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Evidence for Cell-Surface Association Between Fusin and the CD4-gp120 Complex in Human Cell Lines

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Science  25 Oct 1996:
Vol. 274, Issue 5287, pp. 602-605
DOI: 10.1126/science.274.5287.602

Figures

  • Fig. 1.
  • Fig. 2.

    After pretreatment with gp120, antibody to CD4 coprecipitates a 45-kD protein from human cell lines but not from nonhuman cells expressing human CD4. Human cell lines (U937 and MOLT 4) and nonhuman cells (Mink.CD4 and 3T3.CD4.401) were pretreated with or without gp120 (10 μg/ml for 2 hours). After cell lysis, precipitation of CD4 was carried out as described in Fig. 1. Major histocompatibility complex (MHC) class I molecules were precipitated from biotinylated MOLT 4 cells with the W6/32 monoclonal antibody (mAb). Samples were subjected to SDS-PAGE as described in Fig. 1.

  • Fig. 3.

    The 45-kD protein is down-modulated by PMA. A2.01.CD4.401 cells were treated with PMA (100 ng/ml, Sigma) or with control medium for 3 hours at 37°C. The cells were washed extensively, biotinylated, and incubated with gp120 for 2 hours. Cell lysis and immunoprecipitation with OKT4 ascites were conducted as described in Fig. 1, except that the eluates were run on long gels for a longer time to allow good resolution between the 45-kD band and the truncated CD4 molecules.

  • Fig. 4.

    Antibody to fusin recognizes a 45-kD protein that coprecipitates with CD4 after gp120 pretreatment of cells. (A) Surface-biotinylated CEM cells were treated without (lane 1) or with (lane 2) gp120 and lysed in lysis buffer containing 1% Brij 97. Samples were precipitated with OKT4 linked to protein G-Sepharose beads and processed as in Fig. 1. On the same gel, nonbiotinylated whole-cell extracts from cells infected overnight with vCBYF1 (lane 3) or control vSC8 (lane 4) were run in parallel. Vaccinia-infected cells were lysed at a concentration of 5 × 106 cells per milliliter with buffer containing 1% NP-40, 150 mM NaCl, 10 mM tris-HCl (pH 7.4), and protease inhibitors. After 20 min on ice, nuclei were pelleted by centrifugation at 13,000g for 5 min. Cell extracts (10 μl) were mixed with 30 μl of Laemmli sample buffer supplemented with 8 M urea and incubated at 37°C overnight, then boiled for 3 min. Blots were reacted with rabbit polyclonal antiserum to fusin (6) at a 1:500 dilution (lanes 3 and 4) or with normal rabbit serum (no reactivity) (11), followed by HRP-conjugated goat anti-rabbit IgG. The blots were incubated with ultra supersignal chemiluminescent substrate (Pierce) for 5 min and exposed to film. (B) Unlabeled CEM and U937 cells were incubated with gp120 (10 μg/ml for 2 hours at 37°C) or in control medium. Cell lysates were immunoprecipitated with OKT4 antibodies covalently linked to protein G-Sepharose beads. Eluted samples were concentrated, electrophoresed (1 × 108 cell equivalent per lane), and blotted onto nitrocellulose membranes. Blots were reacted with either rabbit polyclonal antiserum to gp120 (Intracel, MA), polyclonal antiserum to CD4, rabbit anti-fusin IgG (6), or normal rabbit IgG, followed by horse HRP-conjugated goat anti-rabbit or anti-mouse IgG (Amersham). The blots were incubated with ultra supersignal substrate for 5 min and exposed to film. All the protein immunoblots shown are from the same immunoprecipitates that were run in adjacent wells on the same gel. The experiment was repeated four times. (C) Fusin can be coprecipitated with CD4.401 molecules from the surface of 3T3.CD4.401 cells infected with vCBYF1. 3T3.CD4.401 cells were infected overnight with vCBYF1 or control vaccinia vSC8 at 10 plaque-forming units (PFU) per cell. Infected cells were biotinylated (1.2 × 107 per milliliter), treated with gp120 (10 μg/ml for 2 hours at 37°C), and lysed in buffer containing 1% Brij 97 (5 × 106 cells per milliliter). Immunoprecipitation with OKT4 mAb and processing of the eluted samples were done as in Fig. 3, except that 5 × 106 cell equivalents were loaded per lane on long 10% SDS gels. The experiment was repeated three times.

Tables

  • Table 1.

    Soluble gp120 can prime 3T3.CD4.401 cells infected with vCBYF1 for PMA-induced down-modulation of CD4. 3T3.CD4.401 cells were infected overnight with control vaccinia virus vSC8 or with vCBYF1 (3) at 10 PFU per cell. Cells (1 × 106 per group) were treated with soluble gp120 (Intracel, MA) (10 μg/ml) or with medium for 45 min and then treated with PMA (100 ng/ml) for three hours at 37°C. Alternatively, cells were pretreated with PMA for 3 hours, washed extensively, and treated with gp120 for 1 hour. All groups were stained with fluorescein isothiocyanate (FITC)-OKT4. Mean fluorescent units (MFU) were determined with fluorescinated beads as described to generate standard curves (5). The percentage of down-modulation of surface CD4 expression was calculated after subtraction of background fluorescence. Dashes indicate no treatment.

    CellTreatmentStaining with FITC-OKT4
    MFU (n)Down-modulation (%)
    A2.01.CD4.4012700
    -PMA27000
    gp120-190030
    gp120 → PMA100063
    PMA → gp120235013
    3T3.CD4.401 (infected with vSC8)3800
    -PMA37003
    gp120-39000
    gp120 → PMA37003
    3T3.CD4.401 (infected with vCBYF1)3300
    -PMA33000
    gp120-270018
    gp120 → PMA130041
    PMA → gp12033000
  • Table 2.

    Rabbit anti-fusin IgG partially blocks syncytium formation and CD4.401 down-modulation. A2.01.CD4.401 cells were treated with gp120 (10 μg/ml), followed by PMA treatment (100 ng/ml) as described in Table 1, in the presence (at 10 μg/ml each) of no serum, of rabbit preimmune IgG, or of IgG derived from a rabbit immunized with a branched peptide containing the NH2-terminal 15 amino acids of fusin (6). Syncytium formation was determined after 6 hours in co-cultures of A2.01.CD4.401 cells and 12E1 cells infected overnight with vPE16 recombinant vaccinia expressing gp120-41 (IIIB) in triplicates as described (4).

    Rabbit IgGCD4 downmodulation after gp120 + PMA treatment (%)Syncytia (n)
    None53157 ± 22
    Preimmune52155 ± 13
    Anti-fusin2868 ± 9