REPLICATION

Human Immunodeficiency Virus Type 1 Envelope Glycoproteins That Lack Cytoplasmic Domain Cysteines: Impact on Association with Membrane Lipid Rafts and Incorporation onto Budding Virus Particles

Jayanta Bhattacharya, Paul J. Peters, Paul R. Clapham
DOI: 10.1128/JVI.78.10.5500-5506.2004

Article Figures & Data

Figures

  • FIG. 1.
    FIG. 1.

    The cytoplasmic domain of gp41. (A) LLP-1, LLP-2, and LLP-3 are putative α-helices that are proposed to interact with lipid bilayers. Palmitate groups covalently attached to C764 and C837 may insert into the lipid bilayer and anchor these regions to the plasma membrane. Palmitoylation may target gp41 into lipid rafts (21). (B) Amino acid substitutions introduced into the gp41 cytoplasmic domain.

  • FIG. 2.
    FIG. 2.

    Envelope expression and function. (A) 293T cells were cotransfected with pNL43env− and pSVIIIenv containing gp41 envelope mutants (C764/Y837 [CY], F764/Y837 [FY], A764/A837 [AA], and S764/S837 [SS]) and were immunostained for envelope. Cells were fixed and permeabilized with methanol-acetone (1:1) 48 h after transfection before immunostaining with anti-gp41 MAb Chessie 8 (1). (B) Cell surface envelope expression. 293T cells (transfected as described for panel A) were stained for surface envelope expression by using anti-gp120 MAb 902 (5, 19) and examined by flow cytometry. (C) Cell-cell fusion induced by NL43 mutant envelopes. 293T cells (transfected as described for panel A) were cocultivated with NP2/CD4/CXCR4. Cells were fixed and stained with 1% methylene blue-0.25% basic fuchsin in methanol.

  • FIG. 3.
    FIG. 3.

    Infectivity conferred by HIV-1 NL43 envelopes lacking gp41 cytoplasmic cysteines. Infectivity was tested with CD4+ indicator cell lines as shown. (A) GHOST parental cells that express endogenous CXCR4; (B) NP2/CD4/CXCR4 cells; (C) GHOST/CXCR4 cells. Cells were fixed 72 h after infection and immunostained for p24. Infectivity titers were then scored as focus-forming units (FFU) per picogram of RT in virus supernatants as measured by ELISA.

  • FIG. 4.
    FIG. 4.

    Incorporation of mutant NL43 envelopes onto virus particles. 293T cells were cotransfected with pNL43env− and pSVIIIenv containing gp41 envelope mutants (C764/Y837 [CY], F764/Y837 [FY], A764/A837 [AA], and S764/S837 [SS]). Supernatants harvested 48 h after transfection were spun at low speed and filtered through a 0.45-μm-pore-size syringe filter. The supernatants were then ultracentrifuged at 100,000 × g for 2 h at 4°C to pellet virus particles. The pelleted virions were resuspended and measured for RT activity. Equivalent amounts of RT activity were then resolved on a sodium dodecyl sulfate-8% polyacrylamide gel electrophoresis gel followed by Western blotting with anti-gp41 Chessie 8 or anti-p24 183-H12-5C as primary antibodies. (A) Western blot analysis of envelope on virions; (B) ratios of envelope to p24 gag concentration evaluated by densitometry of Western blot bands.

  • FIG.5.
    FIG.5.

    Association of mutant NL43 envelopes with lipid rafts. 293T cells were cotransfected with pNL43env− and pSVIIIenv containing gp41 envelope mutants. Cells were lysed with 0.5% Triton X-100 in TNE buffer on ice. Lysates were homogenized, cleared of nuclei, adjusted to 60% sucrose, and applied to the bottom of a sucrose gradient (see text). Gradient fractions were Western blotted and probed for envelope and gag with mouse MAbs. (A) Sucrose gradient fractionation of cold Triton X-100 extracts from 293T cells cotransfected with pNL43env− and pSVIIIenv encoding the NL43 envelope mutants C764/Y837 (CY), F764/Y837 (FY), A764/A837 (AA), and S764/S837 (SS). Note that the NL43 CY envelope associates with both the DRM-L and the DRM-H fractions. FY is excluded from DRM-L but retains an association with DRM-H. The AA and SS envelopes are completely excluded from rafts. (B) Sucrose gradient fractionations, as described for panel A except that Triton X-100 extractions were carried out at 37°C to solubilize lipid rafts. Note that all the gag precursor and envelope detected is present in the fractions representing soluble membranes and not at densities that correspond to lipid rafts. (C) Sucrose gradient of 293T cold Triton X-100 extracts, blotted and probed with a rabbit polyclonal antibody to caveolin (molecular mass, 20 to 22 kDa; DRM-L marker) (BD Biosciences Transduction Laboratories Inc.).

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