FIG 1.

Exposure of the membrane-bound Env trimer to 0°C induces gradual and reversible restructuring and inactivates virus infectivity. (A) Effect of cold on binding of MAbs. Full-length Envs of T/F and chronic-phase (Chr.) viruses were transiently expressed on HOS cells. Samples were incubated with the indicated MAbs or CD4-Ig at 0°C or 37°C for 45 min, and binding was measured by cell-based ELISA. The values represent ratios between binding at 0°C and 37°C (green, high; red, low). “+++” indicates high binding at 0°C but no binding at 37°C. Gray shading signifies no binding at either temperature. The sensitivity of each isolate to cold inactivation (1/IT₅₀; h) is indicated and color coded (blue, sensitive; white, resistant). The right column indicates P values for a Spearman correlation between the IT₅₀s of Envs and their cold-induced change in MAb binding. (B) Rates of cold-induced exposure of Env epitopes. Probe binding to Env-expressing HOS cells was measured after incubation at 0°C for 1.5 or 4.5 h. Binding is expressed relative to that of samples incubated at 37°C. (C) Effect of cold on binding of MAbs to soluble gp120 of strain 89.6. Samples were incubated for 6 h at 0°C with the MAbs, immunoprecipitated, and analyzed by Western blotting. The data represent the intensity of the gp120 band quantified by densitometry. (D) Cold-induced Env restructuring is reversible. HOS cells expressing the indicated Envs were incubated at 0°C for 2 h, reequilibrated to 37°C, and then incubated with probes. The Env labels are color coded by their sensitivity to cold-induced CoR-BS exposure. (E) Cold induces reversible changes in sampling frequencies of epitopes. HOS cells expressing the indicated Envs were incubated at 37°C or 0°C and fixed with glutaraldehyde at the same temperature. In addition, some samples were incubated at 0°C prior to fixation at 37°C. Glutaraldehyde activity was quenched, and binding of probes was measured for all the samples (fixed and nonfixed) at 37°C. The values represent ratios between binding of each probe to fixed and nonfixed samples. (F) Cold-induced inactivation of Env. Viruses were incubated for different times at 0°C and then added to cells at 37°C to quantify residual infectivity. The values represent measured luciferase activity expressed as a percentage of that measured for samples not treated with cold. P values were determined by a two-tailed test. The error bars indicate standard errors of the mean (SEM).