Cholesterol depletion of human immunodeficiency virus type 1 and simian immunodeficiency virus with beta-cyclodextrin inactivates and permeabilizes the virions: evidence for virion-associated lipid rafts

Abstract

Recent evidence suggests that human immunodeficiency virus type 1 (HIV-1) particles assemble and bud selectively through areas in the plasma membrane of cells that are highly enriched with glycosylphosphatidylinositol-anchored proteins and cholesterol, called lipid rafts. Since cholesterol is required to maintain lipid raft structure and function, we proposed that virion-associated cholesterol removal with the compound 2-hydroxy-propyl-beta-cyclodextrin (beta-CD) might be disruptive to HIV-1 and simian immunodeficiency virus (SIV). We examined the effect of beta-CD on the structure and infectivity of cell-free virions. We found that beta-CD inactivated HIV-1 and SIV in a dose-dependent manner and permeabilized the viral membranes, resulting in the loss of mature Gag proteins (capsid, matrix, nucleocapsid, p1, and p6) without loss of the envelope glycoproteins. SIV also lost reverse transcriptase (RT), integrase (IN), and viral RNA. IN appeared to be only slightly diminished in HIV-1, and viral RNA, RT, matrix, and nucleocapsid proteins were retained in HIV-1 but to a much lesser degree. Host proteins located internally in the virus (actin, moesin, and ezrin) and membrane-associated host proteins (major histocompatibility complex classes I and II) remained associated with the treated virions. Electron microscopy revealed that under conditions that permeabilized the viruses, holes were present in the viral membranes and the viral core structure was perturbed. These data provide evidence that an intact viral membrane is required to maintain mature virion core integrity. Since the viruses were not fixed before beta-CD treatment and intact virion particles were recovered, the data suggest that virions may possess a protein scaffold that can maintain overall structure despite disruptions in membrane integrity.

FIG. 1.

Treatment of fixed concentrations of SIV and HIV-1 with increasing concentrations of β-CD results in permeabilization of the viral membrane and loss of capsid protein. A fixed amount of either SIV (A, C, and E) or HIV-1 (B, D, and F) was treated with increasing concentrations of β-CD for 1 h at 37°C. Samples were analyzed by SDS-PAGE and silver staining (A and B). Levels of gp120 and capsid (p28 for SIV; p24 for HIV) were determined by Western blotting (C and D), and band intensity was determined by densitometry analysis (E and F). Bars: ░⃞, densitometry results for gp120; ▪, densitometry results for capsid protein. None, concentrated virus stock prior to processing.

FIG. 2.

Treatment of either SIV or HIV-1 with fixed concentrations of β-CD and decreasing concentrations of virus resulted in permeabilization of the viral membrane and loss of capsid protein. Decreasing concentrations of capsid for either SIV (A, C, and E) or HIV-1 (B, D, and F) were treated with 20 mM β-CD for 1 h at 37°C. Samples were analyzed by SDS-PAGE and silver staining (A and B). Levels of gp120 and capsid (p28 for SIV and p24 for HIV) were determined by immunoblotting (C and D), and the band intensity was determined by densitometry analysis (E and F). Bars: ░⃞, densitometry results for gp120; ▪, the densitometry results for capsid protein. None, concentrated virus stock prior to processing.

FIG. 3.

Permeabilization correlated with total depletion of cholesterol from virus and could be predicted based on the ratio of β-CD to cholesterol present in the sample. Residual cholesterol was measured in both SIV and HIV-1 treated with increasing concentrations of β-CD (A). The ratio of β-CD to the initial cholesterol level was calculated for SIV and HIV for each set of treatment conditions (B), and permeabilization levels estimated from Fig. 1 are indicated by intersecting solid lines. Error bars represent the standard deviation of the mean.

FIG. 4.

Treatment of SIV or HIV-1 under nonpermeabilizing conditions resulted in loss of cholesterol, whereas treatment of SIV or HIV-1 under permeabilizing conditions resulted in loss of proteins found inside the virion. Concentrated SIV was diluted and prepared under nonpermeabilizing (A) or permeabilizing (C) conditions and was examined by HPLC. Concentrated HIV-1 was diluted and prepared under nonpermeabilizing (B) or permeabilizing (D) conditions and examined by HPLC. The upper trace is untreated virus, and the lower trace is β-CD-treated virus in all four panels. Loading was corrected for gp120 content. Peaks were identified by amino acid analysis and are indicated on the upper trace.

FIG. 5.

Differential retention of RT and IN in permeabilized SIV and HIV-1. Stocks of virus prepared for Fig. 1 were normalized to gp120 content and RT, IN, and capsid protein were examined by immunoblotting. (A) Results obtained for SIV; (B) results obtained for HIV-1.

FIG. 6.

SIV and HIV-1 lose viral RNA upon permeabilization. Stocks of virus prepared for Fig. 1 were analyzed for particle-associated viral RNA by real-time RT-PCR. (A) Results obtained for SIV; (B) results obtained for HIV-1. Virus input was normalized to gp120 content.

FIG. 7.

Suppression of viral replication and loss of titer correlate with treatment of either SIV or HIV-1 with increasing doses of β-CD. Stocks of virus prepared for Fig. 1 were analyzed for both replication and infectivity on a cell line (LuSIV) that expresses luciferase upon infection with either SIV or HIV-1. SIV (A and C) or HIV-1 (B and D) was treated with increasing concentrations of β-CD (see legend in figure). Viral replication as measured by luciferase expression was then determined (A and B), and titers were calculated (C and D), under limiting dilution. Luciferase levels were measured 60 h after infection of the indicator lines. The mean luciferase value of three replicate wells is given for each dilution (A and B). The titer is expressed as the reciprocal dilution of the lowest dilution at which a positive well was scored (C and D). NP, concentrated viral stock not processed.

FIG. 8.

Electron microscopy of SIV treated under permeabilizing conditions revealed holes in the membrane of the virus. SIV samples that were untreated (A and D), treated under nonpermeabilizing conditions (B and E), or treated under permeabilizing conditions (C and F to I) were examined by transmission electron microscopy. Panels G and I are larger magnifications of the images in panels F and H, respectively. Magnifications: ×4,000 (A), ×20,000 (B to F and H), and ×40,000 (G and I).

FIG. 9.

Electron microscopy of HIV-1 treated under permeabilizing conditions revealed holes in the membrane of the virus. HIV-1 samples that were untreated (A and D), treated under nonpermeabilizing conditions (B and E), or treated under permeabilizing conditions (C and F to K) were examined by transmission electron microscopy. Magnifications: ×4,000 (A and F) and ×20,000 (B to E and G to K).