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.2013 May 21;110(21):8690-5.
doi: 10.1073/pnas.1300703110. Epub 2013 Apr 22.

Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation

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Allosteric integrase inhibitor potency is determined through the inhibition of HIV-1 particle maturation

Kellie A Jurado et al. Proc Natl Acad Sci U S A..

Abstract

Integration is essential for HIV-1 replication, and the viral integrase (IN) protein is an important therapeutic target. Allosteric IN inhibitors (ALLINIs) that engage the IN dimer interface at the binding site for the host protein lens epithelium-derived growth factor (LEDGF)/transcriptional coactivator p75 are an emerging class of small molecule antagonists. Consistent with the inhibition of a multivalent drug target, ALLINIs display steep antiviral dose-response curves ex vivo. ALLINIs multimerize IN protein and concordantly block its assembly with viral DNA in vitro, indicating that the disruption of two integration-associated functions, IN catalysis and the IN-LEDGF/p75 interaction, determines the multimode mechanism of ALLINI action. We now demonstrate that ALLINI potency is unexpectedly accounted for during the late phase of HIV-1 replication. The compounds promote virion IN multimerization and, reminiscent of class II IN mutations, block the formation of the electron-dense viral core and inhibit reverse transcription and integration in subsequently infected target cells. Mature virions are recalcitrant to ALLINI treatment, and compound potency during virus production is independent of the level of LEDGF/p75 expression. We conclude that cooperative multimerization of IN by ALLINIs together with the inability for LEDGF/p75 to effectively engage the virus during its egress from cells underscores the multimodal mechanism of ALLINI action. Our results highlight the versatile nature of allosteric inhibitors to primarily inhibit viral replication at a step that is distinct from the catalytic requirement for the target enzyme. The vulnerability of IN to small molecules during the late phase of HIV-1 replication unveils a pharmacological Achilles' heel for exploitation in clinical ALLINI development.

Keywords: AIDS; antiretroviral therapy.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
BI-D potency is accounted for during the late phase of HIV-1 replication. (A) Dose–response curves under the indicated conditions of drug treatment. The similarity in late phase and spreading replication EC50 values is statistically significant (P = 0.94); error bars represent the variation obtained from two to three independent experiments. (B) Release of HIV-1NL4-3 from PBMC cultures following 24 h of BI-D (10 µM) or solvent control treatment at peak of virus replication. (C) Infectivity of ultrafiltered particles fromB as assessed in CEMx174 5.25 M7 indicator cells. Results inB andC are averages ± SDs from three independent experiments.
Fig. 2.
Fig. 2.
ALLINIs inhibit the formation of the electron-dense HIV-1 core.Upper, representative images of mature, eccentric, and immature particle morphologies.Lower, quantitation of core morphology frequencies (average ± SD forn = 2 experiments) for ∆IN, V165A, and wild-type HIV-1NL4-3 made in the presence of BI-D (10 µM), BI-1001 (50 µM), or DMSO solvent control. Particles (100 in each experiment) were counted and typed as described in text.
Fig. 3.
Fig. 3.
BI-D–treated HIV-1 is defective for reverse transcription and integration. (A) DNAs (25 ng) isolated from SupT1 cells infected with HIV-Luc in the presence of DMSO (light gray squares), 10 µM BI-D (black circles), or 10 µM RAL (dark gray triangles) were assessed for LRT product formation by quantitative PCR. (B) Time course of two-LTR circle formation. (C) Alu-PCR values at 48 h postinfection (DMSO sample set at 100%) for extracts described inA. (D) DNAs isolated from SupT1 cells infected with IN mutant V165A (dark gray triangles), or wild-type HIV-Luc pretreated during virus production with DMSO (light gray squares) or 10 µM BI-D (black circles), were assessed for ERT product formation. (E andF) Levels of LRT and two-LTR circle product formation, respectively, as a function of time. (G) Alu-PCR values at 48 h postinfection for extracts described inD; values obtained from DMSO-treated HIV-Luc were set at 100%. (H) Percentage of cells positive for beta-lactamase; similar results were found for HIV-Luc carrying the HIV-1 envelope glycoprotein. Results inA andC–G are averages and SDs of two independent infection experiments, with DNA samples queried in duplicate by PCR for each infection;B andH values are averages and SDs from three independent experiments.
Fig. 4.
Fig. 4.
BI-D enhances the formation of IN oligomers. (A) Elution profiles of purified wild-type (WT) and A124D mutant IN (20 µM) in the absence of drug (Upper) or in the presence of 10 µM BI-D. Elution times of indicated peak values, and associated estimates of oligomeric states, are summarized inTable S2. (B) Wild-type HIV-1NL4-3 and IN mutant T124D (position 124 is polymorphic; HIV-1HXB2 carries Ala where HIV-1NL4-3 harbors Thr) made in the presence of DMSO solvent control or 5 µM or 10 µM BI-D as indicated were prepared with and without DTT reducing agent in the SDS/PAGE sample buffer.Upper, blot was probed with anti-IN monoclonal antibody 8E5;Lower, blot probed with anti-p24 antibody. Results are representative of three independent experiments. D, IN dimer; M, IN monomer; O, IN oligomer.
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Comment in

  • Integrating the HIV-1 assembly/maturation pathway.
    Potempa M, Swanstrom R.Potempa M, et al.Proc Natl Acad Sci U S A. 2013 May 21;110(21):8327-8. doi: 10.1073/pnas.1306620110. Epub 2013 May 13.Proc Natl Acad Sci U S A. 2013.PMID:23671082Free PMC article.No abstract available.

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