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. 2001 Nov 20;98(24):13925-30.
doi: 10.1073/pnas.241320298.

Plasma membrane rafts play a critical role in HIV-1 assembly and release

A Ono  1 , E O Freed
Affiliations

Plasma membrane rafts play a critical role in HIV-1 assembly and release

A Ono et al. Proc Natl Acad Sci U S A. .

Abstract

HIV-1 particle production occurs in a series of steps promoted by the viral Gag protein. Although it is well established that assembly and release take place at the plasma membrane, the nature of membrane assembly sites remains poorly understood. We show here that Gag specifically associates with cholesterol-enriched microdomains ("rafts") at the plasma membrane. Kinetic studies demonstrate that raft association follows membrane binding, and the analysis of Gag mutants reveals that, whereas the N terminus of Gag mediates raft binding, this association is greatly enhanced by Gag-Gag interaction domains. We observe that depletion of cellular cholesterol markedly and specifically reduces HIV-1 particle production. Furthermore, treatment of virus-producing cells or virus particles with raft-disrupting agents significantly impairs virus infectivity. These results identify the association of Gag with plasma membrane rafts as an important step in HIV-1 replication. These findings may lead to novel strategies for suppressing HIV-1 replication in vivo.

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Figures

Figure 1
Figure 1
HIV-1 Gag is associated with DRM. (A) Postnuclear supernatants derived from HeLa cells expressing HIV-1 Pr55Gag were treated with or without 0.25% Triton X-100 on ice and subjected to equilibrium flotation centrifugation. Pr55Gag, TfR, and Cav were detected by Western blotting. Membrane (memb.), non-membrane (non-memb.), DRM, and detergent-soluble (DS) fractions are shown. (B) Postnuclear supernatants derived from HeLa cells expressing HIV-1 Pr55Gag were treated with 30 mM octyl glucoside (OG) on ice or with 0.25% Triton X-100 at 37°C and analyzed as in A. (C) Pr55Gag-expressing cells were pulse-labeled for 5 min and chased as indicated. Postnuclear supernatants were treated and fractionated as in A. Fractions 1–5 and 6–10 were pooled, and Pr55Gag in these fractions was recovered by immunoprecipitation. The percentage of membrane-bound Gag that was DRM associated at each time point is indicated.
Figure 2
Figure 2
Determinants of Gag-DRM association. (A) Schematic representation of C-terminally truncated Gag mutants. Positions of N-terminal myristate (m), N-and C-terminal domains of CA (horizontal bars), the two zinc finger motifs in NC (Zn), and the p6 late domain (PTAP) are shown. (B) Cells singly expressing Pr55Gag and CA146 were pooled and analyzed as in Fig. 1A. (C and D) Cells singly expressing Pr55Gag and indicated mutants were metabolically labeled for 90 min, pooled and analyzed as in Fig. 1C.
Figure 3
Figure 3
Effects of cholesterol depletion on virus production. (A and C) HeLa cells expressing NL4-3 (wt virus) were treated with the indicated concentrations of cholesterol-depleting drugs. MβCD treatments were performed for 30 min; cells were treated with simvastatin (simva.) for 2 days before metabolic labeling. Cells were washed and metabolically labeled for 90 min. Labeled viral proteins in cell and virion lysates were analyzed by SDS/PAGE followed by fluorography. Data were quantified by Phosphorimager (Fuji Film) analysis. Viral glycoproteins gp160 and gp120, Pr55Gag, and mature CA (p24) are shown. (B and D) Virus release efficiency represents the amount of virion-associated p24 as a fraction of total Gag (cell-associated Pr55Gag and p24 plus virion-associated p24). The relative virus release efficiencies in drug-treated vs. untreated cultures are indicated. (E) Quantification of virus release from HeLa cells expressing NL4-3/PR−. Virus release efficiency was calculated as the amount of virion-associated Pr55Gag as a fraction of total (cell plus virion) Gag. (F) Quantification of virus release from HeLa cells expressing NL4-3/PR−/PTAP−, calculated as in E.
Figure 4
Figure 4
Models for the role of rafts in HIV-1 assembly and release. Raft and non-raft domains are shown in red and blue, respectively. See text for details.
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