Residues in the gp41 Ectodomain Regulate HIV-1 Envelope Glycoprotein Conformational Transitions Induced by gp120-Directed Inhibitors

Abstract

Interactions between the gp120 and gp41 subunits of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env) trimer maintain the metastable unliganded form of the viral spike. Binding of gp120 to the receptor, CD4, changes the Env conformation to promote gp120 interaction with the second receptor, CCR5 or CXCR4. CD4 binding also induces the transformation of Env into the prehairpin intermediate, in which the gp41 heptad repeat 1 (HR1) coiled coil is assembled at the trimer axis. In nature, HIV-1 Envs must balance the requirements to maintain the noncovalent association of gp120 with gp41 and to evade the host antibody response with the need to respond to CD4 binding. Here we show that the gp41 HR1 region contributes to gp120 association with the unliganded Env trimer. Changes in particular amino acid residues in the gp41 HR1 region decreased the efficiency with which Env moved from the unliganded state. Thus, these gp41 changes decreased the sensitivity of HIV-1 to cold inactivation and ligands that require Env conformational changes to bind efficiently. Conversely, these gp41 changes increased HIV-1 sensitivity to small-molecule entry inhibitors that block Env conformational changes induced by CD4. Changes in particular gp41 HR1 amino acid residues can apparently affect the relative stability of the unliganded state and CD4-induced conformations. Thus, the gp41 HR1 region contributes to the association with gp120 and regulates Env transitions from the unliganded state to downstream conformations.IMPORTANCE The development of an efficient vaccine able to prevent HIV infection is a worldwide priority. Knowledge of the envelope glycoprotein structure and the conformational changes that occur after receptor engagement will help researchers to develop an immunogen able to elicit antibodies that block HIV-1 transmission. Here we identify residues in the HIV-1 transmembrane envelope glycoprotein that stabilize the unliganded state by modulating the transitions from the unliganded state to the CD4-bound state.

Keywords: HIV-1; HR1; antibody resistance; gp120; gp41; intrinsic envelope reactivity; soluble CD4.

FIG 1

Soluble CD4 (sCD4) and CD4mc inhibition of HIV-1_YU2 Env variants with alterations of the gp41 ectodomain. Recombinant HIV-1 strains expressing luciferase and bearing wild-type or mutant HIV-1_YU2 Envs were normalized by reverse transcriptase activity. Equal amounts of viruses were incubated with serial dilutions of sCD4 (A), JP-III-48 (B), or DMJ-II-121 (C) at 37°C for 1 h prior to infection of Cf2Th-CD4 CCR5 cells. Infectivity at each dilution of sCD4 or inhibitor tested is shown as the percentage of infection without sCD4 or inhibitor for each particular mutant. Quadruplicate samples were analyzed in each experiment. Data shown are the means of results obtained in at least 4 (A) or 3 (B and C) independent experiments. The error bars represent the standard deviations.

FIG 2

sCD4 and CD4mc inhibition of HIV-1_HxBc2 Env variants with alterations of the gp41 ectodomain. Recombinant HIV-1 expressing luciferase and bearing wild-type (WT) or mutant HIV-1_HXBc2 Envs were normalized by reverse transcriptase activity. Equal amounts of viruses were incubated with serial dilutions of sCD4 (A) or JP-III-48 (B) at 37°C for 1 h prior to infection of Cf2Th-CD4 CXCR4 cells. Infectivity at each dilution of inhibitor tested is shown as the percentage of infection without inhibitor for each particular mutant. Quadruplicate samples were analyzed in each experiment. Data shown are the means of results from 3 independent experiments. The error bars represent the standard deviations.

FIG 3

Effect of gp41 ectodomain changes on inactivation mediated by ligands that prefer the unbound conformation of HIV-1 Env. Recombinant HIV-1 expressing luciferase and bearing wild-type or mutant Envs from HIV-1_YU2 (A and B) or HIV-1_HXBc2 (C and D) were normalized by reverse transcriptase activity. Equal amounts of viruses were incubated with serial dilutions of BMS806 (A and C) or 484 (C and D) at 37°C for 1 h prior to infection of Cf2Th-CD4 CCR5 (A and B) or Cf2Th-CD4 CXCR4 cells (C and D). Infectivity at each dilution of inhibitor tested is shown as the percentage of infection without inhibitor for each particular mutant. Quadruplicate samples were analyzed in each experiment. Data shown are the means of results from 4 (A), 3 (B), or 2 (C and D) independent experiments. The error bars represent the standard deviations.

FIG 4

sCD4 activation of HIV-1_YU2 Env variants with alterations of the gp41 ectodomain. Effect of soluble CD4 on HIV-1 infection of CD4-negative, CCR5-expressing cells. Recombinant luciferase-expressing HIV-1 with the indicated HIV-1 Env variants were normalized by reverse transcriptase activity and incubated with serial dilutions of sCD4 at 37°C for 1 h prior to infection of Cf2Th-CCR5 cells. The level of infection is reported in relative light units (RLU). For each data point, the infection was performed in quadruplicate; data shown are representative of those obtained in at least two independent experiments.

FIG 5

Sensitivities of HIV-1 variants with alterations of the gp41 ectodomain to neutralization by antibodies. Recombinant HIV-1 strains expressing luciferase and bearing wild-type or mutant HIV-1_HXBc2 Envs were normalized by reverse transcriptase activity. Equal amounts of viruses were incubated with serial dilutions of VRC13 (A), b12 (B), b6 (C), or F105 (D) at 37°C for 1 h prior to infection of Cf2Th-CD4 CXCR4 cells. Infectivity at each concentration of antibody tested is shown as the percentage of infection without inhibitor for each particular mutant. Quadruplicate samples were analyzed in each experiment. Data shown are the means of results from 3 (A, B, and D) or 2 (C) independent experiments. The error bars represent the standard deviations.

FIG 6

Effect of gp41 ectodomain changes on cold inactivation of HIV-1. Recombinant HIV-1 expressing luciferase and bearing wild-type or mutant Envs from HIV-1_HXBc2 were normalized by reverse transcriptase activity. Equal amounts of viruses were kept at −80°C (initially) or incubated on ice for the indicated times, as indicated in Materials and Methods, and used to infect Cf2Th-CD4 CXCR4 cells. The infectivity relative to that observed in the absence of incubation on ice is shown. Quadruplicate samples were analyzed; data shown are representative of those obtained in at least two independent experiments.

FIG 7

Effect of Env changes on gp41 HR1 exposure. 293T cells were transfected with an empty pcDNA3.1 plasmid or a plasmid expressing the indicated cytoplasmic-tail-deleted HIV-1_JR-FL Env variants. (A) Two days posttransfection, Env expression was evaluated with the 2G12 antibody, which recognizes the gp120 outer domain, by flow cytometry as described in Materials and Methods. (B) HR1 exposure was assessed with an Alexa Fluor 647 (AF647)-conjugated C34-Ig recombinant protein in the absence (gray) or presence (green) of 10 μg/ml of sCD4 by flow cytometry, as described in Materials and Methods. (C) Means and standard deviations of AF647-C34-Ig recognition of Env variants in the presence (green) or absence (gray) of sCD4, presented as the mean fluorescence intensity (MFI) detected with AF647-C34-Ig divided by the MFI detected with the 2G12 antibody. Data are the averages from at least two independent experiments. Statistical significance was evaluated using a two-way analysis of variance (ANOVA), with multiple comparisons. ****, P < 0.0001; ns, not significant. (D) Means of CD4-Ig recognition of Env variants are presented as the MFI detected with CD4-Ig divided by the MFI detected with the 2G12 antibody. Data are the averages from four independent experiments. Statistical significance was evaluated using Student's t test. *, P < 0.05; **, P < 0.01; ns, not significant.

FIG 8

(A) Model for modulation of HIV-1 sensitivity to gp120-directed inhibitors by changes in the gp41 ectodomain. (A) In the unliganded conformation, the envelope glycoproteins of primary HIV-1 mostly sample state 1. The proposed effects of the indicated Env changes on Env conformation are shown. The A582T change in gp41 is proposed to stabilize state 1 (indicated by a green arrow), whereas the L587A change in gp41 is suggested to destabilize state 2 (indicated by a red arrow). The H66A change in gp120 decreases CD4 binding by increasing the off rate of the gp120-CD4 interaction (26, 51, 61) (red arrow). All three changes (A582T, L587A, and H66A) act in different ways to predispose Env to assume a state 1 conformation. (B) Sequences of the gp41 ectodomain in primate immunodeficiency virus Envs. Primary sequence alignment of gp41 HR1 region residues from representative HIV-1 B (GenBank accession number K03455), HIV-1 C (accession number U46016), simian immunodeficiency virus strain cpz (SIV_cpz) (accession number DQ373064), HIV-2 (accession number AF082339), SIV_mac/smm (accession number M33262), SIV_tan (accession number U58991), SIV_agm (accession number M30931), and SIV_syk (accession number L06042) isolates (7, 25). The shading highlights residues that are conserved in all primate immunodeficiency virus lineages (85). Heptad repeat positions are shown in lowercase letters on top of the amino acid sequence alignment.