Article Figures & Data
Figures
- Fig. 1.
Structure of the soluble HIV-1 envelope glycoprotein variants. The partial structures of the soluble gp120, gp140, and gp130 envelope glycoproteins used in this paper are shown beneath the corresponding segment of the wild-type gp160 glycoprotein. The junction of gp120 and gp41, the positions of the N-terminal (N36) and C-terminal (C34) gp41 α-helices, and the transmembrane (TM) region are shown. The DP178 peptide used in this study contains sequences corresponding to those of the C34 helix. The positions of the serine substitutions for arginine 508 and arginine 511 are shown (S S). The position of the Cys-Cys-Gly substitution in the N-terminal helical segment (residues 576 to 578) is shown (CCG). The position of the GCN4 trimeric peptide is also shown.
- Fig. 2.
Characterization of the soluble HIV-1 envelope glycoprotein variants. (A) Sucrose density gradient fractions containing the soluble HIV-1 envelope glycoproteins are shown. Fraction 1 was collected from the bottom of the gradient; fraction 10 represents the top of the gradient. Fractions were precipitated by a mixture of sera from HIV-1-infected individuals. Precipitates were analyzed on SDS-polyacrylamide gels under non-reducing conditions, except for the samples [gp130(−/CCG/GCN4) + β-ME] shown in the bottom right panel, which were treated with 1.5% β-ME before gel analysis. Asterisks indicate the expected position of a gp120 monomer in each gel. (B) Radiolabeled envelope glycoproteins were precipitated from transfected cell supernatants by a mixture of sera from HIV-1-infected individuals and were analyzed under nonreducing conditions (in the absence of β-ME). (C) Precipitates of radiolabeled envelope glycoproteins were formed as for panel B and were analyzed on SDS-polyacrylamide gels after treatment with either 1.5% β-ME for 3 min at 100°C or 5% β-ME for 10 min at 100°C.
- Fig. 3.
Molecular exclusion chromatography of soluble envelope glycoproteins. The YU2 gp120 and gp130(−/GCN4) glycoproteins were analyzed on a Superdex 200 column and compared with molecular size standards, the positions of which are indicated by arrows. Elution times are also shown. The aggregate peak for the gp130(−/GCN4) glycoprotein elutes before the highest molecular size standard used (660 kDa).
- Fig. 4.
Recognition of the soluble HIV-1 envelope glycoprotein variants by the DP178 gp41 peptide. Radiolabeled envelope glycoproteins in transfected cell supernatants were normalized for the amount of envelope protein and then precipitated either with a mixture of sera from HIV-1-infected patients (patient sera) or with a mixture of the 1D4 antibody and the PK-C299 peptide (DP178). The PK-C299 peptide contains the residues of the DP178 peptide, which corresponds to the C-terminal gp41 helical region (C34 in Fig. 1), and the residues of the C9 peptide, which are recognized by the 1D4 antibody.
- Fig. 5.
Recognition of the soluble HIV-1 envelope glycoprotein variants by monoclonal antibodies. Immunoprecipitation of the radiolabeled envelope glycoproteins was performed with a mixture of sera from HIV-1-infected individuals (patient sera) or with monoclonal antibodies. The precipitates were analyzed on SDS-polyacrylamide gels under reducing conditions. (A) The envelope glycoproteins were precipitated by a panel of anti-gp120 monoclonal antibodies that recognize discontinuous gp120 epitopes. The 39F antibody recognizes the gp120 V3 variable loop, the F91 antibody recognizes the gp120 CD4 binding site, and the 17b antibody recognizes a CD4-induced gp120 epitope. (B) The envelope glycoproteins were precipitated by monoclonal antibodies against epitopes in the C1 and C5 regions of gp120: a discontinuous epitope involving the C1 and C5 regions (C11), and linear gp120 epitopes within the first (C1) conserved region (4D4#85 and 135/9) or within the fifth (C5) conserved region (670-30D and M91).
- Fig. 6.
CCR5 binding of the soluble HIV-1 envelope glycoproteins. Equivalent amounts of radiolabeled soluble envelope glycoproteins were incubated with Cf2Th/synCCR5 cells in the absence or presence of sCD4. The bound proteins were precipitated and analyzed on SDS-polyacrylamide gels, the autoradiographs of which are shown.
