Palmitoylation of the HIV-1 envelope glycoprotein is critical for viral infectivity

Abstract

Recent studies suggest that HIV-1 budding occurs selectively from detergent-insoluble membrane domains, referred to as lipid rafts. Palmitoylation is thought to be one of the factors responsible for targeting membrane proteins to lipid rafts. The cytoplasmic domain of the HIV-1 envelope glycoprotein (gp160) contains two palmitoylated cysteine residues. In this work, we studied the solubility of gp160 after detergent extraction. We show that wild-type gp160 is mostly insoluble after ice-cold Triton X-100 extraction, but that it becomes almost completely soluble at 37 degrees C. In contrast, we find that a mutant gp160, in which the two palmitoylated cysteine residues are replaced by serine, is Triton X-100 soluble even under ice-cold extraction. These findings are consistent with the properties of proteins that localize to lipid rafts and strongly suggest that gp160 is associated with lipid rafts. Further, removal of both palmitoylation sites results in the formation of virus with low levels of gp160 incorporation as well as a decrease in viral infectivity by 60-fold. Our results strongly support the suggestion that HIV-1 buds from lipid rafts and point to a role for rafts as a viral assembly hub.

Figure 1

A schematic representation of HIV-1 gp160. gp160 is proteolytically processed into the receptor-binding domain, gp120 (green), and the transmembrane domain gp41—composed of an ectodomain (red), transmembrane helix (TM, black), and cytoplasmic domain (blue). The cytoplasmic domain contains two palmitoylated cysteine residues at positions 764 and 837 (22). The location of the gp160 mAb (Chessie-8) recognition sequence (PDRPEG) is indicated (27).

Figure 2

The effect of palmitoylation on gp160 resistance to Triton X-100 extraction. Cells expressing gp160 (WT, C764S, C837S, or C764/837S) were lysed in 0.5% Triton X-100. The soluble (S) and insoluble (I) fractions were immunoprecipitated (after solubilization of the insoluble fraction by sonication) with a gp160 antibody (Chessie-8), and subjected to Western blot analysis (see text).

Figure 3

The state of gp160 palmitoylation affects the amount of Env in virus. Virus-containing supernatants were produced by cotransfection of 293T cells with an Env-deficient, HIV-1 genome (NL43LucR⁻E⁻) and an Env expression vector, as indicated. Supernatants containing equal amounts of virus (as determined by p24 ELISA) then were centrifuged through a 20% sucrose cushion. Virus pellets were resuspended in SDS and subjected to Western blot analysis using a gp160 mAb (Chessie-8).

Figure 4

gp160 palmitoylation is critical for viral infectivity. Luciferase-expressing viruses carrying Env mutants were used to infect HOS T4 (CD4⁺) cells. The infectivity of the virions was measured with a luciferase assay of the infected cell lysates at 48 h postinfection. Raw luciferase data then were normalized to the concentration of the input virus as measured by p24 ELISA (see text). Error bars represent the standard deviation of triplicate experiments.