The human immunodeficiency virus type 1 gp120 V2 domain mediates gp41-independent intersubunit contacts

Abstract

The envelope protein of human immunodeficiency virus type 1 HIV-1 undergoes proteolytic cleavage in the Golgi complex to produce subunits designated gp120 and gp41, which remain noncovalently associated. While gp41 has a well-characterized oligomeric structure, the maintenance of gp41-independent gp120 intersubunit contacts remains a contentious issue. Using recombinant vaccinia virus to achieve high-level expression of gp120 in mammalian cells combined with gel filtration analysis, we were able to isolate a discrete oligomeric form of gp120. Oligomerization of gp120 occurred intracellularly between 30 and 120 min after synthesis. Analysis by sedimentation equilibrium unequivocally identified the oligomeric species as a dimer. In order to identify the domains involved in the intersubunit contact, we expressed a series of gp120 proteins lacking various domains and assessed the effects of mutation on oligomeric structure. Deletion of the V1 or V3 loops had little effect on the relative amounts of monomer and dimer in comparison to wild-type gp120. In contrast, deletion of either all or part of the V2 loop drastically reduced dimer formation, indicating that this domain is required for intersubunit contact formation. Consistent with this, the V2 loop of the dimer was less accessible than that of the monomer to a specific monoclonal antibody. Previous studies have shown that while the V2 loop is not an absolute requirement for viral entry, the absence of this domain reduces viral resistance to neutralization by monoclonal antibodies or sera. We propose that the quaternary structure of gp120 may contribute to resistance to neutralization by limiting the exposure of conserved epitopes.

FIG. 1

Gel filtration analysis of secreted gp120. B-SC-1 cells were infected with a recombinant vaccinia virus encoding gp120 (vPE50). Secreted gp120 was purified by lentil-lectin affinity chromatography and analyzed by gel filtration over Superdex 200. (A) UV trace of gel filtration experiment. The molecular radius standards were given by: ferritin, F, 61.0 Å; catalase C, 52.2 Å; aldolase A, 48.1 Å; and bovine serum albumin, B, 35.5 Å. Void volume (V_o) was given by blue dextran 2000. (B) Aliquots of 1-ml gel filtration fractions were analyzed by SDS-PAGE (10% gel) and immunoblotting with env-specific antiserum. (C) Samples as in panel B but treated with 5 mM EGS prior to SDS-PAGE (6% gel). In panels B and C, the numbers on the right represent the positions and masses (in kilodaltons) of marker proteins.

FIG. 2

Repeat gel filtration of secreted gp120. (A) Signal quantitation of immunoblotted gel filtration fractions. Bars indicate the fractions that were pooled and concentrated prior to repeat gel filtration. (B) Signal quantitation of fractions from repeat gel filtration of pooled monomer. (C) Signal quantitation of fractions from repeat gel filtration of pooled oligomer. Note that panel A is derived from the immunoblot displayed in Fig. 1B.

FIG. 3

Intracellular gp120 oligomer formation. B-SC-1 cells were infected with a recombinant vaccinia virus encoding gp120 (vPE50), metabolically labeled for 15 min, and then lysed immediately (upper panel) or chased for 30 or 120 min prior to lysis (middle and lower panels, respectively). Lysates were sedimented through sucrose density gradients, and gp120 from gradient fractions was immunoprecipitated with an env-specific antiserum and subjected to SDS-PAGE (10% gels). The direction of sedimentation was right to left. Gradients were calibrated with catalase (11.3S), aldolase (7.3S), and bovine serum albumin (4.4S).

FIG. 4

Sedimentation equilibrium concentration profiles for the dimer fraction (top panel) and for the monomer fraction (middle panel). Rotor speeds were 7,600 (○), 9,600 (▵), and 11,600 (▿) rpm for the dimer fraction and 11,000 (+) and 15,00 (×) rpm for the monomer fraction, respectively. Solid lines show the best-fit distributions (root mean square error, 0.0078 optical density) after global modeling with the monomer molecular weight and with the relative amounts of monomer and dimer of each sample as unknowns. The best fit gave an average molecular weight of 92,490 ± 1,445 for the monomer with a corresponding doubling for the dimer. Residuals of the fitted lines to the experimental data are shown in the lower panel. The calculated absorbance contributions of the contaminating species are indicated by dashed lines for the experiments at 11,600 and 15,000 rpm. Their calculated relative total amounts were 5 ± 4% monomer contamination of the dimer fraction and 27 ± 4% dimer contamination of the monomer fraction. Incorporated in each sedimentation equilibrium data panel is an inset in which the highest-centrifugal-speed exponential data have been transformed into a linear plot in the upper panel (▿) and the middle panel (×). This transformation was achieved by subtracting the baseline offset first, converting the x axis to a radius² axis, and then taking the derivative (dlnA/dr²) of the exponential data. Solid lines of increasing slope for either a monomer, dimer, or trimer model are given in order to compare the experimental data with the best model for the quaternary state of the predominant gp120 form under analysis.

FIG. 5

Effect of variable domain deletion on gp120 oligomer formation. B-SC-1 cells were infected with recombinant vaccinia virus encoding wild-type or mutated gp120. Secreted gp120 was purified by lentil-lectin affinity chromatography and analyzed by gel filtration over Superdex 200. Aliquots of 1-ml fractions were immunoblotted with an env-specific antiserum. Signal quantitation is shown for vRCwt (A), vRCΔV1 (B), vRCΔV3 (C), vRCΔV2 (D), and vRCΔV2T (E). D and M indicate the dimer and monomer, respectively.

FIG. 6

Comparison of epitope exposure of monomeric and dimeric gp120. B-SC-1 cells were infected with a recombinant vaccinia virus encoding gp120 (vPE50), metabolically labeled overnight, and lysed. Lysates were sedimented through sucrose density gradients, and fractions containing monomeric or dimeric gp120 were separately pooled. Monomeric and dimeric gp120 were immunoprecipitated with env-specific antiserum (α-env), the anti-V2 loop MAb T54 (α-V2), or the anti-V3 loop MAb D47 (α-V3).