Article Figures & Data
Figures
- FIG. 1.
Mutation sites on YU-2 gp120. Space-filling model of YU-2 gp120, with the conserved bridging sheet domain and two-domain sCD4 in ribbon format. Mutated residues are indicated, as are the V1/V2 and V3 loop stems. All of the mutations lie within or adjacent to the bridging sheet strands. The model was rendered with RasMol 2.7.1 from protein databank file 1G9N; YU-2 gp120 envelope glycoprotein complexed with CD4 and induced neutralizing antibody 17b (29).
- FIG. 2.
Receptor binding efficiencies of YU-2 mutant Envs. The CD4 and CCR5 binding efficiencies of YU-2 and YU-2 mutant Envs were determined by immunostaining and flow cytometry analysis of gp120 binding to the surface of (A) NP2 and NP2/CD4 cells and (B) TREx/CCR5 cells in the presence and absence of sCD4. Results represent the average and standard error of the mean of three independent experiments.
- FIG. 3.
Expression analysis of YU-2 mutant Envs. Env expression was analyzed in QT6 cells transfected with Env expression plasmids and infected with a vaccinia virus encoding T7 polymerase to drive expression. Total Env expression was determined by SDS-PAGE and Western blot analysis of cell lysates, followed by detection of Env with a specific rabbit serum and a horseradish peroxidase-conjugated anti-rabbit immunoglobulin antibody. Expression was quantitated with Fujifilm Science Lab 98 Image Gauge software to analyze chemiluminescent signals imaged on a Fujifilm LAS-1000 Plus luminescent image analyzer. Serial threefold dilutions of cell lysates ensured that detection was within the linear range of the assay. Results represent the average and standard error of the mean of four independent experiments. Cell surface Env expression was detected by immunostaining and flow cytometry analysis. Results represent the mean fluorescence intensity of a representative experiment, expressed as a percentage of the mean intensity obtained with YU-2.
- FIG. 4.
Fusion and infection efficiencies of YU-2 Env mutants. (A) Relative fusion was determined in a cell-cell fusion luciferase reporter assay with QT6 effector cells expressing the indicated Envs and T7 polymerase and QT6 target cells expressing CD4, CCR5, and a T7 luciferase reporter plasmid. Results are expressed as a percentage of YU-2-mediated fusion and represent the average and standard error of the mean of at least seven independent experiments. (B) Relative luciferase reporter pseudotype virus infection of U87/CD4/CCR5 cells. Envs were pseudotyped onto the surface of a reporter virus core, and virus stocks, normalized by p24 assay, were used to infect U87/CD4/CCR5 cells. Cell lysates were assayed for luciferase activity 3 days postinfection. Results are expressed as a percentage of the luciferase activity in YU-2-infected cells and represent the average and standard error of the mean of at least three independent experiments.
- FIG. 5.
Kinetics of YU-2 Env mutant fusion. The efficiency and kinetics of YU-2 Env-mediated cell-cell fusion were determined in a β-lactamase reporter assay with QT6 effector cells expressing the indicated Env, a β-lactamase reporter plasmid and T7 polymerase, and CD4/CCR5+ HeLa target cells loaded with the green fluorescent dye CCF2-AM. Upon cell-cell fusion, β-lactamase cleavage of the CCF2 substrate results in a change from green to blue fluorescent emission that was quantitated in a fluorometer. (A) Fusion expressed as a ratio of blue to green fluorescence. Points represent the average ± standard deviation of triplicate wells from an individual experiment. The data are representative of at least three independent experiments. (B) Fusion (ratio of blue to green fluorescence) expressed as a percentage of maximum fusion for each Env to aid comparison of relative kinetic profiles.
- FIG. 6.
sCD4 triggering of YU-2 mutant Env fusion. sCD4-induced fusion was determined in a cell-cell fusion luciferase reporter assay with QT6 effector cells expressing Env and T7 polymerase and QT6 target cells expressing a T7 luciferase reporter plasmid and CCR5 receptor only. The indicated amount of sCD4 was added to target and effector cells, and luciferase activity was measured 7 h later. Fusion is expressed as a percentage of that observed with YU-2 on target cells expressing a T7 luciferase reporter plasmid and both cellular CD4 and CCR5. Results represent the average and standard error of the mean of four independent experiments.
- FIG. 7.
Entry inhibitor sensitivity of YU-2 Env mutants. Fifty percent inhibition (IC50) values for fusion inhibition of YU-2 mutants by (A) a CD4-specific monoclonal antibody, (B) TAK-779, (C) enfuvirtide, and (D) T-1249, determined in a cell-cell fusion luciferase reporter assay with QT6 effector cells expressing Env and T7 polymerase and QT6 target cells expressing CD4, CCR5 and a T7 luciferase reporter plasmid. The concentration of each inhibitor needed to reduce fusion activity by 50% was determined in at least three independent experiments, and results represent average values and the standard error of the mean.
- FIG. 8.
Replication efficiency of YU-2 mutant viruses. Growth curves of replication-competent YU-2 mutant viruses in the (A) CEMSS-R5 T-cell line and (B) CD8+-T-cell-depleted PBMC cultures, with 1 or 10 ng of p24-normalized input virus/105 cells as indicated. Input virus was removed by extensive washing, and viral replication was evaluated by analyzing culture supernatants for p24 production at the indicated days postinfection. Results represent average p24 values plus standard deviation of the results for triplicate wells from representative experiments.
