A Critical Role of the C-terminal Segment for Allosteric Inhibitor-induced Aberrant Multimerization of HIV-1 Integrase

Shkriabai, Nikoloz; Dharmarajan, Venkatasubramanian; Slaughter, Alison; Kessl, Jacques J.; Larue, Ross C.; Feng, Lei; Fuchs, James R.; Griffin, Patrick R.; Kvaratskhelia, Mamuka

doi:10.1074/jbc.m114.589572

Cited by 28 publications

(48 citation statements)

References 55 publications

(85 reference statements)

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“…We first tested the best characterized archetypal IN inhibitor BI-B2 (also known as ALLINI-2) (Figure S7A) in conjunction with the WT virus and HIV-1 NL4-3 IN(A128T) , a mutation that confers significant resistance to this inhibitor (Figure 7A) (Feng et al, 2013; Fontana et al, 2015; Shkriabai et al, 2014). BI-B2 treatment of virus producer cells substantially inhibited the binding of WT IN to RNA in virions, while the treatment of HIV-1 NL4-3 IN(A128T) with the inhibitor yielded IN-RNA crosslink levels that were comparable with untreated WT virus (Figures 7B and S7B).…”

Section: Resultsmentioning

confidence: 99%

HIV-1 Integrase Binds the Viral RNA Genome and Is Essential during Virion Morphogenesis

Kessl

Kutluay

Townsend

et al. 2016

Cell

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SUMMARY While an essential role of HIV-1 integrase (IN) for integration of viral cDNA into human chromosome is established, studies with IN mutants and allosteric IN inhibitors (ALLINIs) have suggested that IN can also influence viral particle maturation. However, it has remained enigmatic as to how IN contributes to virion morphogenesis. Here we demonstrate that IN directly binds the viral RNA genome in virions. These interactions have specificity as IN exhibits distinct preference for select viral RNA structural elements. We show that IN substitutions that selectively impair its binding to viral RNA result in eccentric, non-infectious virions without affecting nucleocapsid-RNA interactions. Likewise, ALLINIs impair IN binding to viral RNA in virions of wild type but not escape mutant virus. These results reveal an unexpected biological role of IN binding to the viral RNA genome during virion morphogenesis and elucidate the mode of action of ALLINIs.

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Section: Resultsmentioning

confidence: 99%

HIV-1 Integrase Binds the Viral RNA Genome and Is Essential during Virion Morphogenesis

Kessl

Kutluay

Townsend

et al. 2016

Cell

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118

310

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“…Solution-phase amide HDX experiments were carried out as described previously 33 using a fully automated system, and sample handling was done using CTC HTS Twin PAL robots (LEAP Technologies), housed inside a 4 °C cabinet. In parallel reactions, 10 µM of preformed complex between 6 × His-LEDGF/p75 and FLAG-HIV-1 IN or 10 µM 6 × His-HIV-1 IN protein premixed with 1:5 molar excess of BI/D were subjected to HDX analysis.…”

Section: Methodsmentioning

confidence: 99%

“…The sizes of the IN multimers formed in the presence of ALLINIs significantly exceed that of the tetrameric form needed for catalysis. 25,33,34 In infected cells, ALLINIs inhibit both early and late stages of HIV-1 replication with the most potent antiviral activity observed as a result of improper maturation of virus particles. 26,28,35–37 During maturation, the antiviral activity of ALLINIs has been linked to the promotion of aberrant IN multimers, which in turn leads to the mislocalization of ribonucleoprotein complexes outside of the capsid core and results in eccentric, noninfectious virions.…”

mentioning

confidence: 99%

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

et al. 2016

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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.

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“…Recent biophysical studies have revealed the critical role of the CTD in addition to the CCD for inhibitor-induced aberrant IN multimerization (Gupta et al 2014; Shkriabai et al 2014). Sedimentation velocity and turbidity assays with truncated IN variants have shown that the addition of ALLINI stabilized a dimeric form of IN CCD but induced higher order multimerization with the two domain IN CCD–CTD construct or full-length IN (Gupta et al 2014).…”

Section: The Complexity Of In Subunit–subunit Interactionsmentioning

confidence: 99%

“…Sedimentation velocity and turbidity assays with truncated IN variants have shown that the addition of ALLINI stabilized a dimeric form of IN CCD but induced higher order multimerization with the two domain IN CCD–CTD construct or full-length IN (Gupta et al 2014). Mass spectrometry-based protein footprinting of full-length IN in complex with ALLINI has identified protein–protein interactions in the CCD and CTD that extend beyond the inhibitor-binding site and which contribute to higher order IN multimerization (Shkriabai et al 2014). For example, IN CTD residues Lys264 and Lys266, which are significantly distanced from the ALLINI-binding site, were shielded from modification when the inhibitor was added to IN.…”

Section: The Complexity Of In Subunit–subunit Interactionsmentioning

confidence: 99%

HIV-1 Integrase Multimerization as a Therapeutic Target

Feng

Larue

Slaughter

et al. 2015

The Future of HIV-1 Therapeutics

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Multimeric HIV-1 integrase (IN) plays an essential, multifunctional role in virus replication and serves as an important therapeutic target. Structural and biochemical studies have revealed the importance of the ordered interplay between IN molecules for its function. In the presence of viral DNA ends, individual IN subunits assemble into a tetramer and form a stable synaptic complex (SSC), which mediates integration of the reverse transcribed HIV-1 genome into chromatin. Cellular chromatin-associated protein LEDGF/p75 engages the IN tetramer in the SSC and directs HIV-1 integration into active genes. A mechanism to deregulate the productive interplay between IN subunits with small molecule inhibitors has recently received considerable attention. Most notably, allosteric IN inhibitors (ALLINIs) have been shown to bind to the IN dimer interface at the LEDGF/p75 binding pocket, stabilize interacting IN subunits, and promote aberrant, higher order IN multimerization. Consequently, these compounds impair formation of the SSC and associated LEDGF/p75-independent IN catalytic activities as well as inhibit LEDGF/p75 binding to the SSC in vitro. However, in infected cells, ALLINIs more potently impaired correct maturation of virus particles than the integration step. ALLINI treatments induced aberrant, higher order IN multimerization in virions and resulted in eccentric, non-infectious virus particles. These studies have suggested that the correctly ordered IN structure is important for virus particle morphogenesis and highlighted IN multimerization as a plausible therapeutic target for developing new inhibitors to enhance treatment options for HIV-1-infected patients.

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A Critical Role of the C-terminal Segment for Allosteric Inhibitor-induced Aberrant Multimerization of HIV-1 Integrase

Cited by 28 publications

References 55 publications

HIV-1 Integrase Binds the Viral RNA Genome and Is Essential during Virion Morphogenesis

HIV-1 Integrase Binds the Viral RNA Genome and Is Essential during Virion Morphogenesis

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

HIV-1 Integrase Multimerization as a Therapeutic Target

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