Synthesis and processing of HIV-1 Env glycoprotein mutants in 293T cells. Env-expressing 293T cells were lysed at 16 h posttransfection and subjected to reducing SDS-PAGE in 7.5-to-15% polyacrylamide gradient gels. Proteins were transferred to nitrocellulose followed by Western blotting with MAb C8, directed against an epitope in the cytoplasmic domain of gp41. Env proteins were visualized following scanning in a PhosphorImager SF. The asterisk indicates a proteolytic product of gp160. The figure was prepared using Powerpoint software and is representative of three independent experiments. wt, wild type.

gp120-anchoring ability of gp41 mutants. Env-expressing 293T cells were labeled with Tran³⁵S-label for 45 min and chased in complete medium for 4.5 h before lysis. Cell lysates (C) and clarified culture supernatants (S) were immunoprecipitated with human anti-rgp120 and protein G-Sepharose. gp160 and gp120 bands were visualized following SDS-PAGE in 4-to-12% gradient gels under reducing conditions and scanning in a PhosphorImager SF. The relative fusion activities of wild-type and mutated Env glycoproteins (means ± standard errors of the means, taken from Fig. 3) are shown below the corresponding gel lanes. The figure was prepared using Powerpoint software and is representative of three independent experiments.

Recognition of Env mutants by conformation-dependent anti-gp41 MAbs. Env-expressing 293T cells were labeled with Tran³⁵S-label for 45 min and chased in complete medium for 4.5 h before lysis. Cell lysates were immunoprecipitated with the various anti-gp41 MAbs or human anti-rgp120 using protein G-Sepharose. gp160 bands were visualized following SDS-PAGE in 4-to-12% gradient gels under reducing conditions and scanning in a PhosphorImager SF. The figure was prepared using Powerpoint software.

Structural comparison of chain reversal regions from HTLV-1 gp21(354–416) (Protein Data Bank [PDB] accession number 1MG1) (32) (A) and HIV-1 gp41 (559–644) modeled on the SIV_MAC gp41 crystal structure (PDB accession number IQBZ) (60) (B). The structures have been aligned according to the hydrophobic 4-3 repeat sequences of the coiled-coil-forming helices. Coiled-coil-forming helices and C-terminal helices are shown as ribbons colored pink and light blue, respectively, while the rest of the chain is colored grey. The disulfide-bonded loop of gp21 is colored yellow. The region of gp41 that would comprise the disulfide-bonded loop is also shown in yellow. The side chains of residues mutated in this study of gp41 and in a previous study of gp21 (36) are indicated, as are the side chains of some residues interacting with the mutated amino acids. The HIV-1_HXB2R gp160 numbering system is used for gp41. To convert to HIV-1_BH8 numbering, subtract 5; to convert to SIV_MAC numbering, add 13. These panels were prepared using RIBBONS 2.8 software. (C) Structure-based sequence comparison of the chain reversal/disulfide-bonded loop region from HTLV-1 gp21 (residues 374 to 415), MuLV p15E (536 to 581), Ebola virus (Zaire) GP2 (582 to 628), and selected primate lentivirus TM proteins (corresponding to HIV-1 gp41 residues 579 to 644). The lentiviruses used in the alignment include HIV-1_BH8 (group M, GenBank accession number K02011), HIV-1_YBF30 (group N, AJ006022), SIV_CPZGAB (X52154), HIV-1_ANT70 (group O, L20587), SIV_MAC239 (M33262), SIV_MND (S28084), SIV_AGMTAN (U58991), and SIV_SYK (AAA74712). α-helical regions as observed in the crystal structures of HTLV-1 gp21, HIV-1_HXB2R, and SIV_MAC gp41 are shown as cylinders (coiled-coil region, pink; C-terminal helix, light blue). The HIV-1_HXB2R gp160 numbering system is used for the lentiviral gp41 sequences. Residues identical to those of HIV-1_BH8 are highlighted in black; homologous residues are grey. Basic residues in the coiled coil and disulfide-bonded loop are highlighted in blue, and acidic residues are red. The hydrophobic clusters of gp21, p15E, and GP2 are highlighted in green, conserved glycines are dark grey, and the conserved aromatic residue C terminal to the disulfide bridge is light green. Deletions are indicated by dots. The gp41 residues mutated in this study are indicated by asterisks. As a guide, some HTLV-1 gp21 and HIV-1 gp41 residues are numbered.

Cell-cell fusion activities of Env glycoprotein mutants using a luciferase reporter assay. 293T effector cells were transfected with wild-type (wt) and mutated pTMenv.2 expression vectors and then infected with vTF7.3, while target 293T cells were cotransfected with pT4luc and pc.FUSIN. At 16 h postinfection, effectors and targets were mixed and cocultured for 16 h prior to lysis and assay for luciferase activity. The relative fusion activities of Env proteins are expressed as follows: (ratio of luciferase activity induced by mutant Env to luciferase activity induced by wild-type Env) × 100. The means ± standard errors of the means from three independent transfections are shown.

Cell surface expression of Env glycoprotein mutants. 293T cells were transfected with wild-type (wt) and mutated pTMenv.2/EC vectors followed by infection with vTF7.3. At 16 h postinfection, cells were washed twice with PBS, incubated with 1.25 mM sulfo-NHS-long-chain biotin for 30 min, and then quenched with 50 mM glycine–PBS. Envelope proteins were immunoprecipitated from precleared cell lysates with IgG from an HIV-1-positive individual and protein G-Sepharose. Biotinylated Env proteins were visualized following SDS-PAGE on 4-to-12% polyacrylamide gradient gels under reducing conditions, transfer to nitrocellulose, and blotting with neutravidin-horseradish peroxidase. The figure was prepared using Powerpoint software from a single experiment.