Structural Basis for Inhibitor-Induced Aggregation of HIV Integrase

Abstract

The allosteric inhibitors of integrase (termed ALLINIs) interfere with HIV replication by binding to the viral-encoded integrase (IN) protein. Surprisingly, ALLINIs interfere not with DNA integration but with viral particle assembly late during HIV replication. To investigate the ALLINI inhibitory mechanism, we crystallized full-length HIV-1 IN bound to the ALLINI GSK1264 and determined the structure of the complex at 4.4 Å resolution. The structure shows GSK1264 buried between the IN C-terminal domain (CTD) and the catalytic core domain. In the crystal lattice, the interacting domains are contributed by two different dimers so that IN forms an open polymer mediated by inhibitor-bridged contacts; the N-terminal domains do not participate and are structurally disordered. Engineered amino acid substitutions at the inhibitor interface blocked ALLINI-induced multimerization. HIV escape mutants with reduced sensitivity to ALLINIs commonly altered amino acids at or near the inhibitor-bound interface, and these substitutions also diminished IN multimerization. We propose that ALLINIs inhibit particle assembly by stimulating inappropriate polymerization of IN via interactions between the catalytic core domain and the CTD and that understanding the interface involved offers new routes to inhibitor optimization.

Fig 1. Allosteric inhibitors of HIV IN.

(A) Domain organization of IN. (B) Chemical structures of the ALLINIs used in this study. (C) Disruption of assembly by the ALLINIs GSK1264 and GSK002. Viral particles produced in the presence of 1,000 nM GSK1264 or GSK002 were visualized by transmission electron microscopy, and morphology was scored (see S1 Data). The p-value is the probability of obtaining the observed (or greater) differences in numbers of nonmature particles (immature, deformed, or ambiguous) between treated and nontreated samples, given the null hypothesis of no inhibitor-induced changes.

Fig 2. Crystal structure of HIV-1 IN^Y15A,F185H•GSK1264 complex.

(A) Shown is a simulated annealing composite-omit 2F_o-F_c electron density map contoured to 1.5 σ (shown in blue). Contiguous electron density is observed for the catalytic core domains, the CTDs, and GSK1264 (red), but the NTDs are disordered. (B) Simulated annealing F_o-F_c map density for GSK1264, contoured to 3σ (shown in blue). (C) The ALLINI-bound HIV-1 IN polymer observed in crystals. One dimer of HIV-1 IN comprises the asymmetric unit. Bound GSK1264 is shown by the red spheres, two interacting dimers are highlighted in the dotted box, and additional subunits of the open polymer are shown in light grey. (D) Cartoon of the CTD–catalytic core domain interaction, where the hand represents the CTD, the ball represents the catalytic core domain dimer, and GSK1264 is shown in red. See also S2 Fig and S2 Table.

Fig 3. The GSK1264 binding interface.

(A) Orthogonal views of the GSK1264-induced polymer interface. GSK1264 is shown in red, packed between the CTD (grey) and the catalytic core domain dimer (tan). The positions of the side chains rendered are inferred from the high-resolution crystal structures used in the DEN refinement procedure. (B) Sphere rendering of the protein–ALLINI-protein interface. Colored as in A. (C) Schematic of IN-GSK1264 contacts. GSK1264 (red) is predominantly buried via van der Waals contacts with 13 residues from the CTD (grey) and catalytic core domain (tan). Thr¹⁷⁴, Lys²⁶⁶, and His¹⁷¹ are predicted to hydrogen bond with the tert-butoxy and carboxylic acid moieties. This panel was generated using LIGPLOT [30]. See also S2 Fig, S3 Fig and S1 Table.

Fig 4. Experimental probing of the GSK1264 interface.

Models of the catalytic domain–CTD interface with bound small molecules (A, C, E, and G) are shown beside aggregation time course assays for the same compounds (B, D, F, and H). Aggregation assays contained one and two-domain fragments of IN^F185H as indicated (“CCD” denotes catalytic core domain). Compounds studied are GSK1264 (A, B; red, PDB 4OJR [7]), GSK002 (C,D; blue, PDB 5HRN [this work]), BI-D (E, F; green, PDB 4ID1 [8]), and tetraphenylarsonium (TPA; G, H; magenta, PDB 1HYV [10]). Models were generated by docking the indicated CCD/compound structures into the GSK1264/ IN^F185H structure studied here. Aggregation assays were carried out using light scattering at 405 nm at 25°C with 10 μM IN (see S1 Data). In panel G, arrows indicate steric clashes predicted by modeling of the CTD–catalytic core domain interface with TPA, based on the TPA binding mode in PDB 1HYV [10].

Fig 5. CTD substitutions affect IN oligomerization and ALLINI-induced aggregation.

(A) CTD residues (shown in blue) at the IN^F185H–ALLINI interface mapped onto the IN^F185H–GSK1264 structure. GSK1264 is shown in red. Mutations at K264 and K266 that affect ALLINI function were previously reported [24]; the effects of mutations at Y226 and W235 are reported here. (B) Aggregation assays. The time-dependent aggregation of IN by GSK1264 and GSK002 was monitored using absorbance optics. To initiate the reaction, either DMSO or drug was added to recombinant IN^F185H for final concentrations of ~40–61 μM IN^F185H monomer and 44 μM drug at room temperature. For each panel in this figure, “IN” denotes “IN^F185H”. (C) Analytical ultracentrifugation sedimentation equilibrium analysis. Data were recorded at 12,000 RPM, at a concentration of ~10 μM IN^F185H, at 4°C. Linearized radial distributions are shown. The slopes are proportional to M_w at a given value of r². Single-species plots with calculated slopes for idealized IN^F185H monomer, dimer, and tetramer are shown for the same rotor speed and temperature as black lines. (D) SEC-MALS analysis. The IN^F185H SEC trace is superimposed on each panel in the dotted red line. Retention times for molecular weight markers are shown at the top of each panel, and retention times for globular molecular weight standards are shown as open circles. Elution concentrations by refractive index approached ~0.1 mg/mL. Experiments were performed at room temperature using a Superdex 200 10/300 column. Data shown in panels B–D are provided in S1 Data.

Fig 6. HIV IN mutations conferring reduced sensitivity to GSK1264 and GSK002 after serial passage.

(A) Amino acid substitutions resulting from resistance mutations identified by serial passage in the presence of GSK1264 (blue spheres). (B) Substitutions identified in the presence of GSK 002 (orange spheres). Bound GSK1264 is shown in red. (C) The A205T resistance substitutions, which arose in both experiments. S4 Fig provide structural insights into the mechanism of resistance conferred by polymorphisms at residues 124 and 125.

Fig 7. Sensitivity of HIV strains to GSK1264 and GSK002.

(A) Distributions of IC₅₀ values for GSK1264 and GSK002 in infections of multiple subtypes of HIV, colored by subtype and sorted by small molecule. The y-axis shows IC₅₀ values. Data are provided in tabular form in S1 Data. (B) Polymorphisms identified by lasso logistic regression linked to resistance to GSK1264 (blue spheres) or GSK002 (green spheres) mapped onto the IN-GSK1264 structure. GSK1264 is shown in red. See S3 Table.