Finally, we tested whether envelope mutants that lacked cysteines in the cytoplasmic domain were associated with lipid rafts. The procedures used to analyze lipid rafts were based on those described previously by others (7
). 293T cells (1.2 × 106
cells) were transfected with pNL43env−
and pSVIIIenv constructs carrying each envelope mutant. Transfected cells were lysed with Triton X-100 at a final concentration of 0.5% in TNE (10 mM Tris, 100 mM sodium chloride, 10 mM EGTA [pH 7.5], protease inhibitor cocktail [Sigma Inc.]) for 30 min. Lysates were homogenized and centrifuged at low speed to remove nuclei. Supernatants were adjusted to 60% sucrose in a 1-ml volume and loaded on top of 250 μl of 80% sucrose. The sucrose gradient was completed by layering 1 ml of 50% sucrose, 3 ml of 38% sucrose, and 750 μl of 10% sucrose on top of the lysate sample. These steps were carried out with the sample either on ice or at a temperature below 4°C. Sucrose gradients were centrifuged for 18 h at 100,000 × g
(4°C) in an SW 50.1 Ti rotor. Fourteen fractions were collected, and each fraction was immunoprecipitated with HIV-1+
human serum (1:1,000). Immunoprecipitated proteins were analyzed by polyacrylamide gel electrophoresis followed by Western blotting. Blots were probed with anti-p55gag
MAb 183-H12-5C (6
) and anti-gp41 MAb Chessie 8 (1
) (Fig. ). DRM-L and DRM-H float at densities of 1.09 to 1.13 and 1.16 to 1.2 g/ml, respectively. The p55gag
precursor was detected throughout the gradient but was concentrated in both DRM-H and DRM-L, consistent with previously reported observations (18
). The envelope precursor gp160 and the processed gp41 were consistently both located in the same fractions. The wild-type C764/Y837 envelope was detected in fractions with densities consistent with both DRM-L and DRM-H lipid rafts. In contrast, envelopes that lacked gp41 cytoplasmic cysteines were not associated with DRM-L fractions. However, the F764/Y837 envelope was detected in fractions with densities consistent with DRM-H, indicating that association with these heavier rafts was retained.
FIG.5. Association of mutant NL43 envelopes with lipid rafts. 293T cells were cotransfected with pNL43env− and pSVIIIenv containing gp41 envelope mutants. Cells were lysed with 0.5% Triton X-100 in TNE buffer on ice. Lysates were homogenized, cleared (more ...)
Envelopes that did not contain gp41 cytoplasmic cysteines or residues with bulky hydrophobic side chains at positions 764/837 (A764/A837 and S764/S837) were almost completely excluded from both DRM-L and DRM-H. Treatment of Triton X-100 cell lysates at 37°C disrupted the DRM relocating envelope and the p55gag precursor into the soluble fraction at the bottom of the gradient (Fig. ). Western blots of cold Triton X-100 extracts of 293T cells, fractionated on sucrose gradients (prepared as described for Fig. ), indicated that caveolin (a marker for lipid rafts) was contained almost exclusively in fractions with densities consistent with DRM-L.
These results show that (i) gp41 cytoplasmic cysteines, which are targets for palmitoylation, are required for the association of the envelope with DRM-L; (ii) envelopes that contained residues with bulky hydrophobic side chains (instead of cysteines) retained their association with DRM-H and were efficiently assembled on virions; and (iii) envelopes with C764A/C837A and C764S/C837S substitutions were excluded from lipid rafts yet were incorporated onto virions with efficiencies of up to 40% compared to wild-type envelopes.
Previously, Yang et al. (26
) reported that gp41 cytoplasmic cysteines (C764 and C837) were palmitoylated and that the replacement of these residues completely abolished palmitoylation. Subsequently, Rousso et al. showed that HIV-1 envelopes carrying double C764S/C837S substitutions lost their association with lipid rafts, were inefficiently incorporated onto budding viruses, and conferred only low levels of infectivity (21
). Nevertheless, several infectious molecular clones of HIV-1, e.g., JRCSF, lack cysteines in the cytoplasmic domain of gp41 and yet are apparently fully functional and replication competent. Since no other gp41 amino acid has been shown to be a substrate for palmitic acid addition (26
), this observation implied that palmitoylation of gp41 was not essential for envelope assembly on virions or infectivity.
Palmitoylation occurs almost exclusively via the covalent attachment of palmitate groups to cysteine residues via thioester bonds (12
). A single exception was recently reported by Kleuss and Krause (12
), who demonstrated that an N-terminal glycine of the G-protein α subunit Gsα was palmitoylated via an amide linkage. The mechanism of glycine palmitoylation is unclear, although it is possible that the palmitate group is transferred from an adjacent palmitoylated cysteine in Gsα (13
). Here, we further examined the role of gp41 cytoplasmic cysteine residues in envelope assembly. Using HIV-1 NL43, which has only a single gp41 cytoplasmic cysteine at position 764 (C764), we showed that gp41 cytoplasmic cysteines are not required for HIV-1 envelope incorporation onto virions and infectivity. The replacement of C764 and C837 with residues carrying bulky hydrophobic side chains may therefore compensate for the lack of a palmitate group. Here, we showed that cytoplasmic cysteines which are targets for palmitoylation are required for the localization of envelope glycoproteins to light lipid rafts (DRM-L). The replacement of cysteines with residues that carry bulky hydrophobic side chains resulted in the retention of the association with heavy lipid rafts (DRM-H) and conferred near-wild-type levels of envelope assembly on virions. The replacement of cysteines with alanines or serines eliminated raft association and reduced envelope incorporation onto virions and the infectivity of virions. Nevertheless, the A764/A837 mutant envelope retained nearly 40% infectivity compared to the wild type, even though this envelope was excluded from lipid rafts. In summary, our data show that the presence of cysteine residues in the gp41 cytoplasmic domain is not essential for envelope incorporation onto virions or the infectivity of virions.