Background: Membrane rafts are cholesterol-enriched microdomains originally characterized by their insolubility at 4°C in non-ionic detergent as Triton x-100. Rafts incorporate specific proteins such GPI-anchored proteins. They are involved in several intracellular sorting and signal transduction events. Before virus budding, HIV-1 assembly occurs by associating structural proteins (Pr55^Gag/Pr160^Gag-Pol), accessory proteins (Vpr, Vif, Nef) and virus genome to a region of the plasma membrane enriched in envelope glycoproteins (gp120/gp41). Rafts have been identified as assembly and/or budding site of several enveloped virus. Are membrane rafts useful for HIV assembly?

Methods: We work with simian cell lines, which stably express structural and enzymatic precursors of HIV, envelope glycoproteins, and accessory proteins. These cell lines permit the study of the assembly and budding of pseudo-viral particles. Raft microdomains of these cells are isolated by flotation onto sucrose gradient after a lysis in cold Triton X-100 1% buffer. Raft localization of HIV proteins are studied by western blot. Patching experiment using biotinylated cholera toxin and Alexa⁴⁸⁸-streptavidin cross-linking agent are performed at 37°C. Co-localization of GM1 with anti-p24 antibodies is determined by confocal microscopy.

Results: Pr55^Gag/Pr160^Gag-Pol precursors of HIV have the intrinsic ability to localize into raft. Co-patching experiments reveal a strong co-localization of anti-p24 antibody with the raft lipid constituent GM1. All but one Pr55^Gag cleavage products, including the mature p24^CA and p17^MA, are not associated with rafts. Only p49 cleavage product was also found associated with raft. In the absence of a functional HIV protease, a significant increase in the amount of Pr55^Gag is found to be associated with rafts. Using CD46/gp41 chimeric protein, we also show that the cytoplasmic part of the envelope glycoproteins enables the chimera to localize into rafts.

Conclusions: Our data indicate that membrane rafts act as intermediate assembly platform for HIV-1. The perspective is to define the kinetic of raft association/disassociation of Pr55^Gag cleavage product and to identify any cellular factor possibly involved in the localization of Pr55^Gag in rafts.