Stabilization of human immunodeficiency virus type 1 envelope glycoprotein trimers by disulfide bonds introduced into the gp41 glycoprotein ectodomain

Abstract

Biochemical and structural studies of fragments of the ectodomain of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane envelope glycoprotein have demonstrated that the molecular contacts between alpha helices allow the formation of a trimeric coiled coil. By introducing cysteine residues into specific locations along these alpha helices, the normally labile HIV-1 gp160 envelope glycoprotein was converted into a stable disulfide-linked oligomer. Although proteolytic cleavage into gp120 and gp41 glycoproteins was largely blocked, the disulfide-linked oligomer was efficiently transported to the cell surface and was recognized by a series of conformationally dependent antibodies. The pattern of hetero-oligomer formation between this construct and an analogous construct lacking portions of the gp120 variable loops and of the gp41 cytoplasmic tail demonstrates that these oligomers are trimers. These results support the relevance of the proposed gp41 structure and intersubunit contacts to the native, complete HIV-1 envelope glycoprotein. Disulfide-mediated stabilization of the labile HIV-1 envelope glycoprotein oligomer, which has been suggested to possess advantages as an immunogen, may assist attempts to develop vaccines.

FIG. 1

Location of the introduced cysteines in the gp41 ectodomain. (a) Sequence of the coiled-coil region of gp120, with the position along the heptad repeat indicated beneath. (b) X-ray structure of the HIV-1 gp41 coiled coil (38), with the location of the LQA/CCG change shown in black. The amino-terminal α-helical coiled coil is white, and the carboxy-terminal helices are grey. The gp41 residue numbers are indicated. (c) View of the LQA region of the gp41 coiled coil. The perspective is from the threefold symmetry axis of the coiled coil. Only the main chain and Cβ atoms are depicted. The d position leucine is indicated in black. Dark dashed lines are drawn between the Cβ atoms of leucine 576 (d in the heptad repeat) and glutamine 577 (e in the heptad repeat) to indicate where trimeric cross-links might form. The Cβ–Cβ distance is 6.84 Å, outside of the ideal distance for introducing a disulfide bond (5.28 Å) (33, 35). A possible alternative cross-link, between adjacent d position leucines, is shown by a light dashed line.

FIG. 1

Location of the introduced cysteines in the gp41 ectodomain. (a) Sequence of the coiled-coil region of gp120, with the position along the heptad repeat indicated beneath. (b) X-ray structure of the HIV-1 gp41 coiled coil (38), with the location of the LQA/CCG change shown in black. The amino-terminal α-helical coiled coil is white, and the carboxy-terminal helices are grey. The gp41 residue numbers are indicated. (c) View of the LQA region of the gp41 coiled coil. The perspective is from the threefold symmetry axis of the coiled coil. Only the main chain and Cβ atoms are depicted. The d position leucine is indicated in black. Dark dashed lines are drawn between the Cβ atoms of leucine 576 (d in the heptad repeat) and glutamine 577 (e in the heptad repeat) to indicate where trimeric cross-links might form. The Cβ–Cβ distance is 6.84 Å, outside of the ideal distance for introducing a disulfide bond (5.28 Å) (33, 35). A possible alternative cross-link, between adjacent d position leucines, is shown by a light dashed line.

FIG. 1

Location of the introduced cysteines in the gp41 ectodomain. (a) Sequence of the coiled-coil region of gp120, with the position along the heptad repeat indicated beneath. (b) X-ray structure of the HIV-1 gp41 coiled coil (38), with the location of the LQA/CCG change shown in black. The amino-terminal α-helical coiled coil is white, and the carboxy-terminal helices are grey. The gp41 residue numbers are indicated. (c) View of the LQA region of the gp41 coiled coil. The perspective is from the threefold symmetry axis of the coiled coil. Only the main chain and Cβ atoms are depicted. The d position leucine is indicated in black. Dark dashed lines are drawn between the Cβ atoms of leucine 576 (d in the heptad repeat) and glutamine 577 (e in the heptad repeat) to indicate where trimeric cross-links might form. The Cβ–Cβ distance is 6.84 Å, outside of the ideal distance for introducing a disulfide bond (5.28 Å) (33, 35). A possible alternative cross-link, between adjacent d position leucines, is shown by a light dashed line.

FIG. 2

Immunoprecipitation of HIV-1 envelope glycoprotein variants. Plasmids encoding the wild-type HIV-1 envelope glycoproteins and three of the mutant envelope glycoproteins described in Table 1 were transfected into COS-1 cells. Cell lysates were immunoprecipitated with the anti-gp41 antibody D61, and the precipitates were boiled in 2% β-mercaptoethanol for 3 min before being analyzed on an SDS–8% polyacrylamide gel.

FIG. 3

Analysis of wild-type and LQA/CCG envelope glycoproteins. Lysates were immunoprecipitated with the anti-gp41 antibody D61 and boiled in either 2 or 5% β-mercaptoethanol (βME) for 3 or 10 min, as indicated, before being analyzed on an SDS–8% polyacrylamide gel.

FIG. 4

Formation of the LQA/CCG higher-order forms in the presence of iodoacetamide. Lysates of 293T cells expressing the LQA/CCG construct were immunoprecipitated with the anti-CD4 binding site antibody F105 and boiled for 3 min with the indicated percentage of β-mercaptoethanol in the presence (left lane) or absence (other lanes) of 10 mM iodoacetamide. In the experiment in the left lane, iodoacetamide was present during cell lysis and throughout the sample preparation.

FIG. 5

Cell surface expression of the LQA/CCG envelope glycoproteins. 293T cells were transfected with a control plasmid or with plasmids expressing a mutant HIV-1 envelope glycoprotein with amino acid changes at the proteolytic cleavage site (3) or LQA/CCG envelope glycoproteins. The transfected cells were incubated with radiolabeled F105 antibody for 2 h, washed, and lysed. The antibody was precipitated and analyzed by SDS-PAGE. The antibody heavy chain is shown.

FIG. 6

Precipitation of LQA/CCG and ΔLQA/CCG envelope glycoproteins with antibodies. 293T cells expressing the LQA/CCG and the ΔLQA/CCG envelope glycoproteins were lysed in Nonidet P-40 buffer. Cell lysates were precipitated with HIV-1-infected patient sera (PS1, PS2), the F105 antibody, the 17b antibody in the presence or absence of soluble CD4, the C11 antibody, or the G3-519 antibody (28). The A32 antibody and the D61, T3, and T4 anti-gp41 antibodies (4) all recognized both monomeric and higher-order forms of LQA/CCG and ΔLQA/CCG envelope glycoproteins (data not shown). The 110.4 antibody, directed against the third variable (V3) loop of gp120, also recognized the LQA/CCG glycoproteins (Fig. 7, lane 1:1, αV3). The LQA/CCG and ΔLQA/CCG glycoproteins were not precipitated by monoclonal antibodies against unrelated proteins (data not shown).

FIG. 7

Formation of hetero-oligomers between LQA/CCG and ΔLQA/CCG envelope glycoproteins. Serum from an HIV-1-infected individual was used to precipitate lysates of 293T cells transfected with plasmids encoding LQA/CCG and ΔLQA/CCG envelope glycoproteins. In lane 2:1, plasmids expressing the LQA/CCG and ΔLQA/CCG envelope glycoproteins were transfected at a 2:1 ratio, while in lane 1:1, the LQA/CCG- and ΔLQA/CCG-expressing plasmids were transfected in equal amounts. In lane 1:1, αV3, the same cell lysates as those used for the experiment in lane 1:1 were used for precipitation by the anti-V3 loop antibody 110.4.