HIV-1 Escape from a Peptidic Anchor Inhibitor through Stabilization of the Envelope Glycoprotein Spike

Abstract

The trimeric HIV-1 envelope glycoprotein spike (Env) mediates viral entry into cells by using a spring-loaded mechanism that allows for the controlled insertion of the Env fusion peptide into the target membrane, followed by membrane fusion. Env is the focus of vaccine research aimed at inducing protective immunity by antibodies as well as efforts to develop drugs that inhibit the viral entry process. The molecular factors contributing to Env stability and decay need to be understood better in order to optimally design vaccines and therapeutics. We generated viruses with resistance to VIR165, a peptidic inhibitor that binds the fusion peptide of the gp41 subunit and prevents its insertion into the target membrane. Interestingly, a number of escape viruses acquired substitutions in the C1 domain of the gp120 subunit (A60E, E64K, and H66R) that rendered these viruses dependent on the inhibitor. These viruses could infect target cells only when VIR165 was present after CD4 binding. Furthermore, the VIR165-dependent viruses were resistant to soluble CD4-induced Env destabilization and decay. These data suggest that VIR165-dependent Env proteins are kinetically trapped in the unliganded state and require the drug to negotiate CD4-induced conformational changes. These studies provide mechanistic insight into the action of the gp41 fusion peptide and its inhibitors and provide new ways to stabilize Env trimer vaccines.

Importance: Because of the rapid development of HIV-1 drug resistance, new drug targets need to be explored continuously. The fusion peptide of the envelope glycoprotein can be targeted by anchor inhibitors. Here we describe virus escape from the anchor inhibitor VIR165. Interestingly, some escape viruses became dependent on the inhibitor for cell entry. We show that the identified escape mutations stabilize the ground state of the envelope glycoprotein and should thus be useful in the design of stabilized envelope-based HIV vaccines.

FIG 1

Escape of HIV-1_LAI from the anchor inhibitor VIR165. (A) Sequences of the natural peptide VIRIP and the more potent and stable derivatives VIR165 and VIR353, both cyclized by a disulfide bond, and the dipeptide VIR576. The “p” in the VIR353 sequence indicates the introduction of a d-proline residue. (B) Stepwise increases of the VIR165 concentration during passage experiments. Cultures were passaged twice a week for 2 1/2 months with increasing concentrations of VIR165. (C) Linear representation of HIV_LAI gp160 showing positions of VIR165 escape mutations in C1 and HR1, as well as previously identified escape mutations conferring resistance to the VIRIP analogue VIR353 (26). (D) The residues involved in VIR165 resistance and dependence were mapped onto a structure of the BG505 SOSIP.664 trimer containing the complete gp41 interactive domain (PDB accession code 5CEZ [9]) by use of Pymol (DeLano Scientific [http://pymol.sourceforge.net]). The residues are shown as spheres on a cartoon model of one of the protomers, with gp41 in sand and gp120 in green. A surface model of the other two protomers is shown in white and gray. Residues at positions 42, 58, 558, and 577, which are involved in VIR165 resistance, are indicated in red. The control position 62 is indicated in cyan, and residues 60, 64, and 66, which are involved in VIR165 dependence, are depicted in yellow. For comparison, residues involved in resistance to the VIRIP analogue VIR353 are indicated in blue (26). The right panel shows a detail of the region that includes residues 58, 60, 64, and 66, all situated in layer 1 of gp120. gp41 is depicted in orange, and gp120 is in green, with layer 1 in magenta and layer 2 in blue.

FIG 2

HIV-1_LAI VIR165 escape variants can be resistant to or dependent on VIR165. Single-cycle infection experiments were performed as described in Materials and Methods. Inhibition data are shown for HIV-1_LAI variants containing VIR165 resistance mutations in gp41 (A), VIR165 resistance mutations in the C1 domain (B), and VIR165 dependence mutations in C1 (C). A D62N control virus, the T20-resistant V549A variant, and the T20-dependent V549A/N637K virus (44) were included as control viruses. (D) Infectivities of VIR165-resistant and -dependent viruses in the absence of VIR165 relative to that of WT virus in a single-cycle infection assay. The virus mutants indicated with asterisks were VIR165 dependent, i.e., they were not infectious in the absence of VIR165. (E) VIR165-dependent virus variants are inhibited at high VIR165 concentrations. Single-cycle infection experiments were performed using concentrations of VIR165 of up to 300 μg/ml, revealing bell-shaped dose-response curves. (F) Maximum infectivities of VIR165-dependent mutants relative to that of the WT in the presence of VIR165. The infectivities of VIR165-dependent viruses (indicated with asterisks) were obtained in the presence of VIR165 at 10 μg/ml (A60E and E64K mutants) or 30 μg/ml (H66R mutant). WT infectivity was measured in the absence of VIR165 (WT) or in the presence of 10 μg/ml VIR165 (WT*). (G) VIR165 dose response curves with WT HIV-1_BG505 and E64K and H66R variants.

FIG 3

Modeling of trimer occupancy by VIR165 explains the bell-shaped dose-response curves of VIR165-dependent viruses. The enhancement and inhibition of WT and VIR165-dependent viruses were modeled mathematically to assess whether enhancing or inhibitory effects of VIR165 could be associated with occupancy levels of 1, 2, or 3 VIR165 molecules per trimer. Different mathematical occupancy models were fitted to the experimentally obtained bell-shaped stimulation-inhibition data from Fig. 2E. The best-fitting models are shown.

FIG 4

VIR165-dependent viruses are less prone to CD4-induced decay. (A) Data from a representative experiment of at least three independent experiments in which VIR165-resistant and VIR165-dependent viruses were incubated at physiological temperature (37°C) in the absence or presence of sCD4 for different times, followed by assessment of the remaining infectivity on TZM-bl cells in the absence of VIR165 for WT virus or in the presence of 10 or 30 μg/ml VIR165 for the dependent variants. (B) Half-lives (t_1/2) of WT and VIR165-resistant and -dependent mutant viruses, measured in at least three independent experiments performed in duplicate. (C) Differences in t_1/2 for viruses incubated in the absence or presence of sCD4 at 37°C. Statistical significance is indicated as follows: *, P < 0.05; and **, P < 0.005.

FIG 5

VIR165-dependent viruses show increased thermostability in the presence of sCD4. (A) The thermostability of WT, VIR165-resistant, and VIR165-dependent viruses was tested by incubating them for 1 h each at escalating temperatures, followed by testing of the remaining infectivity on TZM-bl reporter cells in the absence of VIR165 (WT) or the presence of 10 or 30 μg/ml VIR165 (dependent variants). Data shown are from a representative experiment of at least three independent experiments. (B) Midpoints of thermal denaturation (T_m) of WT and mutant viruses, measured in at least three independent experiments performed in duplicate. (C) Changes in T_m in the presence of sCD4. Statistical significance is indicated as follows: **, P < 0.005; and ***, P < 0.0005.

FIG 6

Hypothetical models of the inhibiting and enhancing modes of action of VIRIP-derived peptides on WT and VIR165-dependent viruses. (A) The FP becomes fully exposed upon binding of the CD4 receptor and coreceptor and is inserted into the target membrane, followed by subsequent fusion of the two membranes. (B) VIR165 binds to the FP in the short-lived intermediate state that is induced by CD4 binding, thereby inhibiting insertion of the FP into the target membrane. (C) In VIR165-dependent viruses, a hyperstable gp120-gp41 interaction cannot be weakened sufficiently by CD4 binding, and CD4-induced conformational changes in the gp120-gp41 complex and subsequent entry steps are blocked. (D) VIR165 in subsaturating amounts acts as a “wedge” between gp120 and gp41, destabilizing the gp120-gp41 interaction and facilitating CD4-induced conformational changes that allow subsequent entry steps. We do not know whether, in this scenario, VIR165 remains associated with the one or two FPs that it occupies and only the free FP(s) inserts into the cell membrane or if it dissociates to allow all three FPs to insert. (E) High concentrations of VIR165 result in occupancy of all three FPs of the Env trimer, effectively inhibiting infection.