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Abstract

Assembly of Gag, the main structural polyprotein of HIV-1, drives virion biogenesis by recruiting and assembling the necessary factors at the plasma membrane (PM) of infected cells. Lipid rafts, microdomains rich in Cholesterol and Sphingomyelin (SM), are believed to serve as platforms for HIV-1 assembly and release. However, the localization, arrangement, composition, interactions, and diversity of these lipid domains remain poorly understood. In this work, I combined labeling of native Cholesterol and SM using recombinant fluorescence probes with stimulated emission depletion (STED) microscopy, to investigate lipid distribution at individual HIV-1 assembly sites within the plasma membrane of cells. Gag assembly sites highly colocalized with Cholesterol- and SM-rich domains. Notably, one third of Gag domains were associated with a distinct ring-shaped lipid domain localized at the border of Gag assembly sites – coinciding with the extended ring-shaped envelope glycoprotein (Env) domain. Gag oligomerization at the PM in absence of all other viral proteins, including Env, was sufficient to induce formation of this lipid ring. Semi-automated image analyses and classification of Gag assembly sites based on their fluorescence intensity indicated a progressive recruitment of lipid microdomains. Punctate lipid domains more often associated with low-intensity Gag sites while half-ring and full-ring domains associated with respectively higher intensity and larger Gag domains. The Env ring domain largely overlapped with the lipid ring, but quantitative image analyses revealed the lipid ring to be on average ~15 nm closer towards the center of assembly. Punctate Cholesterol signal was also detected in released virus-like particles (VLPs), indicating that a portion of the lipid ring domain gets incorporated during budding. GagCA3, a mutant that is unable to generate PM curvature, lacked recruitment of Cholesterol-rich domains while Env recruitment was retained. However, only punctate Env domains were observed in GagCA3, which may result from altered interaction with the GagCA3 lattice compared to Gag wild type. Together these data indicate that the induction of negative curvature of the PM during Gag immature lattice formation and assembly is essential for the recruitment of punctate lipid microdomains, which transition into a distinct ring-shaped domain surrounding Gag. In conclusion, this study deepened our mechanistic understanding of HIV-1 assembly and provided a basis for further investigations of lipid-protein interactions in viral assembly and in a broader context, vesicle-mediated release events.