Fig. 1. Schematic diagram of the HIV-1 JRCSF Env and cleavage sites in the V3 domain. The amino acid positions of HIV Env sequence are based on HIV-1 HXB2 numbering system. Sequences of various motifs and cleavage sites are indicated, based on previous studies [16], [17], [19], [20], [21], [24] and [30]. The positions of amino acid residues (P1, P1′, P2, and P2′) relative to cleavage by thrombin are also indicated above the V3 sequences to facilitate discussion in the text.

Fig. 2. Characterization of the cleavage of gp120 expressed in CHO-K1 cells. In all gel and Western blot images, the solid bar to the left indicates intact gp120 and the open bars to the left indicate degradation products. Molecular masses in kiloDaltons (kDa) are indicated on the right side of the images. (A) Time course study of gp120 degradation during cell growth in roller bottles. CHO-K1 cells stably expressing gp120 were expanded to roller bottles in serum-free medium. Culture supernatants were harvested and fresh medium was added every 24 h for 14 days. Ten microliters of the daily supernatant harvests were analyzed by Western blots using a polyclonal anti-gp120 serum. (B) Gel analysis of purified gp120 under reducing and non-reducing conditions. Two micrograms of partially degraded gp120 were denatured under reducing and non-reducing conditions and analyzed separately on SDS–PAGE gels followed by SimplyBlue staining. (C) Identification of the N-terminus-derived degradation product. Partially degraded gp120 was analyzed by Western blot using a mouse polyclonal anti-gp120 serum or a mouse anti-His monoclonal antibody.

Fig. 3. Cleaved gp120 cannot be separated from intact gp120. (A) High Q anion-exchange chromatography was performed using partially degraded gp120 (Fig. 2B). Twenty-six fractions were collected and analyzed on two SDS–PAGE gels and stained. The two gel images were merged and chromatographic steps were marked for comparison. Both the leftmost and rightmost lanes contain the starting material. V₀ represents the void volume of the system. Gradient elution from 0% to 50% HQ-B buffer is illustrated as a black triangle. (B) Hydroxyapatite chromatography was performed on a CHT-II Econo-Pac cartridge using partially degraded gp120 recovered from the High Q anion-exchange run in (A). All 26 fractions were analyzed and presented as in (A).

Fig. 4. Analysis of gp120 cleavage. In all Western blot analyses presented here, a polyclonal anti-gp120 serum was used to detect both intact and degraded gp120. The lane numbers are indicated at the bottom of each image. (A) The effects of supernatant concentration and a protein inhibitor cocktail on gp120 cleavage were analyzed as follows. Day 14 supernatant from roller-bottle production (Fig. 2A) was concentrated to various extents as indicated, followed by incubation for 10 h at room temperature and overnight at 4 °C. The effect of the Sigma Protease Inhibitor Cocktail was evaluated by adding the inhibitors after concentration. Equal amounts were analyzed on Western blot. (B) Serine protease inhibitors were evaluated for their ability to prevent gp120 degradation. An aliquot of day 13 supernatant (Fig. 2A) was pre-incubated with individual or pooled protease inhibitors at two different concentrations as indicated, concentrated by 100-fold, and incubated at room temperature for 16 h. The terms “Low” and “High” indicate that inhibitors were pooled at their lower and higher concentrations, respectively, as in individual tests. Equal amounts were analyzed on Western blot. Un-concentrated and 100-fold concentrated supernatant with no inhibitor were used as controls and loaded in lanes 1 and 2, respectively. (C) The effect of cell culture media on gp120 cleavage was evaluated. Cell growth in roller bottles was performed using two different serum-free media, CHO III (A) and OptiMEM I, as described in Materials and methods. Supernatants with or without a 30-fold concentration were incubated at room temperature for 6 h and overnight at 4 °C. Equal amounts were analyzed on Western blot.