TABLE 1

Phenotypes of SIVmac239 gp120 layer 3 variantsa

Env mutationAssociation indexbProcessing indexcInfectivitydCell-to-cell fusioneCD4-Ig bindingfCCR5 bindinggCD4-Ig binding by SPRh
Without sCD4With sCD4On-rate (M−1s−1)Off-rate (s−1)KD, M (fold change)
None (WT)1.001.001.001.001.001.001.001.63 × 1043.39 × 10−42.09 × 10−8 (1.0)
T248A1.250.370.030.770.030.980.641.91 × 1043.15 × 10−31.65 × 10−7 (7.93)
R249A1.070.280.050.810.02NDNDNDNDND
E252A0.720.840.681.290.32NDNDNDNDND
T253A0.770.860.901.150.10NDNDNDNDND
Q254A1.120.771.201.190.64NDNDNDNDND
A476G0.961.191.211.240.521.040.959.85 × 1034.16 × 10−44.22 × 10−8 (2.02)
E477A0.980.930.071.300.101.290.676.62 × 1036.88 × 10−41.04 × 10−7 (4.98)
L478A0.420.940.010.380.030.390.09NANANA
Y479A0.460.650.041.470.350.910.498.37 × 1035.89 × 10−47.04 × 10−8 (3.37)
R480A0.500.470.181.020.34NDNDNDNDND
L481A0.480.230.021.200.04NDNDNDNDND
E482A0.620.620.091.000.190.520.666.62 × 1035.98 × 10−49.03 × 10−8 (4.33)
L483A0.920.380.140.930.140.780.58NANANA
  • a Values represent the means of data from at least three independent experiments, with experimental variation typically not more than 20% of the value reported. In the case that WT values were normalized to 1, signals of ≤0.5 are in italics. ND, not determined.

  • b 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 calculation method is described in Materials and Methods.

  • c 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 calculation method is described in Materials and Methods.

  • d The infectivity was assessed on Cf2Th-CD4/CCR5 cells using RT-normalized amounts of pseudoviruses of WT and SIVmac239 layer 3 variants. Data are the ratios of mutant to wild-type virus infectivity.

  • e Cell-to-cell fusion activity was assessed by coincubation between 293T cells expressing Env variants and TZM-bl cells for 6 h at 37°C. Luciferase activity in the mixture of cell lysate was measured to determine the cell-to-cell fusion efficiency.

  • f Comparable amounts of radiolabeled monomeric gp120 from WT and layer 3 variants were incubated with 2 μg/ml of CD4-Ig for 1 h at 37°C. The precipitates were washed, run on SDS-polyacrylamide gels, and analyzed by densitometry.

  • g Comparable amounts of radiolabeled monomeric gp120 from WT and selected layer 3 variants were incubated in the presence or absence of 200 nM sCD4 at 37°C for 1 h prior to addition to Cf2Th-CCR5 cells. The amount of bound mutant gp120 was determined and normalized to the observed amount of bound WT gp120.

  • h CD4-Ig was immobilized directly onto a CM5 sensor chip and the binding of the indicated gp120 protein was evaluated as described in Materials and Methods. KD, equilibrium dissociation constant. NA, not applicable (signal below the limit of detection).