G.-K. was supported by a grant from the Deutsche Forschungsgemeischaft within TRR86

Cholesterol in the Viral Membrane is a Molecular Switch Governing HIV‐1 Env Clustering

HIV‐1 entry requires the redistribution of envelope glycoproteins (Env) into a cluster and the presence of cholesterol (chol) in the viral membrane. However, the molecular mechanisms underlying the specific role of chol in infectivity and the driving force behind Env clustering remain unknown. Here, gp41 is demonstrated to directly interact with chol in the viral membrane via residues 751–854 in the cytoplasmic tail (CT751–854). Super‐resolution stimulated emission depletion (STED) nanoscopy analysis of Env distribution further demonstrates that both truncation of gp41 CT751–854 and depletion of chol leads to dispersion of Env clusters in the viral membrane and inhibition of virus entry. This work reveals a direct interaction of gp41 CT with chol and indicates that this interaction is an important orchestrator of Env clustering.The authors are grateful to Barbara Müller, N. Landau, and Tom Hope for providing the plasmids pCHIV and pCAGGS NL4‐3 Env, pMM310, and peGFP‐Vpr, respectively. Proteomic analysis was performed by the SGIKER service of the University of the Basque Country. The authors would like to thank Advanced Light Microscopy Unit at the Centre for Genomic Regulation (CRG), Barcelona, Spain for the access to Leica STED microscope. The following reagents were obtained through the NIH AIDS Reagent Program (Division of NIAID, NIH): Anti‐HIV‐1 gp41 Hybridome (Chessie 8) (Cat# 526) from Dr. George Lewis; Antiviral bicyclam JM‐2987 (hydrobromide salt of AMD‐3100) from NIAID, DAIDS (cat# 8128). This project was supported by the Basque Government (grant number IT1264‐19 to M.L. and F.‐X.C.) and the Spanish Ministry of Science, Innovation, and Universities (BFU‐2015‐68981‐P). This work was supported in part by the Fundación Biofísica Bizkaia and the Basque Excellence Research Centre (BERC) program of the Basque Government. J.A.N.‐G. was supported by a FI predoctoral fellowship from the Basque Government and currently by Fundación Biofísica Bizkaia. A.A. was supported by Fundación Biofísica Bizkaia. S.O. was supported by an IKASIKER fellowship from the Basque Government. J.C. was supported by European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska‐Curie grant agreement No. 793830. H.G.‐K. was supported by a grant from the Deutsche Forschungsgemeischaft within TRR86.Peer reviewe