Article Figures & Data
Figures
- FIG. 1.
Schematic of MLV Env protein. Sequences are the C-terminal cytoplasmic tails of MLV Env, GaLV Env, and human CD4. GaLV sequences in boldface are residues that have been shown to modulate the HIV-1 incompatibility (4). Underlined sequences in CD4 are amino acids required for Vpu-mediated downmodulation (2, 15). Arrows denote the location of MLV/GaLV tail substitution. SU, surface domain; TM, transmembrane domain.
- FIG. 2.
Distribution of MLV Env relative to HIV-1 assembly sites. 293T mCAT-1 cells were cotransfected with a plasmid expressing late domain-defective HIV-1 Gag and a plasmid expressing YFP-tagged MLV Env (A) or YFP-tagged MLV/GaLV Env (B). Env was labeled with 12-nm gold and imaged by SEM. Left, secondary electron images of HIV-1 assembly sites. Right, backscatter electron images of gold-labeled Env. Scale bars, 200 nm.
- FIG. 3.
HIV-1 accessory genes modulate MLV/GaLV Env restriction. (A) Schematic of HIV-1 packaging constructs ΔR8.2. (B) ΔR8.2 or its derivatives containing the accessory genes shown were cotransfected with reporter vector pSIN18.cPPT.hEF1a.EGFP.WPRE and either MLV Env or MLV/GaLV Env. Viral supernatant was transferred from transfected 293FT cells to 293T mCAT-1 cells, and infectivity was determined by flow cytometry. (C) Output of the same experiment with the infectivity of each construct expressed as the ratio of infections with MLV/GaLV Env to infections with MLV Env.
- FIG. 4.
Vpu modulates HIV-1 infectivity with MLV/GaLV Env. Infectivity of MLV Env, MLV/GaLV Env, or an empty DNA vector (control) pseudotyped with HIV-1 was determined in the absence (▪) or presence (□) of Vpu. Infectivity was measured as infectious units (I.U.) per ml of viral supernatant transferred from transfected 293FT cells to 293T mCAT-1 cells 48 h posttransfection. The averages and standard deviations of infectious units per milliliter of seven independent experiments with the same combinations are shown. Significant differences (*, P < 0.05) and nonsignificant differences (NS) were determined by two-tailed Student's t test for each envelope type in the presence or absence of Vpu.
- FIG. 5.
Vpu prevents MLV/GaLV Env from being incorporated into HIV-1 particles. (A) 293FT cells were transfected with HIV-CMV-GFP containing no Vpu (Vpu−), Vpu wt, VpuRD, or Vpu52/56, and MLV Env or MLV/GaLV Env. For the upper panel, Western blot analysis performed on the transfected cells and pelleted viral supernatants. The lower panel shows the infectivity output from the same experiment. Infectious units per ml were normalized to p24 levels. (B) The averages and standard deviations of infectious units per milliliter of six independent experiments with the same combinations are shown. Significantly different means for each Env treatment are indicated by unique letters (Tukey-Kramer HSD, P < 0.05).
- FIG. 6.
Vpu does not prevent MLV/GaLV Env surface expression. (A) Schematic of surface labeling experiment. 293FT cells were transfected HIV-CMV-GFP (+/− Vpu) in the presence or absence of MLV Env-GFP or MLV/GaLV Env-GFP. At 48 h posttransfection, cells were collected and stained live for surface GFP expression with Alexa Fluor 647-conjugated anti-GFP antibody, and the supernatant was transferred to 293T mCAT-1 cells and assayed for infectivity. (B) Surface expression and infectivity of Env proteins. The average surface GFP intensity/total GFP intensity of transfected cells is reported in the upper right-hand corner of surface expression scatter plots. Infectivity scatter plots show infections on the x axis; the y axis (FL2) is not relevant for this experiment, but is used to maintain visual consistency. Infectivity is shown in each plot as the percentage of the 293T mCAT-1 cells infected with HIV-CMV-GFP.
- FIG. 7.
Vpu does not prevent the production of infectious MLV particles. (A) Schematic of dual infection assay. 293FT cells were transfected with HIV-1 and MLV assembly components, along with MLV or MLV/GaLV Env. At 48 h posttransfection the supernatant was transferred to 293T mCAT-1 cells. The ratio of HIV-1 to MLV was adjusted so that similar HIV-1 and MLV infectious particles were produced with MLV/GaLV Env and so that same ratio was used in each of the four transfections. (B) Flow cytometry output of 293T mCAT-1 infections. MLV infections display red fluorescence (y axis), and HIV-1 infections display green fluorescence (x axis). Infectivity is shown in each plot as percentage of the 293T mCAT-1 cells infected, excluding double-positive cells.
