Background:  Assembly of the HIV-1 envelope glycoprotein onto budding virus particles is important for efficient infection of target cells. In infected cells, detergent resistant membranes (DRM or lipid rafts) have been proposed to form platforms for virus assembly and budding. Gag precursors partly associate with lipid rafts. The cytoplasmic domain of the envelope gp41 usually carries palmitate groups that were also reported to confer lipid raft association. Gag precursors confer budding and carry envelope glycoproteins onto virions via specific gag-envelope interactions. Thus, specific mutations both in matrix domain of gag precursor and gp41 cytoplasmic domain abrogate envelope incorporation onto virions. Here, we show that HIV-1 envelope association with DRM is directly influenced by its interaction with gag.

Methods:  The NL4.3 envelope and env mutants were expressed in 293T cells by cotransfection of env⁺ pSVIIIenv in combination with pSV2tat72, env^- pNL4.3, or pCMV gag constructs. After 48 hours, transfected 293T cells were extracted with 0.5% Triton-X on ice. Cell extracts were subjected to sucrose gradient flotation centrifigation. Fractions collected were immunoprecipitated for gag and env and analyzed by Western blot. Fractions with a density of 1.09 to 1.13 g/mL represented DRM. 

Results:  Our results show that in the absence of gag, envelope fails to associate with DRM. A mutation in the p17 matrix (L30E) domain in gag that abrogates envelope incorporation onto virions also eliminated envelope assembly in DRM, consistent with a requirement for an env-gag interaction for raft association and subsequent assembly onto virions. In addition, we found that mutations in gp41 cytoplasmic domain that prevented a patient-derived envelope from assembling onto virions and impaired infectivity of cell-free virus, also eliminated envelope association with DRM.

Conclusions:  Our observations indicate that gag-envelope interactions are essential for efficient envelope association with lipid rafts, which in turn is required for optimal assembly of envelope onto virus particles.