Oligomeric structure of the human immunodeficiency virus type 1 envelope protein on the virion surface

Abstract

The envelope protein (Env) of human immunodeficiency virus type 1 forms homo-oligomers in the endoplasmic reticulum. The oligomeric structure of Env is maintained after cleavage in a Golgi compartment and transport to the surfaces of infected cells, where incorporation into budding virions takes place. Here, we use biophysical techniques to assess the oligomeric valency of virion-associated Env prior to fusion activation. Virion-associated Env oligomers were stabilized by chemical cross-linking prior to detergent extraction and were purified by immunoaffinity chromatography. Gel filtration revealed a single predominant oligomeric species, and sedimentation equilibrium analysis-derived mass values indicated a trimeric structure. Determination of the masses of individual Env molecules by scanning transmission electron microscopy demonstrated that virion-associated Env was trimeric, and a triangular morphology was observed in 20 to 30% of the molecules. These results, which firmly establish the oligomeric structure of human immunodeficiency virus virion-associated Env, parallel those of our previous analysis of the simian immunodeficiency virus Env.

FIG. 1.

Gel filtration analysis of virion-derived HIV-1 Env. (A) Aliquots of gel filtration fractions were subjected to SDS-8% PAGE in the presence of reducing agent (cross-links broken) and sequentially immunoblotted with an HIV-1 gp120-specific MAb, a mouse immunoglobulin G-specific rabbit polyclonal serum, and iodinated protein A. gp120 was quantified by phosphor-screen autoradiography. Blue Dextran 2000 (giving the void volume) had an elution peak in fraction 43. (Inset) Pool of fractions 48 to 51 analyzed by SDS-4 to 20% PAGE in the presence of reducing agent and Coomassie blue staining. (B) Aliquots of gel filtration fractions analyzed by SDS-5% PAGE in the absence of reducing agent (cross-links maintained) and immunoblotted as described above. The bars in panels A (inset) and B indicate the electrophoretic mobility of a 250-kDa marker protein.

FIG. 2.

Mass distribution of virion-derived HIV-1 Env determined by quantitative analysis of STEM images. Shown are the results of an analysis of 795 molecules within a pool of gel filtration fractions 48 and 49 (A) and 753 molecules within a pool of fractions 50 and 51 (B).

FIG. 3.

Montage of STEM images of virion-derived HIV-1 Env molecules exhibiting triangular morphology. Depicted are images from pooled gel filtration fractions 48 and 49. Smoothing was used to reduce pixelation. Bar = 50 nm.

FIG. 4.

Sedimentation equilibrium concentration profiles of individual gel filtration fractions of virion-derived HIV-1 Env. Solid lines show the best-fit distributions after global modeling of data obtained at four different rotor speeds. For clarity, only data obtained at 7,000 rpm are shown. The lower panel shows the residuals of the fitted lines to the experimental data.