Allosteric HIV Integrase Inhibitors Promote Formation of Inactive Branched Polymers via Homomeric Carboxy-Terminal Domain Interactions

Abstract

The major effect of allosteric HIV integrase (IN) inhibitors (ALLINIs) is observed during virion maturation, where ALLINI treatment interrupts IN-RNA interactions via drug-induced IN aggregation, leading to the formation of aberrant virions. To understand the structural changes that accompany drug-induced aggregation, we determined the soft matter properties of ALLINI-induced IN aggregates. Using small-angle neutron scattering, SEM, and rheology, we have discovered that the higher-order aggregates induced by ALLINIs have the characteristics of weak three-dimensional gels with a fractal-like character. Their formation is inhibited by the host factor LEDGF/p75, as well as ex vivo resistance substitutions. Mutagenesis and biophysical analyses reveal that homomeric carboxy-terminal domain interactions are required to achieve the branched-polymer nature of the ALLINI-induced aggregates. These studies provide key insight into the mechanisms of ALLINI action and resistance in the context of the crowded virion environment where ALLINIs exert their effect.

Keywords: ALLINI; HIV; analytical ultracentrifugation; host factors; oligomerization; polymer network; retroviral integration; rheology; small-angle X-ray scattering; small-angle neutron scattering.

Figure 1.. HIV-1 Integrase.

A. Domain Map of HIV-1 Integrase. B. Chemical structures of small molecules used in these studies. C. Open polymer configuration of ALLINI-bound HIV integrase as observed in the IN-GSK1264 crystal structure. D. ALLINI binding site and CCD-CTD interface.

Figure 2.. Properties of ALLINI-Induced Aggregation.

A. Concentration-dependent aggregation of HIV Integrase with BI-224436. In the absence of drug, protein at 221 μM concentration appears transparent. With the increasing addition of BI-224436, the evolution of an opaque aggregate is readily apparent. B. Time-course kinetics of drug-induced aggregation for BI-224436 (open black circles) and BI-D (grey circles). Protein aggregation without drug is shown with closed black circles. C. Dose response profile for BI-224436 after a 1-hour incubation at room temperature. D. Scanning Electron Microscopy (SEM) analyses of BI-224436 and BI-D-induced IN aggregates. E. Dynamic Light Scattering (DLS) analyses of drug-induced aggregates after an overnight incubation in 1 mM drug.

Figure 3.. Viscoelastic Properties of Drug-Induced Aggregates.

A. Shear stress at increasing levels of shear strain for a sample of 10 mg/mL HIV-1 IN^F185H•1 mM BI-224436 deformed at a constant shear rate of 0.15 s⁻¹ showing the initial elastic response followed by an abrupt fracture of the sample. B. Viscosity vs shear rate at steady shear deformation for very large strains at which intermolecular bonds are continuously broken and reforming, and elongated structures orient in the direction of flow to an extent that depends on the rate of deformation.

Figure 4.. SANS Analysis of ALLINI-induced aggregates of HIV IN.

A. Two-dimensional Neutron Scattering of BI-224436-induced aggregates of HIV-1 IN^F185H. B. Porod Models for Polymer Networks with different shapes. The slope of the Porod region can be directly related to different surface and mass fractal dimensions. For mass fractals, a lower figure correlates with Gaussian chains where larger figures indicate collapsed polymer coils; values of 3-4 indicated branched systems (gels). For surface fractals, values between 3-4 indicate rough interfaces. C. Representative small-angle neutron scattering (SANS) of BI-224436 induced drug aggregate of HIV IN^F185H. Shown in grey is the experimental data where 0.003 A⁻¹ < q < 0.84 Å⁻¹. Shown in inset is a Power law fit to data obtained from 100 μM IN^F185H-BI-224436, in the range where 0.003 < q < 0.01 Å⁻¹. Shown in the main plot is the same data, fit with a Fractal model in the range where 0.003 < q < 0.05 Å⁻¹. A summary of the parameters derived from model fitting of SANS data can be found in STable 1. D. Schematic representation of the mesoscopic structure of the network formed by the ALLINI-mediated branched polymers of HIV IN, where ζ is the correlation length obtained from SANS data at 20°C. The small spheres are a simplified view of the basic units of the network backbone (IN dimers). The scattering centers (grey circles) are formed by regions of clustered protein that give rise to the scattering curves presented. The properties derived from SANS of IN^Y15A,F185H in the absence of drug are provided in STable 2. E. Dose-response curve for the drug-induced aggregation of 80 μM HIV IN^Y15A,F185H. Each data point represents one recorded SANS profile. Using the zero-angle intensity from each experiment, a dose-response curve could be obtained, revealing the concentration for half-maximal aggregation (EC₅₀). The data were fit with a standard Hill equation, yielding an apparent EC₅₀ of 22.1 μM ± 2.33 and a cooperativity factor of 5.6 ± 2.35. Error bars shown on the graph represent the error associated with the determination of I(0).

Figure 5.. Effects of ex vivo resistance mutations and host factors on drug-induced aggregation.

A. W131C and N222K at the CCD-CTD-ALLINI interface. B-D. Small-angle neutron scattering profiles for 212 μM IN^Y15A,F185H (B, red), 205 μM in^W131C,F185H (C, blue), and 200 μM IN^F185H,N222K (D, pink) in the presence and absence of BI-224436 (DMSO controls are shown in grey). Arrows indicate I(0) in each condition. E. The LEDGF(IBD)•CCD•NTD interface, as seen in the PDB 3F9K HIV-2 structure. F-G. SANS profiles for 100 μM IN^F185H or 100 μM co-expressed IN^F185H•LEDGF(IBD) in the presence of 100 μM BI-224436 (F) or 100 μM BI-D (G). Arrows indicate I(0) in each condition.

Figure 6.. Disruption of the CTD-CTD Dimer interface.

A. Models for linear vs. branched polymer networks of ALLINI-induced HIV IN interactions. B. Superposition of the CTD Dimer (PDB 1IHV) with the CCD-CTD-ALLINI interaction observed in the IN-ALINI crystal structure (PDB 5HOT). C. CTD-CTD dimer interface. The quaternary structure of the CTD dimer was confirmed by SEC-SAXS (see SFig1A and STable 3) and was best captured by PDB 5TC2. Shown in spheres are residues that were mutated for effects on oligomerization. D. SEC-MALS analyses of His₆-FLAG-SUMO (HFS)-tagged CTD^220-270 to assess the effects of interfacial mutations on oligomerization. Relative to wild-type CTD fusion (dimers, red), the mutants L241A (orange), L242A (green), and the more extensive quadruply mutated W243Q/V250T/I257N/V259T (blue) disrupted dimerization of the isolated domain. E. Sedimentation velocity (SV-AUC) analysis of isolated CTD^220-270(L242A) (green), indicating a single species with a calculated mass consistent with a monomer.

Figure 7.. Role of the CTD in branched polymer formation.

A. Turbidity time courses for IN^F185H,L242A in the presence of BI-224436. No aggregation was observed in absence of drug, nor the presence of up to three-fold excess of ALLINI. Shown in green is IN^Y15A,F185H. B. SV-AUC analysis of 15 μM IN^F185H,L242A in the presence (purple) and absence (orange) of 100 μM BI-224436. In the absence of drug, species consistent with monomers and dimers are observed (confirmed by SEC-MALS; see SFig1B). In the presence of ALLINI, a ladder of species is observed consistent with the formation of higher-order oligomers. C. Shown is a cartoon schematic depicting the SV-AUC analysis of drug-mediated CCD-CTD interactions using Alexafluor647-labelled HFS-CTD. D. SV-AUC results at 657 nm is shown, with control data in the absence of any drug for both HFS-CTD^L242A alone (red) and a mixture with excess CCD (orange). No evidence of complex formation is observed. F. In the presence of BI-224436, a new ~5S species is formed that is consistent with a higher order HFS-CTD₂•CCD₂•ALLINI₂ complex (~77.8 kD suggested by SE-AUC analysis (See SFig 2 and STable 4). The breadth of the species observed in the presence of drug are consistent with an associating system.