Role for human immunodeficiency virus type 1 membrane cholesterol in viral internalization

Abstract

The membrane of human immunodeficiency virus type 1 (HIV-1) virions contains high levels of cholesterol and sphingomyelin, an enrichment that is explained by the preferential budding of the virus through raft microdomains of the plasma membrane. Upon depletion of cholesterol from HIV-1 virions with methyl-beta-cyclodextrin, infectivity was almost completely abolished. In contrast, this treatment had only a mild effect on the infectiousness of particles pseudotyped with the G envelope of vesicular stomatitis virus. The cholesterol-chelating compound nystatin had a similar effect. Cholesterol-depleted HIV-1 virions exhibited wild-type patterns of viral proteins and contained normal levels of cyclophilin A and glycosylphosphatidylinositol-anchored proteins. Nevertheless, and although they could still bind target cells, these virions were markedly defective for internalization. These results indicate that the cholesterol present in the HIV-1 membrane plays a prominent role in the fusion process that is key to viral entry and suggest that drugs capable of disturbing the lipid composition of virions could serve as a basis for the development of microbicides.

Figures

FIG. 1.

Alteration of viral membrane cholesterol strongly decreases HIV-1 infectivity. (A and B) [3H]cholesterol-labeled wild-type (R9) and VSV G-pseudotyped (R9ΔE/VSV G) virions were treated with increasing concentrations of methyl-β-cyclodextrin (CD). (A) Virions were subsequently concentrated by ultracentrifugation, and the amounts of pelletable and free [3H]cholesterol were determined by scintillation counting. For “pellets,” the numbers on the ordinate correspond to the ratio of pelleted to pelleted plus free cholesterol. (B) Infectivity of the above virions was determined in a single-round assay on CD4+ HeLa P4 cells. Results are representative of at least five experiments. Infectivity of the untreated virus was given in each case the arbitrary value of 100%. (C) Infectivity of wild-type (R9) and VSV G-pseudotyped (R9ΔE/VSV G) virions produced by transfection with decreasing amounts of VSV G-expressing plasmid: 10 μg and 0.5 μg for R9ΔE/VSV G0.1 and R9ΔE/VSV G.2, respectively. Viral supernatants were treated or not with 10 mM methyl-β-cyclodextrin and concentrated by ultracentrifugation, and virion infectivity was determined on HeLa P4 cells. (D) Infectivity of control and cholesterol-depleted virions purified on a sucrose gradient after treatment with 10 mM methyl-β-cyclodextrin, expressed in infectious units per unit of reverse transcriptase activity. Infectivity of R9 and R9ΔE/VSV G virions was measured on HeLa P4 cells, while R8BaL virions were tested on HeLa P4-CCR5 cells. (E) Virus supernatants were treated with increasing concentrations of nystatin and purified by ultracentrifugation through a 20% sucrose cushion. Infectivity was determined as described for panel B.

FIG. 2.

Cholesterol-depleted purified particles exhibit normal protein profile. (A) Reverse transcriptase (RT) activity in sucrose density gradient fractions of native and methyl-β-cyclodextrin-treated R9 HIV-1 virions. (B) Immunoblot analysis with the indicated antisera of pooled fractions corresponding to intact virions of untreated (0 mM methyl-β-cyclodextrin [CD]) and methyl-β-cyclodextrin-treated (10 mM methyl-β-cyclodextrin) R9. Cyp A, cyclophilin A. (C) GPI-anchored protein profile of untreated, methyl-β-cyclodextrin-treated, and phospholipase C (PLipase)-treated virions with the proaerolysin/anti-proaerolysin overlay procedure (top). The same membrane was probed by Western blotting with an anti-CA antibody (bottom).

FIG. 3.

Cholesterol-depleted particles show a strong internalization defect. Virus attachment and internalization measured on HeLa P4 cells for wild-type (R9) and envelope-defective (R9ΔE) viruses after treatment of the virions with cyclodextrin (CD) where indicated. Results are representative of four independent experiments. The multiplicity of infection was 0.1. After binding (at 4°C) or internalization (at 37°C), cells were exposed to trypsin (+TRYP) or to PBS (−TRYP). The amount of cell-associated p24 after incubation with wild-type virus at 4°C was given the arbitrary value of 100%.

FIG. 4.

Confocal microscopic analysis of virus attachment and internalization. GFP-labeled wild-type (A and B), methyl-β-cyclodextrin-treated (C and D), and envelope-defective (E and F) viruses were used to infect CD4+ HeLa cells. Inocula contained equivalent amounts of reverse transcriptase corresponding to a multiplicity of infection of 1 for the wild-type untreated control. (A, C, and E) Virus attachment, assessed after incubating the virus with the cells at 4°C for 1 h. (B, D, and F) Virus internalization, examined after 2 h at 37°C. CD4 staining (red) was performed after fixation and without permeabilization to label only molecules present at the cell surface.

FIG. 5.

Confocal microscopic analysis of intracellular virus. GFP-labeled wild-type (A) and methyl-β-cyclodextrin-treated (B) HIV-1 particles and R9ΔE/VSV G (C) and methyl-β-cyclodextrin-treated R9ΔE/VSV G (D) viruses were used to infect CD4+ HeLa cells. Inocula contained equivalent amounts of reverse transcriptase corresponding to a multiplicity of infection of 1 for the wild-type untreated control. Virus internalization was examined after 2 h at 37°C. TAMRA-ConA was used to stain cell membranes.

FIG. 6.

Assessment of viral endocytosis. GFP-labeled wild-type R9ΔE/VSV G (A), methyl-β-cyclodextrin-treated R9ΔE/VSV G (B), wild-type HIV-1 (C), and methyl-β-cyclodextrin-treated HIV-1 (D) viruses were used to infect CD4+ HeLa cells. Inocula contained equivalent amounts of reverse transcriptase corresponding to a multiplicity of infection of 10 for the wild-type untreated control. Virus internalization was performed in the presence of Texas Red-conjugated transferrin to label endosomes and examined after 20 min (A and B) or 2 h (C and D) at 37°C.

FIG. 7.

PCR-based monitoring of reverse transcription. HeLa P4 cells were infected with wild-type HIV-1 in the absence (R9) or presence (R9/AZT) of AZT or with cholesterol-depleted (R9+CD) virus. The R/U5 primer pair amplifies the minus-strand strong-stop DNA, while R/Gag detects late products generated after the second template switch. β-Globin primers were used as a control to monitor the cellular DNA content of lysate samples.