TABLE 1.
CCR5-expressing clones derived from HI-J and HI-R HeLa CD4 cells
| Clonea | Mean rabbit anti-CCR5 binding (cpm/μg of protein) ± SDb | CCR5 expression (molecules/cell)c |
|---|---|---|
| HI-J control | 3 ± 1 | 0 |
| JC.20 | 4 ± 1 | <7.0 × 102 |
| JC.10 | 6 ± 1 | 2.0 × 103 |
| JC.57 | 17 ± 2 | 9.2 × 103 |
| JC.58 | 23 ± 8 | 1.3 × 104 |
| JC.37 | 26 ± 4 | 1.5 × 104 |
| JC.55 | 33 ± 3 | 2.0 × 104 |
| JC.47 | 34 ± 9 | 2.0 × 104 |
| JC.6 | 44 ± 18 | 2.7 × 104 |
| JC.54 | 49 ± 10 | 3.0 × 104 |
| JC.48 | 79 ± 9 | 5.0 × 104 |
| JC.24 | 141 ± 22 | 9.1 × 104 |
| JC.53 | 201 ± 27 | 1.3 × 105 |
| HI-R control | 3 ± 0.1 | 0 |
| RC.30 | 6 ± 2 | 2.4 × 103 |
| RC.33 | 7 ± 1 | 3.2 × 103 |
| RC.6 | 7 ± 0.4 | 3.2 × 103 |
| RC.15 | 8 ± 1 | 4.8 × 103 |
| RC.10 | 11 ± 3 | 7.1 × 103 |
| RC.56 | 11 ± 1 | 7.9 × 103 |
| RC.23 | 13 ± 3 | 8.7 × 103 |
| RC.4 | 13 ± 1 | 9.5 × 103 |
| RC.55 | 25 ± 4 | 2.1 × 104 |
| RC.28 | 39 ± 9 | 3.3 × 104 |
| RC.12 | 57 ± 10 | 4.3 × 104 |
| RC.17 | 62 ± 16 | 5.6 × 104 |
| RC.25 | 85 ± 15 | 7.8 × 104 |
| RC.49 | 92 ± 16 | 8.5 × 104 |
Cells stably expressing CCR5 were generated by retroviral transduction of HeLa-CD4 clones HI-J and HI-R. See Materials and Methods for further details.
CCR5 expression levels on cell surfaces were measured for all clones by binding rabbit antiserum to CCR5 followed by [125I]protein A as described in Materials and Methods. Protein content per sample equaled approximately 50 μg. Values represent the means of three or four assays (JC and RC clones, respectively), with each assay performed in duplicate. HI-R cells were assayed twice; the range is displayed. The RC and JC clones were analyzed at different times using different batches of [125I]protein A and different bleeds of rabbit anti-CCR5. Multiple cultures of the JC.53 clone assayed simultaneously with RC clones yielded an average value of 167 cpm/μg of protein, and the results for the RC clones were normalized relative to this internal JC.53 standard. A conversion factor of 1.2 (representing the ratio of the two anti-CCR5-specific binding values on JC.53 cells obtained while assaying JC [198 cpm/μg of protein] and RC clones [164 cpm/μg of protein]) was used to adjust RC clone cell surface CCR5 values to the JC scale. The number of CCR5 molecules per cell for the JC.53 cells did not change during this time period (see Fig. 2 and Results).
The JC.53 CCR5 expression level, determined by titration with [125I]MIP1β, was approximately 1.3 × 105 molecules per cell (see Fig. 2 and Results). The cell surface CCR5 values shown were calculated by subtracting control anti-CCR5 binding values from the binding value for each clone, dividing this difference by the comparable difference obtained with clone JC.53, and then multiplying the ratio by 1.3 × 105 CCR5 molecules per cell (see Fig. 2 and results).