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. 2000 Nov 7;97(23):12794-9.
doi: 10.1073/pnas.230438497.

Cooperative subunit interactions within the oligomeric envelope glycoprotein of HIV-1: functional complementation of specific defects in gp120 and gp41

K Salzwedel  1 , E A Berger
Affiliations

Cooperative subunit interactions within the oligomeric envelope glycoprotein of HIV-1: functional complementation of specific defects in gp120 and gp41

K Salzwedel et al. Proc Natl Acad Sci U S A. .

Abstract

The envelope glycoprotein (Env) of HIV-1 is displayed on the surface of the virion or infected cell as an oligomer of multiple gp120/gp41 complexes. We sought to unravel the relationships between this oligomeric structure and the requirements for sequential interactions with CD4 and coreceptor (CCR5 or CXCR4). We used a quantitative cell fusion assay to examine the effects of coexpressing pairs of Envs, each nonfunctional because of a specific defect in one of the essential properties. We observed efficient fusion activity upon coexpression of two Env variants, one containing a gp41 subunit with a mutated fusion peptide and the other containing a gp120 subunit with a mutated CD4 binding site or a mismatched coreceptor specificity. We also observed fusion upon coexpression of two Env variants with distinct gp120 defects, i.e., a CD4 binding site mutation and the incorrect coreceptor specificity determinants. Coimmunoprecipitation experiments verified the efficient formation of mixed oligomers, suggesting that the observed fusion reflected subunit complementation within the oligomeric complex. These results support a model in which cooperative subunit interactions within the Env oligomer result in concerted conformational changes upon receptor binding, resulting in activation for fusion. The implications of these findings for Env function and virus neutralization are discussed.

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Figures

Figure 1
Figure 1
Schematic of HIV-1 Env and variants. The locations of relevant functional domains are shown at the top; genetic modifications are indicated at the bottom, including CD4 binding site mutation (BS), fusion peptide mutation (FP), cytoplasmic tail deletion (Δ), and C-terminal FLAG epitope tag (*).
Figure 2
Figure 2
Complementation between Env variants for CCR5-dependent fusion. Effector cells expressing the indicated Envs (alone or in combination) were mixed with target cells expressing CD4 and either CCR5 (filled bars) or no coreceptor (open bars). Cell fusion was quantitated by measurement of β-galactosidase activity (β-gal) after 2.5 h. Error bars indicate the standard errors of the mean of duplicate samples. wt, Wild type.
Figure 3
Figure 3
Effect of DNA transfection ratio on complementation efficiency. The Env DNA transfection ratios were varied, keeping total amount of DNA at 5 μg. Ratios are shown for SF162-FP:SF162-BS (closed symbols) and SF162-FP:LAV (open symbols). Target cells expressed CD4 and either CCR5 (squares) or no coreceptor (circles). Error bars indicate the standard errors of the mean of duplicate samples.
Figure 4
Figure 4
Mixed oligomer formation detected by coimmunoprecipitation of Env variants. Effector cells prepared as in Fig. 2 were lysed in 1% Nonidet P-40, and the lysates were divided into two tubes. HIV-1 Env was immunoprecipitated as designated on the left with either the broadly cross-reactive T8 anti-gp120 mAb, the anti-FLAG epitope tag mAb, or the D47 anti-gp120 mAb that specifically recognizes the V3 loop of LAV Env but not SF162. As a control (mixed lysates), lysates from cells separately transfected with either SF162-BSΔ or SF162-FP* alone were mixed before immunoprecipitation. Immunoprecipitates were analyzed by SDS/PAGE and immunoblotting with the broadly cross-reactive T8 anti-gp120 mAb, as described in Materials and Methods. Another control involved treatment under the identical transfection conditions without DNA (Mock). The symbols Δ and * indicate truncated and FLAG-tagged Envs, respectively. The positions of full-length gp160 (gp160) and truncated gp160 (gp160Δ) are indicated on the right.
Figure 5
Figure 5
Complementation between Env variants for CXCR4-dependent fusion. Effector cells expressing the indicated Envs (alone or in combination) were mixed with target cells expressing CD4 and either CXCR4 (filled bars) or no coreceptor (open bars). Cell fusion was quantitated by measurement of β-galactosidase activity (β-gal) after 2.5 h. Error bars indicate the standard errors of the mean of duplicate samples. wt, Wild type.
Figure 6
Figure 6
Model for cooperative subunit interactions within the HIV-1 Env oligomer. For simplicity, only two gp120/gp41 complexes are shown. Labeling indicates gp120, gp41, CD4, and coreceptor (CoR); inactive Env regions are designated by X. Different shapes distinguish the preactivated and activated states of gp120; different shading distinguishes the gp120 subunits activated directly by CD4 binding (black) versus indirectly by subunit interactions (gray). (A) Wild-type Env. gp120 subunits on both complexes bind to CD4 and undergo a conformational change to expose determinants critical for coreceptor interaction. Both gp120 subunits bind to coreceptor, leading to activation of both gp41 subunits for fusion peptide insertion. (B) Complementation between one Env variant with a defective fusion peptide and another with a defective CD4 binding site. The gp120 subunit on one complex binds to CD4 and undergoes the conformational change exposing coreceptor interaction determinants. The functional gp41 subunit on the other complex is activated for fusion peptide insertion. We also presume that the coreceptor interaction determinants are indirectly exposed on the other gp120 subunit via concerted subunit interactions and contribute to gp41 activation (as described for D below). (C) Complementation between one Env variant with a defective fusion peptide and another with inactive coreceptor interaction determinants. The gp120 subunits on both complexes bind to CD4 and undergo the associated conformational changes. The coreceptor interaction determinants are functional on only one gp120 subunit; this is sufficient to activate the functional gp41 subunit on the other complex for fusion peptide insertion. (D) Complementation between one Env variant with a defective CD4 binding site and another with inactive coreceptor binding determinants. The gp120 subunit on one complex binds to CD4 and undergoes the associated conformational change. Although the coreceptor binding determinants on this subunit are inactive, cooperative interactions lead to a concerted conformational change in the other gp120 subunit, which then interacts with coreceptor and activates both gp41 subunits for fusion peptide insertion.
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