TABLE 4.
Env processing and stability in LAI Env mutants
| Clone | Processing indexa | Association indexa | Env glycoprotein concn (ng/ml) inb:
|
|
|---|---|---|---|---|
| Lysates | Supernatants | |||
| Wild type | 1.00 | 1.00 | 953 ± 92 | 61 ± 5 |
| FivL | 0.44 | 0.01 | ND | ND |
| SivL | 0.26 | 0.16 | ND | ND |
| t35L | 0.66 | 0.05 | ND | ND |
| p34L | 0.75 | 0.08 | 1,220 ± 34 | 280 ± 52 |
| t7L | 0.88 | 0.07 | 1,220 ± 51 | 186 ± 46 |
| t24L | 1.35 | 0.13 | 1,000 ± 69 | 188 ± 2 |
| t25L | 1.06 | 0.19 | 903 ± 106 | 336 ± 5 |
The amounts of Env glycoproteins immunoprecipitated after transfection of HeLa P5 cells were evaluated by PhosphorImager analysis of sodium dodecyl sulfate-polyacrylamide gels. Background radioactivity was subtracted from specific band radioactivity. The processing index (4) was calculated as follows: [(total gp120)mutant × (gp160)wild type]/[(gp160)mutant × (total gp120)wild type]. The association index (4), calculated as [(mutant gp120)cell × (wild-type gp120)supernatant]/[(mutant gp120)supernatant × (wild-type gp120)cell], reflects the stability of the SU-TM association relative to that of wild-type glycoproteins.
To measure the release of SU gp120 from HIV-1 LAI Env mutants, HeLa P5 cells plated in six-well plates were transfected in triplicate with wild-type or mutant env expression vectors, and Env glycoproteins were quantified in cell lysates and in supernatants by an ELISA with anti–HIV-1 gp120 D7324 as the capture antibody. ELISA values were normalized in relation to the protein content of the cell lysates. The anti-gp120 reactivity in cell lysates reflects both gp160 and gp120 reactivities; in culture supernatants, it reflects only the presence of gp120. This finding was confirmed by an ELISA with anti–HIV-1 gp41 D7323: no reactivity was detected in the supernatants, indicating that no detectable gp41 or gp160 was present. ND, not done.