The Competitive Interplay between Allosteric HIV-1 Integrase Inhibitor BI/D and LEDGF/p75 during the Early Stage of HIV-1 Replication Adversely Affects Inhibitor Potency

Abstract

Allosteric HIV-1 integrase inhibitors (ALLINIs) have recently emerged as a promising class of antiretroviral agents and are currently in clinical trials. In infected cells, ALLINIs potently inhibit viral replication by impairing virus particle maturation but surprisingly exhibit a reduced EC50 for inhibiting HIV-1 integration in target cells. To better understand the reduced antiviral activity of ALLINIs during the early stage of HIV-1 replication, we investigated the competitive interplay between a potent representative ALLINI, BI/D, and LEDGF/p75 with HIV-1 integrase. While the principal binding sites of BI/D and LEDGF/p75 overlap at the integrase catalytic core domain dimer interface, we show that the inhibitor and the cellular cofactor induce markedly different multimerization patterns of full-length integrase. LEDGF/p75 stabilizes an integrase tetramer through the additional interactions with the integrase N-terminal domain, whereas BI/D induces protein–protein interactions in C-terminal segments that lead to aberrant, higher-order integrase multimerization. We demonstrate that LEDGF/p75 binds HIV-1 integrase with significantly higher affinity than BI/D and that the cellular protein is able to reverse the inhibitor induced aberrant, higher-order integrase multimerization in a dose-dependent manner in vitro. Consistent with these observations, alterations of the cellular levels of LEDGF/p75 markedly affected BI/D EC50 values during the early steps of HIV-1 replication. Furthermore, genome-wide sequencing of HIV-1 integration sites in infected cells demonstrate that LEDGF/p75-dependent integration site selection is adversely affected by BI/D treatment. Taken together, our studies elucidate structural and mechanistic details of the interplay between LEDGF/p75 and BI/D during the early stage of HIV-1 replication.

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

“…In our experiments, the relative inhibition of LEDGF/p75 interaction with IN polymorphs strikingly matches the corresponding ARV activity at integration of INLAIs. This finding is also in agreement with the previous reports showing that competition by LEDGF/p75 controls the INLAImediated inhibition of integration and weakens its magnitude compared to the ARV potency at late stage that does not suffer from such competition (15,(37)(38)(39).…”

“…The close correlation that we observed between the level of IN multimerization and the potency of INLAI ARV activity in multipleround infection assays provides further evidence that aberrant IN multimerization is the primary biochemical mechanism for the of INLAIdependent viral maturation defect. It was also reported that modulating LEDGF/p75 levels in target cells (by knockout or overexpression) determines INLAI BI-D potency at integration (37). In our experiments, the relative inhibition of LEDGF/p75 interaction with IN polymorphs strikingly matches the corresponding ARV activity at integration of INLAIs.…”

“…However, if this binding is sufficient to inhibit IN-LEDGF/p75 interaction, it is not sufficient, even if fully conserved, to promote the second activity on IN, aberrant multimerization, and consequently the late effect on virus maturation. Some other effects, additional to the binding to the LEDGF-binding pocket, are required to promote IN multimerization, presumably, conformational changes of allosteric nature between CCD-CTD interactions as previously described (36,37). In this report, we demonstrate that the two ARV activities of INLAIs can indeed be fully dissociated for several compounds such as MUT-A and some of its derivatives.…”

A New Class of Allosteric HIV-1 Integrase Inhibitors Identified by Crystallographic Fragment Screening of the Catalytic Core Domain

“…The ligand efficiency (LE) for compound 5 was calculated to be 0.38 kcal/mol per non-hydrogen atom (40,41). This compares favorably to the LE of 0.31 kcal/mol per non-hydrogen atom for direct binding of a highly potent representative ALLINI, BI-D, to IN (42). The inhibition of LEDGF/ p75-dependent activity could be due to (i) compound-induced aberrant protein multimerization of IN resulting in inactivation, (ii) the compound competing with IN binding to LEDGF/ p75, or (iii) a combination of these activities.…”

Computational and synthetic approaches for developing Lavendustin B derivatives as allosteric inhibitors of HIV-1 integrase

“…Surprisingly, ALLINIs exhibit higher potency when present during virion morphogenesis compared with the early stage of viral replication [29,34–37]. In the virions, where due to the lack of competing LEDGF/p75 binding to the IN dimer, ALLINIs potently induce aberrant IN multimerization and result in eccentric, non-infectious virions; whereas during the early stage of HIV-1 replication LEDGF/p75 effectively competes with ALLINI binding to IN and reduces the inhibitor potency [38]. Selection of HIV-1 phenotypes under the genetic pressure of various ALLINIs have identified substitutions at the IN dimer interface at the inhibitor binding sites that confer resistance to these compounds [25,28,39,40].…”