Mutations That Increase the Stability of the Postfusion gp41 Conformation of the HIV-1 Envelope Glycoprotein Are Selected by both an X4 and R5 HIV-1 Virus To Escape Fusion Inhibitors Corresponding to Heptad Repeat 1 of gp41, but the gp120 Adaptive Mutations Differ between the Two Viruses

Abstract

Binding of the gp120 surface subunit of the envelope glycoprotein (Env) of HIV-1 to CD4 and chemokine receptors on target cells triggers refolding of the gp41 transmembrane subunit into a six-helix bundle (6HB) that promotes fusion between virus and host cell membranes. To elucidate details of Env entry and potential differences between viruses that use CXCR4 (X4) or CCR5 (R5) coreceptors, we generated viruses that are resistant to peptide fusion inhibitors corresponding to the first heptad repeat region (HR1) of gp41 that target fusion-intermediate conformations of Env. Previously we reported that an R5 virus selected two resistance pathways, each defined by an early gp41 resistance mutation in either HR1 or the second heptad repeat (HR2), to escape inhibition by an HR1 peptide, but preferentially selected the HR1 pathway to escape inhibition by a trimer-stabilized HR1 peptide. Here, we report that an X4 virus selected the same HR1 and HR2 resistance pathways as the R5 virus to escape inhibition by the HR1 peptide. However, in contrast to the R5 virus, the X4 virus selected a unique mutation in HR2 to escape inhibition by the trimer-stabilized peptide. Significantly, both of these X4 and R5 viruses acquired gp41 resistance mutations that improved the thermostability of the six-helix bundle, but they selected different gp120 adaptive mutations. These findings show that these X4 and R5 viruses use a similar resistance mechanism to escape from HR1 peptide inhibition but different gp120-gp41 interactions to regulate Env conformational changes.IMPORTANCE HIV-1 fuses with cells when the gp41 subunit of Env refolds into a 6HB after binding to cellular receptors. Peptides corresponding to HR1 or HR2 interrupt gp41 refolding and inhibit HIV infection. Previously, we found that a CCR5 coreceptor-tropic HIV-1 acquired a key HR1 or HR2 resistance mutation to escape HR1 peptide inhibitors but only the key HR1 mutation to escape a trimer-stabilized HR1 peptide inhibitor. Here, we report that a CXCR4 coreceptor-tropic HIV-1 selected the same key HR1 or HR2 mutations to escape inhibition by the HR1 peptide but different combinations of HR1 and HR2 mutations to escape the trimer-stabilized HR1 peptide. All gp41 mutations enhance 6HB stability to outcompete inhibitors, but gp120 adaptive mutations differed between these R5 and X4 viruses, providing new insights into gp120-gp41 functional interactions affecting Env refolding during HIV entry.

Keywords: HIV-1; conformational changes; fusion; fusion inhibitor; gp41; resistance.

FIG 1

Resistance pathways of LAI HIV-1 after selection with N36 and IZN36 peptide fusion inhibitors. Mutations are noted from top to bottom as they emerged in each culture. Passage number (P) and fold increase (x) in inhibitor concentration over the initial concentration are shown. Control cultures shown were passaged without peptides. The glutamic acid-to-lysine substitutions at residues 560 (E560K) and 648 (E648K) define pathways 1 and 2, respectively. The asparagine-to-lysine substitution at residue 637 (N637K) defines pathway 3. C, culture. Con, control.

FIG 2

N36 and IZN36 peptide inhibition of pseudovirus bearing Envs with mutations that emerged in the resistance cultures. The IC₅₀ values for the N36 and IZN36 peptides were determined using pseudoviruses with Envs containing various combinations of the mutations found in the resistance cultures and control cultures, normalized to the IC₅₀ for the wild-type pseudovirus (WT). Pseudoviruses bearing Envs containing all mutations from each culture (A and C) or specified gp41 mutations (B and D) are shown. Averages and standard deviations (error bars) from at least three independent experiments are shown. *, P < 0.05 compared with the WT.

FIG 3

Thermal denaturation studies of the α-helical complexes formed with HR1 and HR2 peptides. The α-helical content was calculated from the circular dichroism (CD) spectroscopy signal at the indicated wavelengths. Unfolding was recorded at 222 nm by CD spectroscopy at the indicated temperatures, with calculated T_m values shown. (A and B) The CD of the complexes formed by mixing the C34 peptide corresponding to the LAI strain with the N36 peptide corresponding to the JR_CSF or LAI strain (A) and their melting curves (B). (C) The melting curves of the complexes formed by mixing the C34 or C34-637K peptide with the N36 or N36-560K peptide. (D and E) The melting curves of the complexes formed by mixing the N36 peptide with the indicated C34 peptide. (F) The melting curves of the complexes formed by mixing the IZN36 peptide with the indicated C34 peptides. Results shown are representative of two experiments.

FIG 4

Correlation between stability of 6HB and resistance to HR1 peptide inhibitors. Correlations between T_m and the logarithmic mean IC₅₀ of Env clones and single mutants relative to that of the WT (relative resistance) selected by N36 (A) and IZN36 (B). r_s, Spearman correlation coefficient.

FIG 5

Six-helix bundle complexes formed by mixing HR1 and HR2 peptides visualized using native PAGE electrophoresis. HR1 N36 peptides migrated off the gel due to their net positive charge. N36 and C34 peptides corresponding to mutations selected in pathway 3 (A) and pathways 2 and 3 (B). The upward migration of the C34 peptide bands reflects interactions with the N36 peptide. In panel A, both images are from the same gel, but lanes with irrelevant peptides were removed.

FIG 6

Pathway 3 resistance mutations modeled in the six-helix bundle conformation. Mutations N637K, T639I, N651S, and K655E are modeled in the six-helix bundle conformation (PDB entry 1AIK) in a ribbon model in longitudinal view (A) and cross-section view (B) and in a hydrophobic surface rendition (Kyte & Doolittle scale) in longitudinal view (C), with the inset showing a close-up (D). Mutations N637K and K655E occupy positions c and g of the heptad repeat without obvious interactions affecting six-helix bundle stability. The T639I mutation in HR2 likely contributes additional hydrophobic interactions, with the hydrophobic cleft running between HR1 protomers. (D) HR2 hydrophobic residues are colored magenta and labeled. The T639I is labeled green.

FIG 7

Mutations from the HR1 resistance cultures modeled in Env trimers. Fusion inhibitor resistance mutations are modeled in the unliganded Env SOSIP trimer (PDB entry 5CEZ) (A) and the CD4- and 17b-liganded Env SOSIP trimer (PDB entry 5VN3) (B). In both panels, one protomer of gp120 is colored tan and one protomer of gp41 is colored light blue. The remaining protomers are colored gray. Mutations in gp120 are colored magenta, and mutations in gp41 are colored cyan. Mutations affecting N-linked glycosylation are colored yellow. Zoomed-in views of the gp120 inner domain and gp41 HR1 region highlight gp120 mutations located in and around its potential interaction sites with gp41 and gp120 topological layers (upper insets). The following color scheme was used for gp120 inner domain topological layers: layer 1, purple; layer 2, red; layer 3, orange. The HR2 gp41 mutations are amphipathic, perhaps allowing different interactions in different Env conformations (lower insets, shown rotated 180° from the trimer).