The existence of lipid rafts on cell membranes has been controversial. Lipid rafts are usually characterized by ultracentrifugation of detergent-lysed cells in sucrose density gradients. It is possible that this method may affect the structure of cell membranes in unknown ways and create the phenomenon of rafts (40
). Nevertheless, the association of particular membrane proteins with DRMs has physiological relevance. Thus, putative lipid rafts have been implicated in the traffic and sorting of membrane proteins throughout the cell, including the endoplasmic reticulum, Golgi complex, cell membrane, and vesicles (18
). The role of lipid rafts in the trafficking of HIV-1 envelope to the sites of virus budding is unclear; however, recent studies have provided evidence that rafts support budding and assembly of many enveloped viruses, including HIV-1 (29
). Previously, we showed that envelope incorporation into lipid rafts conferred optimal assembly onto virus particles and infectivity (4
). In this study, we investigated whether the Gag precursor was required for envelope association with DRMs. Our results show that Gag is required for HIV-1 envelopes to associate with DRMs. When envelope was coexpressed with a mutated p55gag
(L30E) that does not interact with envelope, then envelope association with DRMs was eliminated. Several studies have previously supported an interaction between Gag and envelope during assembly. Deletions and mutations in the matrix domain of gag
abrogate envelope incorporation onto mature virions (12
). In addition, association of gp41 with p55gag
prevents envelope from interacting with endocytic machinery (10
). Wyma et al. (44
) showed that in immature HIV-1 particles, the p55gag
interaction with gp41 was shown to be persistent even in the presence of Triton X-100, suggesting that envelope-Gag interactions are stable in detergent. In addition, cores isolated from immature HIV-1 virions containing uncleaved p55 precursors partially retain gp41. Thus, Gag-envelope interactions are intricately involved in the recruitment of envelope proteins for assembly onto budding virions.
To further examine the role of the gp41 cytoplasmic domain in envelope association with lipid rafts, we investigated a patient-derived envelope (NA420 LN40) that was found to be defective for envelope incorporation onto virions. The NA420 LN40 envelope was derived by PCR from lymph node tissue of an AIDS patient (36
). Sequence analyses revealed that LN40 envelope gp41 has several amino acid changes compared to B33, a functional envelope encoded by sequences amplified from brain tissue of the same patient. The amino acid substitutions included (i) C764F that eliminates the single (palmitoylation targeted) cysteine residue from the gp41 cytoplasmic region of this envelope and (ii) RR and HS substitutions at positions 787 and 788, respectively. These latter substitutions are located in the LLP-2 domain of gp41, a region previously implicated in envelope-Gag interactions and assembly onto virus particles (12
). LN40 envelope failed to associate with DRMs, even in the presence of Gag, thus firmly implicating the importance of this envelope region for assembly.
The envelope must traffic from the site of synthesis in the endoplasmic reticulum to sites of virus budding. However, the roles of envelope-Gag interactions and lipid raft association in this process are unknown. Neither is it known where Gag and envelope interact, whether at the sites of virus budding or during envelope traffic. Nor it is known if the envelope-Gag interaction is required for envelope trafficking before association with lipid rafts. The site of envelope palmitoylation is also unclear, and it will be interesting to test whether envelope excluded from rafts is palmitoylated. We cannot rule out whether H787/S788 or other LN40 mutations might interfere with envelope trafficking to a site in the cell where it meets Gag. Interestingly, substitution of F764C in the gp41 cytoplasmic domain of LN40 envelope did not rescue the envelope incorporation and infectivity nor facilitate envelope association with rafts in the presence of H787 and S788 (not shown). These observations are consistent with our previous conclusions that gp41 cysteines are neither sufficient nor entirely essential for envelope association with DRMs and assembly onto virions.
Our observations show that HIV-1 Gag is required for envelope glycoproteins to associate with DRMs. In contrast, the reciprocal arrangement has been reported for other viruses. Thus, for respiratory syncytial virus, the association of the matrix protein with DRMs is dependent on the expression of envelope glycoproteins (19
). Similarly, influenza virus matrix protein associated with DRMs only if hemagglutinin and neuraminidase glycoproteins were coexpressed (1
In conclusion, we have demonstrated that the p55gag protein is required to recruit envelope into DRMs. Identification of critical motifs regulating envelope trafficking and assembly onto virions will help the design of strategies to prevent release of infectious virions.