Evidence for budding of human immunodeficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts

Abstract

A number of recent studies have demonstrated the significance of detergent-insoluble, glycolipid-enriched membrane domains or lipid rafts, especially in regard to activation and signaling in T lymphocytes. These domains can be viewed as floating rafts composed of sphingolipids and cholesterol which sequester glycosylphosphatidylinositol (GPI)-linked proteins, such as Thy-1 and CD59. CD45, a 200-kDa transmembrane phosphatase protein, is excluded from these domains. We have found that human immunodeficiency virus type 1 (HIV-1) particles produced by infected T-cell lines acquire the GPI-linked proteins Thy-1 and CD59, as well as the ganglioside GM1, which is known to partition preferentially into lipid rafts. In contrast, despite its high expression on the cell surface, CD45 was poorly incorporated into virus particles. Confocal fluorescence microscopy revealed that HIV-1 proteins colocalized with Thy-1, CD59, GM1, and a lipid raft-specific fluorescent lipid, DiIC(16)(3), in uropods of infected Jurkat cells. CD45 did not colocalize with HIV-1 proteins and was excluded from uropods. Dot immunoassay of Triton X-100-extracted membrane fractions revealed that HIV-1 p17 matrix protein and gp41 were present in the detergent-resistant fractions and that [(3)H]myristic acid-labeled HIV Gag showed a nine-to-one enrichment in lipid rafts. We propose a model for the budding of HIV virions through lipid rafts whereby host cell cholesterol, sphingolipids, and GPI-linked proteins within these domains are incorporated into the viral envelope, perhaps as a result of preferential sorting of HIV Gag to lipid rafts.

FIG. 1

Flow cytometry and virus capture. (A) Jurkat cells were fixed with 2% paraformaldehyde and stained with MAbs specific for gp41, MHCI, CD45, Thy-1, and CD59. Antibodies were then detected with FITC-conjugated GAM IgG. FITC log fluorescence intensity is plotted on the x axis, and the number of counts is plotted on the y axis. % Pos, percentage of cells with >7.15 fluorescence intensity; MTF, mean total fluorescence of all cells in the sample. (B) Virus capture immunoassays to determine relative viral phenotypes were performed with MAbs specific for gp41, MHCI, CD45, Thy-1, and CD59 as described in Materials and Methods. (C) Jurkat cells labeled with soluble HRP were captured with the indicated MAbs in an assay similar to the virus capture as described in the Materials and Methods. Error bars represent the standard deviation in panels B and C.

FIG. 2

βCD effects on GPI-linked protein incorporation. Jurkat cells were treated with or without 20 mM βCD in cRPMI for 1 h at 37°C. Virions produced for 2 h posttreatment were precipitated with MAbs to CD59, Thy-1, CD45, and gp41. IgG2b was used as a nonspecific control. The captured p24 was measured and expressed as a percentage of the total p24 in the supernatant. Error bars represent the standard deviation.

FIG. 3

CTB captures GM1 on HIV-1. CTB in cRPMI was added to viral supernatants to the indicated concentrations. Goat anti-CTB and SaC were added to precipitate viral CTB complexes. The captured virus was lysed and p24 was measured and expressed as a percentage of the total p24 from the original supernatant. NV, no virus control. Error bars represent the standard deviation.

FIG. 4

Colocalization of Thy-1, CD59, and GM1 into areas of HIV protein expression and the exclusion of CD45 from the site of HIV surface protein localization. Infected Jurkat cells were labeled with a primary antibody to a host antigen, followed by an appropriate FITC-conjugated secondary (GAM or goat anti-rabbit) IgG, and then labeled with biotinylated anti-HIV human polyclonal antibody and detected with streptavidin-Texas red. Superimposed FITC and Texas red images are viewed in column 3 of all panels. Colocalization is indicated by the yellow color. Each panel was labeled with the following antibodies: A, anti-CD59 MAb p282(H19); B, anti-Thy-1 MAb 5E10; C, anti-CD45 MAb H5A5; and D, rabbit anti-GM1 polyclonal antibody. The figure was prepared on a Silicon Graphics Workstation with Showcase software.

FIG. 5

DiIC₁₆(3) colocalizes with HIV, while DiIC₁₂(3) does not. Infected Jurkat cells were incubated with DiIC₁₆(3) (A) or DiIC₁₂(3) (B) for 15 min on ice before being fixed in 2% paraformaldehyde-PBS. Cells were then labeled with CD4Ig or anti-HIV IgG and FITC-labeled sheep anti-human IgG. Dialkylindocarbocyanine lipids are shown in red, FITC staining is shown in green, and the overlap is indicated in yellow. All of the micrographs in this figure were labeled with CD4Ig, except the last micrograph in panel A, which was labeled with anti-HIV IgG.

FIG. 6

HIV matrix, p17, and gp41 are colocalized with GEM domains upon equilibrium centrifugation of Triton X-100-treated Jurkat cells. Jurkat cells were treated with 1% Triton X-100 at 4°C as described in Materials and Methods. The lysates were equilibrium centrifuged on a discontinuous sucrose gradient, and 1-ml fractions were collected from the bottom and labeled 1 through 10. (A) Fraction 10 is the bottom fraction, while fraction 1 corresponds to the top of the tube. GEM domains are indicated as fractions 3 through 5 as previously identified (36). For immunoblots, each MAb was incubated with lysates from uninfected Jurkat cells (top row) and infected Jurkat cells (bottom row). The figure was prepared from scanned blots in Adobe Photoshop on a Power Macintosh 7600. (B) Blots were quantitated as described in Materials and Methods, and the amounts in the GEM domains or soluble fractions were determined as a percentage of the total of all the dots.

FIG. 7

[³H]myristic acid-labeled Gag partitions to GEM domains. Jurkat cells labeled with [³H]myristic acid were treated with 1% Triton X-100 at 4°C and then equilibrium centrifuged. GEM domain fractions 3 to 5 were pooled and precipitated for Gag. IgG was used as a negative control. Soluble fractions 8 to 10 were pooled and precipitated for Gag as well. Precipitated proteins were blotted onto nitrocellulose and exposed to film. Blots were quantitated as described in Materials and Methods and expressed as a percentage of the total.