Étude par FRET-FLIM de l'interaction de la protéine Gag du VIH-1 avec l'ARN génomique et les domaines lipidiques de la membrane plasmique

La protéine Gag du VIH-1 participe aux différentes étapes de l'assemblage du virion qui comprennent la sélection de l'ARN génomique (ARNg), l'oligomérisation de Gag via le domaine capside (CA) et la liaison à la membrane plasmique (PM) via le domaine de la matrice (MA) pour l'assemblage du virion. La sélection de l'ARNg est médiée par le domaine de la nucléocapside (NC) de Gag via ses deux doigts de zinc (ZF). Le domaine p6 à l'extrémité C-terminale aide le virion naissant à bourgeonner à partir de la PM. On sait que les virions produits ont une composition unique de leur bicouche lipidique, différente de la PM d'origine. Malgré des efforts considérables, les rôles de chaque ZF, des acides aminés aromatiques (AA), de l'architecture des ZF, de l'oligomérisation de Gag et de la myristylation du domaine MA dans l'interaction Gag-ARNg sont encore mal connus. On ignore également si l'interaction Gag-PM réorganise les domaines lipidiques de la PM. Nos résultats montrent que la délétion des deux ZF ou du domaine NC complet abolit complètement l'interaction Gag-gRNA. La délétion d’un seul ZF retarde l’adressage de l'ARNg à la PM tout en maintenant l'interaction Gag-ARNg. Cependant, le ZF2 et tout particulièrement le tryptophane en position 37 joue un rôle plus important que le ZF1 dans l'interaction Gag-gRNA. De même, la structure repliée du domaine NCp7 joue un rôle primordial. Il est à noter que la Gag non myristoylée interagit avec l'ARNg au niveau cytoplasmique, alors que la Gag non oligomérisée interagit avec l'ARNg uniquement au niveau de la PM. D’autres résultats indiquent que la Gag liée au feuillet interne de la PM colocalise avec les domaines riches en sphingomyéline (SM) du feuillet externe et que les domaines riches en SM liés par Gag sont plus grands que les domaines correspondants en absence de Gag. Une analyse plus poussée a révélé que la liaison de Gag au feuillet interne de la PM restreint la diffusion latérale et induit la coalescence des domaines riches en SM du feuillet externe. Nous avons finalement montré que l'oligomérisation de Gag induit la coalescence des domaines lipidiques riches en SM et ceux riches en cholestérol.HIV-1 Gag protein orchestrates various steps of virion assembly which include genomic RNA (gRNA) selection accompanied by Gag oligomerization via capsid (CA) domain and plasma membrane (PM) binding via matrix (MA) domain for virion assembly. The selection of gRNA relies on its interaction with the nucleocapsid (NC) domain of Gag bearing two zinc fingers (ZFs). The p6 domain at the C-terminus helps the nascent virion to bud from the PM. It is known that HIV viruses have unique lipid bilayer composition, different from the originating PM. Despite substantial efforts, the roles of each ZF, aromatic amino acid (AA) residues, ZF architecture, Gag oligomerization and MA domain myristylation in Gag-gRNA interaction are still not fully understood. It is also unknown whether the Gag-PM interaction reorganizes the lipid domains of the PM. Our results showed that deletion of both ZFs or the complete NC domain completely abolished the Gag-gRNA interaction. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interaction. However, ZF2 played a more prominent role than ZF1 in establishing Gag-gRNA interaction. Furthermore, our data also indicate that gRNA recognition and trafficking to the PM, are governed by ZF motifs with a key role of the Tryptophan 37 in the second ZF and the ZFs architecture. Interestingly, non-myristoylated Gag was found to interact with the gRNA, whereas, non-oligomerized Gag was found to interact with the gRNA only at the PM. Furthermore, our data indicate that the Gag bound to the PM inner leaflet colocalized well with outer leaflet sphingomyelin (SM)-rich domains. Moreover, Gag-bound SM rich domains were larger than the SM domains in the absence of Gag. Further analysis revealed that the binding of Gag to the inner leaflet of the PM restricted the lateral diffusion and induced the coalescence of outer leaflet SM-rich domains. Finally, we showed that Gag oligomerization induces the coalescence of SM-rich and cholesterol-rich lipid domains

HIV-1 Gag targeting to the plasma membrane reorganizes sphingomyelin-rich and cholesterol-rich lipid domains

Abstract Although the human immunodeficiency virus type 1 lipid envelope has been reported to be enriched with host cell sphingomyelin and cholesterol, the molecular mechanism of the enrichment is not well understood. Viral Gag protein plays a central role in virus budding. Here, we report the interaction between Gag and host cell lipids using different quantitative and super-resolution microscopy techniques in combination with specific probes that bind endogenous sphingomyelin and cholesterol. Our results indicate that Gag in the inner leaflet of the plasma membrane colocalizes with the outer leaflet sphingomyelin-rich domains and cholesterol-rich domains, enlarges sphingomyelin-rich domains, and strongly restricts the mobility of sphingomyelin-rich domains. Moreover, Gag multimerization induces sphingomyelin-rich and cholesterol-rich lipid domains to be in close proximity in a curvature-dependent manner. Our study suggests that Gag binds, coalesces, and reorganizes pre-existing lipid domains during assembly

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Source Data file for the study entitled 'HIV-1 Gag targeting to the plasma membrane reorganizes sphingomyelin-rich and cholesterol-rich lipid domains'</p

Zinc Fingers in HIV-1 Gag precursor are not equivalent for gRNA Recruitment at the Plasma Membrane

International audienceThe human immunodeficiency virus type 1 Gag precursor specifically selects the unspliced viral genomic RNA (gRNA) from the bulk of cellular and spliced viral RNAs via its nucleocapsid (NC) domain and drives gRNA encapsidation at the plasma membrane (PM). To further identify the determinants governing the intracellular trafficking of Gag-gRNA complexes and their accumulation at the PM, we compared, in living and fixed cells, the interactions between gRNA and wild-type Gag or Gag mutants carrying deletions in NC zinc fingers (ZFs) or a nonmyristoylated version of Gag. Our data showed that the deletion of both ZFs simultaneously or the complete NC domain completely abolished intracytoplasmic Gag-gRNA interactions. Deletion of either ZF delayed the delivery of gRNA to the PM but did not prevent Gag-gRNA interactions in the cytoplasm, indicating that the two ZFs display redundant roles in this respect. However, ZF2 played a more prominent role than ZF1 in the accumulation of the ribonucleoprotein complexes at the PM. Finally, the myristate group, which is mandatory for anchoring the complexes at the PM, was found to be dispensable for the association of Gag with the gRNA in the cytosol