Apoptosis induction by the binding of the carboxyl terminus of human immunodeficiency virus type 1 gp160 to calmodulin

Abstract

The role of calmodulin (CaM) in apoptosis induced by gp160 of human immunodeficiency virus type 1 was investigated with cells undergoing single-cell killing. These cells were found to express, under the control of an inducible promoter, wild-type gp160 or mutant gp160 devoid of various lengths of the carboxyl terminus. Immunoprecipitation accompanied by immunoblotting revealed binding of CaM to wild-type gp160 but not to mutant gp160 bearing a carboxyl terminus with a deletion spanning more than five amino acid residues. A significant coenzyme activity was detected in the CaM bound to gp160 even in the presence of a Ca2+ chelater, EGTA. The cells forming this gp160-CaM complex exhibited an elevated intracellular Ca2+ level followed by DNA fragmentation, which is a hallmark of apoptosis, and finally cell killing, while the cells not forming this complex did not show any significant elevation in Ca2+ level or DNA fragmentation. These results thus indicated that CaM plays a key role in gp160-induced apoptosis.

FIG. 1

Immunoblot and immunoprecipitation analyses. For the immunoblot analysis, the cell lysate from each cell clone, containing 100 μg of protein, was loaded as indicated at the top and then was examined with an anti-Env MAb (A) or anti-CaM MAb (C). For the detection of the gp160-CaM complex, the cell lysate was either (i) immunoprecipitated with the anti-CaM MAb, electrophoresed on a polyacrylamide gel, and then analyzed by immunoblotting with anti-Env MAb (B) or (ii) subjected to the same procedure with the MAbs reversed (E). The lysate was also subjected to immunoprecipitation with MOL171 or anti-CaM MAb and then analyzed by immunoblotting with 0.5β (D). As the standard for gp160 in panel B, the lysate from UE160 cells containing 100 μg of protein was loaded (lane S). As the standard for CaM in panel E, 0.5 or 2.5 ng of a purified CaM was loaded. The relative densities of bands corresponding to CaM as determined by densitometry were 1.0, 4.4, 0.46, and 1.1 in the first four lanes, respectively. The bands at around 49 kDa in panels B and D represent mouse IgG.

FIG. 2

Time course of intracellular Ca²⁺ level. (a) UE160 cells were treated with Fluo 3-AM, and the Ca²⁺ concentration over time was measured after the addition of various amounts of A23187, as indicated at the top, at 0 s in the presence of 0.4 mM CaCl₂. (b) Each cell clone, as indicated at the top of each panel, was treated with Fluo 3-AM, and the Ca²⁺ concentration over time was measured after the addition of 10 μM CdCl₂ at 0 s. In one case, W7 (10 μM) was added 5 min before the induction with CdCl₂ (UE160+W7). Each curve represents the mean of the fluorescence curves from 20 to 30 individual cells.

FIG. 3

CaM activity analyzed with the PDE assay system. Env-CaM complex bound to Sepharose beads was obtained from the cell lysate of each cell clone at 1, 10, and 240 min after induction with CdCl₂ as indicated on the left, and its CaM coenzyme activity was examined by using the PDE assay system (23). Where indicated, W7 was added to the cell culture 5 min before induction. As a standard, 0, 1, 2, and 4 ng of purified CaM was added to the PDE assay mixture with nontreated Sepharose beads. ND, not determined. The bars represent the standard errors (n = 3).

FIG. 4

Detection of apoptosis by cell cycle analysis. (a) The proportion of cells undergoing apoptosis was examined by cell cycle analysis with each cell clone, as indicated to the right, at 0 and 48 h after induction. The numbers indicate the percentages of subdiploid cells and the percentages of apoptotic cells. (b) UE160 cells were treated with CH-11 (A) or CH-11 plus ZB-4 (B) for 24 h in the absence of induction. In addition, UE160 cells were cultured either without (C) or with (D) the addition of CdCl₂ as an inducer for 48 h. (E and F) Same experiments as in panels C and D, respectively, except in the presence of ZB-4. The numbers represent the percentages of subdiploid cells.

FIG. 5

Cell growth after induction. Each cell clone, as indicated at the top of each panel, was adjusted to 5 × 10⁵ cells in 10 ml of RPMI 1640 medium on day 0 and cultured either with (closed circles) or without (open circles) 10 μM CdCl₂. On each day, 1 ml of cell suspension was removed from the cultures, and the number of viable cells in the cell suspension was measured.

FIG. 6

Fluorescence-activated cell sorter analysis of surface CD4. UE160 or Δ859 cells were cultured for 4 h either without (−) or with (+) the addition of 10 μM CdCl₂. The cells were then examined for their levels of expression of surface CD4 with a FACScan with MAb Leu3a (anti-CD4; Becton Dickinson, San Jose, Calif.), OKT4 (anti-CD4; Ortho Diagnostic Inc., Raritan, N.J.), or NU-Lpan (anti-CD45; Nichirei Corp., Tokyo, Japan) as indicated on the right. The amounts of these MAbs which bound to the cell surface were then revealed by secondary staining with fluorescein isothiocyanate–anti-mouse IgG antibody (Tago Inc., Burlingame, Calif.). Cells treated only with fluorescein isothiocyanate–anti-mouse IgG antibody were used as an autofluorescence control (C).