Reduced monomeric CD4 is the preferred receptor for HIV

Abstract

CD4 is a co-receptor for binding of T cells to antigen-presenting cells and the primary receptor for the human immunodeficiency virus type 1 (HIV). CD4 exists in three different forms on the cell surface defined by the state of the domain 2 cysteine residues: an oxidized monomer, a reduced monomer, and a covalent dimer linked through the domain 2 cysteines. The disulfide-linked dimer is the preferred immune co-receptor. The form of CD4 that is preferred by HIV was examined in this study. HIV entry and envelope-mediated cell-cell fusion were tested using cells expressing comparable levels of wild-type or disulfide bond mutant CD4 in which the domain 2 cysteines were mutated to alanine. Eliminating the domain 2 disulfide bond increased entry of HIV reporter viruses and enhanced HIV envelope-mediated cell-cell fusion 2-4-fold. These observations suggest that HIV enters susceptible cells preferably through monomeric reduced CD4, whereas dimeric CD4 is the preferred receptor for binding to antigen-presenting cells. Cleavage of the domain 2 disulfide bond is possibly involved in the conformational change in CD4 associated with fusion of the HIV and cell membranes.

FIGURE 1.

Redox potential of the CD4 domain 2 disulfide bond. A, CD4 domain 2 (residues 125–202) was incubated with 75 mm DTT_ox and different concentrations of DTT_red. The reactions were resolved on SDS-PAGE and the protein stained with colloidal Coomassie. Differential staining with the dye was used to calculate the ratio of oxidized to reduced protein. B, plot of the fraction of reduced CD4 domain 2 protein as a function of the ratio of DTT_red to DTT_ox is shown. The solid line represents the best fit of the data to Equation 1. The calculated equilibrium constant is 5.1 ± 0.7 × 10⁻³, and standard redox potential of the Cys¹³⁰-Cys¹⁵⁹ disulfide is −239 mV. The bars and errors are the mean ± S.D. of three experiments.

FIGURE 2.

Mechanism-based kinetic trapping of the Cys¹³⁰-Cys¹⁵⁹ disulfide bond with human thioredoxin 1. CD4 domain 2 (7.0 μm) was incubated with wild-type thioredoxin 1 (Trx) or C35S trapping mutant (2.6 μm) and the proteins were resolved on SDS-PAGE under nonreducing and reducing conditions and stained with colloidal Coomassie. A complex of domain 2 with the thioredoxin trapping mutant is apparent, which resolves upon reduction by 20 mm DTT.

FIGURE 3.

Inducible cell expression of wild-type and C130A,C159A mutant CD4. CD4 expression was induced with doxycycline in HEK Tet-On cells stably transfected with wild-type (0.5 μm doxycycline) or C130A,C159A mutant (2 μm doxycycline) protein. A, Western blot of HEK Tet-On cell lysates showing comparable, doxycycline-dependent expression of wild-type and mutant CD4 protein. A blot for GAPDH is shown as loading control. The positions of molecular weight markers are shown on the left. B, flow cytometry of HEK Tet-On cells showing comparable, doxycycline-dependent surface expression of wild-type (mean fluorescence = 226) and mutant (mean fluorescence = 207) CD4 protein. CD4 expression in cells transfected with empty vector is shown as control. C, flow cytometry of HEK Tet-On cells showing comparable surface expression of X4 or R5 chemokine receptor in cells expressing either wild-type or mutant CD4 protein. X4 and R5 mean fluorescence is 734 and 1282, and 2942 and 2346 in cells expressing wild-type or mutant CD4 protein, respectively. Nil is cells transfected with empty chemokine vector.

FIGURE 4.

Elimination of the CD4 domain 2 disulfide bond enhances HIV env-mediated cell-cell fusion. Fusion of HEK Tet-On target cells expressing comparable levels of wild-type or C130A,C159A mutant CD4 and X4 or R5 chemokine receptor with HEK 293T effector cells expressing either 89.6 or Q1521.34 HIV env is shown. Fusion is reported as luciferase units. The bars and errors are the mean ± S.D. of four experiments. ***, p < 0.001.

FIGURE 5.

Elimination of the CD4 domain 2 disulfide bond enhances HIV entry. A, flow cytometry of HEK 293T cells showing comparable transient surface expression of wild-type (mean fluorescence = 243) and mutant (mean fluorescence = 176) CD4 protein. CD4 expression in cells transfected with empty vector is shown as control. B, entry of HIV UG024 luciferase reporter virus into HEK 293T cells expressing X4 chemokine receptor and wild-type or C130A,C159A mutant CD4. Fusion is reported as luciferase units, and results from two separate experiments are shown. The bars and errors are the mean ± S.D. of three or four experiments. *, p < 0.05; ***, p < 0.001.

FIGURE 6.

Summary model for control of CD4 function by the domain 2 disulfide bond. CD4 exists in three different forms on the cell surface defined by the state of the domain 2 cysteine residues: an oxidized monomer, a reduced monomer, and a disulfide-linked dimer. The observations to date imply that the disulfide-linked dimer is the immune co-receptor, whereas the reduced monomer is the preferred receptor for HIV entry.