Structure-function analyses unravel distinct effects of allosteric inhibitors of HIV-1 integrase on viral maturation and integration

Abstract

Recently, a new class of HIV-1 integrase (IN) inhibitors with a dual mode of action, called IN-LEDGF/p75 allosteric inhibitors (INLAIs), was described. Designed to interfere with the IN-LEDGF/p75 interaction during viral integration, unexpectedly, their major impact was on virus maturation. This activity has been linked to induction of aberrant IN multimerization, whereas inhibition of the IN-LEDGF/p75 interaction accounts for weaker antiretroviral effect at integration. Because these dual activities result from INLAI binding to IN at a single binding site, we expected that these activities co-evolved together, driven by the affinity for IN. Using an original INLAI, MUT-A, and its activity on an Ala-125 (A125) IN variant, we found that these two activities on A125-IN can be fully dissociated: MUT-A-induced IN multimerization and the formation of eccentric condensates in viral particles, which are responsible for inhibition of virus maturation, were lost, whereas inhibition of the IN-LEDGF/p75 interaction and consequently integration was fully retained. Hence, the mere binding of INLAI to A125 IN is insufficient to promote the conformational changes of IN required for aberrant multimerization. By analyzing the X-ray structures of MUT-A bound to the IN catalytic core domain (CCD) with or without the Ala-125 polymorphism, we discovered that the loss of IN multimerization is due to stabilization of the A125-IN variant CCD dimer, highlighting the importance of the CCD dimerization energy for IN multimerization. Our study reveals that affinity for the LEDGF/p75-binding pocket is not sufficient to induce INLAI-dependent IN multimerization and the associated inhibition of viral maturation.

Keywords: INLAI; LEDGF; allosteric regulation; crystal structure; human immunodeficiency virus (HIV); inhibitor; integrase; multimerization; polymorphism; viral replication.

Figure 1.

Effect of IN 124–125 TT/AA polymorphism on biochemical activities of indicated INLAIs. A, setup of IN-LEDGF/p75 interaction and IN multimerization HTRF assays. B, HTRF signal of interactions with IN TT (WT NL4-3) or AA variant in the absence of inhibitor. C–H, dose-response curves of IN-LEDGF/p75 inhibition (C–E) or IN multimerization (F–H) with IN TT (in black) or IN AA variant (in orange) for the studied compounds. Results shown as average ± S.D. of at least three independent experiments performed in duplicate.

Figure 2.

Cryo-EM images of HIV-1 NL4-3 WT (IN TT 124/125) and HIV-1 NL4-3 polymorphic IN AA 124/125 virus particles treated in the presence or absence of MUT-A. Red arrows indicate the formation of eccentric condensates; blue arrows indicate normal conical cores; and green arrows show nonconical cores. A, NL4-3 IN AA 124/125 polymorphic virus produced from 293T cells, transfected with pNL4-3 IN AA 124/125, in the presence of DMSO and in the absence of MUT-A (negative control). B, NL4-3 WT (IN TT 124/125) virus produced in the presence of 1 μm MUT-A (positive control showing eccentric condensate). C, NL4-3 IN AA 124/125 polymorphic virus produced in the presence of 5 μm MUT-A. Scale bars, 100 nm.

Figure 3.

Structure-activity relationship study of the substituent at position 5 of the thienyl core in MUT-A series. Shown are activities of racemic MUT-A and analogs with various modifications on the chemical group at position 5 of the thiophene core on IN T124/T125 and A124/A125 variant in biochemical interaction assays and multiple-round and single-round infection assays. Means are from at least three experiments (standard deviation omitted for clarity). The two compounds with better susceptibility of IN AA (fold-change closest to 1) are boxed. ^a, IC₅₀ or EC₅₀ fold-change AA/TT. ^b, for IN multimerization, the fold-change takes the AC₅₀ and plateau shifts into account, according to Equation 1. nt, not tested; nr, not reliable (marginal effect).

Figure 4.

Structures of IN CCD TT and AA ± MUT-A. A, global view of the IN CCD TT structure. B, superposition of the IN CCD TT ± MUT-A structures, close view of the binding pocket (gold = + ligand; light gold = − ligand). Large displacement of Thr-124 and Glu-170 between + and − MUT-A. C, 2D view of MUT-A interactions with IN CCD TT. D, global view of the IN-CCD AA structure. E, superposition of the IN-CCD AA ± MUT-A structures, close view of the binding pocket (light blue = + ligand, blue = − ligand). F, 2D view of MUT-A interactions with IN-CCD AA.

Figure 5.

Surface potential of the ligand-binding pockets of IN TT and AA variants with MUT-A and MUT-A03. A, close view in the binding pocket of the IN CCD TT/MUT-A structure. Negative potential is in red, and positive potential is in blue. B, close view in the IN CCD TT MUT-A03-binding pocket. C, 2D view of MUT-A03 interactions with IN CCD TT. D, close view in the binding pocket of the IN CCD AA/MUT-A structure. E, close view in the IN CCD AA MUT-A03-binding pocket. F, 2D view of MUT-A03 interactions with IN CCD TT. All the ligands are contoured with the experimental map at 1.5 σ.