The Allosteric HIV-1 Integrase Inhibitor BI-D Affects Virion Maturation but Does Not Influence Packaging of a Functional RNA Genome

Bel, Nikki van; Velden, Yme van der; Bonnard, Damien; Rouzic, Erwann Le; Das, Atze T.; Bénarous, Richard; Berkhout, Ben

doi:10.1371/journal.pone.0103552

Cited by 22 publications

(31 citation statements)

References 57 publications

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“…Allosteric IN inhibitors (ALLINIs, which are also known as LEDGINs, NCINIs or INLAIs) have recently emerged as a promising class of antiretroviral agents and select compounds are currently in clinical trials (Christ et al, 2010; Fader et al, 2014; Gupta et al, 2014; Kessl et al, 2012; Le Rouzic et al, 2013; van Bel et al, 2014). ALLINIs bind at the IN catalytic core domain (CCD) dimer interface and induce aberrant IN multimerization.…”

Section: Introductionmentioning

confidence: 99%

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

Kessl

Kutluay

Townsend

et al. 2016

Cell

125

310

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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: Introductionmentioning

confidence: 99%

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

Kessl

Kutluay

Townsend

et al. 2016

Cell

125

310

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“…In these particles, the viral ribonucleoprotein complexes (vRNPs), mainly composed of the viral genomic RNA and NC, are eccentrically localized between the empty CA lattice and the viral membrane (7,9,17). A remarkably similar morphological defect is observed in particles generated in the presence of allosteric integrase inhibitors (ALLINIs) (also known as LEDGINs, NCINIs, or INLAIs) (18)(19)(20)(21)(22)(23). Proposed mechanisms that underlie this morphogenesis defect include ALLINI-induced aberrant IN multimerization (9,17,(24)(25)(26)(27) and inhibition of IN binding to the viral RNA genome (4).…”

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confidence: 95%

“…While a large fraction of eccentric particles contain irregularly shaped nonconical CA assemblies (9,17,24), they appear to retain all of the components necessary for reverse transcription, i.e., a dimeric viral RNA genome primed with tRNA-Lys, functional reverse transcriptase (RT) (23), and normal levels of NC-RNA complexes (4,17). Nevertheless, these particles are blocked at an early reverse transcription stage in target cells (9,11,13,24), the basis of which remains unknown.…”

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confidence: 99%

Allosteric HIV-1 Integrase Inhibitors Lead to Premature Degradation of the Viral RNA Genome and Integrase in Target Cells

Madison

Lawson

Elliott

et al. 2017

J Virol

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Recent evidence indicates that inhibition of HIV-1 integrase (IN) binding to the viral RNA genome by allosteric integrase inhibitors (ALLINIs) or through mutations within IN yields aberrant particles in which the viral ribonucleoprotein complexes (vRNPs) are eccentrically localized outside the capsid lattice. These particles are noninfectious and are blocked at an early reverse transcription stage in target cells. However, the basis of this reverse transcription defect is unknown. Here, we show that the viral RNA genome and IN from ALLINI-treated virions are prematurely degraded in target cells, whereas reverse transcriptase remains active and stably associated with the capsid lattice. The aberrantly shaped cores in ALLINI-treated particles can efficiently saturate and be degraded by a restricting TRIM5 protein, indicating that they are still composed of capsid proteins arranged in a hexagonal lattice. Notably, the fates of viral core components follow a similar pattern in cells infected with eccentric particles generated by mutations within IN that inhibit its binding to the viral RNA genome. We propose that IN-RNA interactions allow packaging of both the viral RNA genome and IN within the protective capsid lattice to ensure subsequent reverse transcription and productive infection in target cells. Conversely, disruption of these interactions by ALLINIs or mutations in IN leads to premature degradation of both the viral RNA genome and IN, as well as the spatial separation of reverse transcriptase from the viral genome during early steps of infection.IMPORTANCE Recent evidence indicates that HIV-1 integrase (IN) plays a key role during particle maturation by binding to the viral RNA genome. Inhibition of IN-RNA interactions yields aberrant particles with the viral ribonucleoprotein complexes (vRNPs) eccentrically localized outside the conical capsid lattice. Although these particles contain all of the components necessary for reverse transcription, they are blocked at an early reverse transcription stage in target cells. To explain the basis of this defect, we tracked the fates of multiple viral components in infected cells. Here, we show that the viral RNA genome and IN in eccentric particles are prematurely degraded, whereas reverse transcriptase remains active and stably associated within the capsid lattice. We propose that IN-RNA interactions ensure the packaging of both vRNPs and IN within the protective capsid cores to facilitate subsequent reverse transcription and productive infection in target cells.KEYWORDS ALLINIs, capsid, HIV-1, integrase, maturation, protein-RNA interaction, RNA packaging, TRIM5, reverse transcriptase

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“…Investigating the molecular bases of the observed infectivity defects, we found that HIV-1 virions produced in the presence of the quinoline INLAI compound BI-D (developed by Boehringer Ingelheim) package normal levels of genomic RNA dimer and harbor a properly placed tRNA lys3 primer that could be extended ex vivo. In addition, reverse transcriptase extracted from these virions is fully active (21).…”

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confidence: 99%

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

Bonnard

Rouzic

Eiler

et al. 2018

Journal of Biological Chemistry

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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, while inhibition of the IN-LEDGF/p75 interaction accounts for weaker antiretroviral effect at integration. Since 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, that are responsible for inhibition of virus maturation, were lost, while 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 Xray structures of MUT-A bound to the IN catalytic core domain (CCD) with or without the A125 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.The integrase (IN) protein of Human Immunodeficiency Virus type 1 (HIV-1) catalyzes the stable insertion of the viral cDNA genome into the host cell chromatin, a step of the viral life cycle that is required for efficient viral gene expression. Integration occurs via a two-step reaction where IN initially cleaves after a conserved CA dinucleotide at the 3' end of the viral cDNA genome to free a 3'-OH group (3' processing), which is next used to carry out a nucleophilic attack on cellular chromosomal DNA (strand transfer).IN is one of the preferred targets for the development of antiretroviral (ARV) drugs. However, given the high genetic variability of HIV-1, IN mutations conferring cross-resistance to the first generation INSTIs, RAL and EVG, were described in patients receiving INSTI-containing regimens (2). The second generation INSTI DTG has a higher genetic barrier and conserves good ARV activity against a number of RAL-and EVGresistant strains. Recent reports showed that Bictegravir, a second generation INSTI still in development from Gilead Sciences, has a resistance profile similar to DTG (3). Nevertheless, DTG and Bictegravir are sensitive to the most detrimental INSTI resistant mutations albeit at lower levels than first generation INSTIs (4). Therefore, the development of small molecule inhibitors impairing IN functions with dis...

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The Allosteric HIV-1 Integrase Inhibitor BI-D Affects Virion Maturation but Does Not Influence Packaging of a Functional RNA Genome

Cited by 22 publications

References 57 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

Allosteric HIV-1 Integrase Inhibitors Lead to Premature Degradation of the Viral RNA Genome and Integrase in Target Cells

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

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