TABLE 1.

Phenotypes of HIV-1YU2 mutants

Envelope glycoproteinResidue locationIndexaBindingCell-to-cell fusiondG3-299 recognitione
AssociationProcessingCD4bCCR5c
Wild-type YU21.001.001.00++++1.001.00
V120Sβ20.291.281.20NDND1.82
L122Rβ20.441.150.77NDND1.62
V200Aβ3NDND1.10NDND0.93
V200Sβ30.71.690.60NDND1.21
I307AV3 loop0.251.10ND++0.05ND
I307EV3 loop0.301.030.86+0.020.20
I307LV3 loop0.631.340.79++++1.000.26
I307SV3 loopNDND0.69NDND0.10
I309AV3 loop0.341.450.71+++0.810.32
I309SV3 loop0.281.250.81+++0.480.10
I309LV3 loop1.071.310.98++++1.010.81
L317AV3 loop0.321.52ND+++0.84ND
L317EV3 loop0.291.450.88++0.020.56
L317SV3 loop0.211.830.780.030.60
L317FV3 loop0.821.050.72NDND0.61
L317IV3 loop1.291.070.66++++1.050.35
I420Sβ190.281.650.30+0.791.83
I423Sβ200.192.160.51++++0.031.75
I423Rβ200.142.540.60++++0.010.83
I423Lβ200.152.590.82++++0.910.91
I424Sβ200.211.910.45++++0.441.22
M434Sβ210.271.280.47NDND0.15
M434Rβ210.680.500.45NDND0.16
  • a The processing and association indices were determined by precipitation of radiolabeled cell lysates and supernatants with mixtures of sera from HIV-1-infected individuals. The association index is a measure of the ability of the mutant gp120 molecule to remain associated with the envelope glycoprotein complex on the expressing cell, relative to that of the wild-type envelope glycoproteins. The association index is calculated as follows: association index = ([mutant gp120]cell × [wild-type gp120]supernatant)/([mutant gp120]supernatant × [wild-type gp120]cell). The processing index is a measure of the conversion of the mutant gp160 envelope glycoprotein precursor to mature gp120, relative to that of the wild-type envelope glycoproteins. The processing index was calculated by the formula: processing index = ([total gp120]mutant × [gp160]wildtype)/([gp160]mutant × [total gp120]wildtype). Additional HIV-1 YU2 gp120 mutants that exhibited association indices of <0.20 were K121D, R298G, E381R, K421A, and P437A (data not shown). ND, not determined.

  • b Radiolabeled wild-type and mutant gp120 glycoproteins in the supernatants of envelope-expressing 293T cells were incubated with various amounts of sCD4-Ig for 2 h at 37°C in the presence of 70 μl of 10% protein A-Sepharose (American Biosciences). At a near-saturating concentration of sCD4-Ig for the wild-type gp120 glycoprotein, the relative ratio of the mutant gp120 glycoprotein precipitated is reported. ND, not determined.

  • c The CCR5-binding ability of the HIV-1 gp120 envelope glycoprotein variants was determined as described in Materials and Methods. The amount of bound mutant gp120 glycoprotein was compared to the amount of bound wild-type gp120 glycoprotein. The relative CCR5-binding ability is reported as follows: ++++, 75 to 100% of the wild-type gp120 level; +++, 50 to 74% of the wild-type level; ++, 25 to 49% of the wild-type level; +, 5 to 24% of the wild-type level; and -, <5% of the wild-type level. ND, not determined.

  • d To assess cell-to-cell fusion, 3 × 105 293T cells were cotransfected by the calcium phosphate method with an HIV-1 Tat-expressing plasmid, pLTR-Tat, and the pSVIIIenv plasmid expressing the HIV-1YU2 envelope glycoproteins. At 2 days after transfection, 3 × 104 293T cells were added to TZM-bl target cells that were seeded at a density of 3 × 104 cells/well in 96-well luminometer-compatible tissue culture plates (Dynex) 24 h before the assay. Cells were coincubated for 6 h at 37°C, after which they were lysed by the addition of 30 μl of passive lysis buffer (Promega) and three freeze-thaw cycles. The luciferase activity in each well was measured as described above. The reported value represents the ratio of the luciferase activity observed for the mutant envelope glycoproteins relative to that of the wild-type envelope glycoproteins. ND, not determined.

  • e Radiolabeled wild-type and mutant gp120 glycoproteins in the supernatants of 293T cells expressing the HIV-1 YU2 envelope glycoproteins were precipitated by the G3-299 antibody for 2 h at 4°C in the presence of Complete protease inhibitor cocktail (Roche Applied Science). Precipitates were analyzed as described in Materials and Methods. At a near-saturating concentration of G3-299 antibody for the wild-type gp120 glycoprotein, the relative ratio of the mutant gp120 glycoprotein precipitated is reported. Relative values for G3-299 recognition for mutant HIV-1 YU2 gp120 glycoproteins not shown in the table were as follows: m1, 1.07; m2, 0.77; H66A, 1.91; W69L, 2.17; L111A, 0.59; S375W, 0.26; H66A/S375W, 0.55; W69L/S375W, 0.73; and L111A/S375W, 0.60. ND, not determined.