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1.
Cell ; 166(5): 1257-1268.e12, 2016 Aug 25.
Artículo en Inglés | MEDLINE | ID: mdl-27565348

RESUMEN

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.


Asunto(s)
Genoma Viral , Integrasa de VIH/metabolismo , VIH-1/crecimiento & desarrollo , ARN Viral/metabolismo , Virión/crecimiento & desarrollo , Células HEK293 , Integrasa de VIH/genética , Inhibidores de Integrasa VIH/farmacología , VIH-1/efectos de los fármacos , VIH-1/enzimología , Humanos , Morfogénesis , Nucleocápside/efectos de los fármacos , Unión Proteica , Virión/efectos de los fármacos , Virión/enzimología , Integración Viral/efectos de los fármacos
2.
PLoS Pathog ; 10(5): e1004171, 2014 May.
Artículo en Inglés | MEDLINE | ID: mdl-24874515

RESUMEN

The quinoline-based allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are promising candidates for clinically useful antiviral agents. Studies using these compounds have highlighted the role of IN in both early and late stages of virus replication. However, dissecting the exact mechanism of action of the quinoline-based ALLINIs has been complicated by the multifunctional nature of these inhibitors because they both inhibit IN binding with its cofactor LEDGF/p75 and promote aberrant IN multimerization with similar potencies in vitro. Here we report design of small molecules that allowed us to probe the role of HIV-1 IN multimerization independently from IN-LEDGF/p75 interactions in infected cells. We altered the rigid quinoline moiety in ALLINIs and designed pyridine-based molecules with a rotatable single bond to allow these compounds to bridge between interacting IN subunits optimally and promote oligomerization. The most potent pyridine-based inhibitor, KF116, potently (EC50 of 0.024 µM) blocked HIV-1 replication by inducing aberrant IN multimerization in virus particles, whereas it was not effective when added to target cells. Furthermore, KF116 inhibited the HIV-1 IN variant with the A128T substitution, which confers resistance to the majority of quinoline-based ALLINIs. A genome-wide HIV-1 integration site analysis demonstrated that addition of KF116 to target or producer cells did not affect LEDGF/p75-dependent HIV-1 integration in host chromosomes, indicating that this compound is not detectably inhibiting IN-LEDGF/p75 binding. These findings delineate the significance of correctly ordered IN structure for HIV-1 particle morphogenesis and demonstrate feasibility of exploiting IN multimerization as a therapeutic target. Furthermore, pyridine-based compounds present a novel class of multimerization selective IN inhibitors as investigational probes for HIV-1 molecular biology.


Asunto(s)
Antivirales/farmacología , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/metabolismo , Péptidos y Proteínas de Señalización Intercelular/farmacología , Multimerización de Proteína/efectos de los fármacos , Integración Viral/efectos de los fármacos , Línea Celular , VIH-1/efectos de los fármacos , Humanos , Unión Proteica/efectos de los fármacos , Quinolinas/farmacología , Replicación Viral/efectos de los fármacos
3.
Curr Top Microbiol Immunol ; 389: 93-119, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25778682

RESUMEN

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.


Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/tratamiento farmacológico , Inhibidores de Integrasa VIH/uso terapéutico , Integrasa de VIH/fisiología , Multimerización de Proteína/efectos de los fármacos , Integrasa de VIH/química , Humanos , Subunidades de Proteína
4.
Proc Natl Acad Sci U S A ; 110(29): 12036-41, 2013 Jul 16.
Artículo en Inglés | MEDLINE | ID: mdl-23818621

RESUMEN

The selection of chromosomal targets for retroviral integration varies markedly, tracking with the genus of the retrovirus, suggestive of targeting by binding to cellular factors. γ-Retroviral murine leukemia virus (MLV) DNA integration into the host genome is favored at transcription start sites, but the underlying mechanism for this preference is unknown. Here, we have identified bromodomain and extraterminal domain (BET) proteins (Brd2, -3, -4) as cellular-binding partners of MLV integrase. We show that purified recombinant Brd4(1-720) binds with high affinity to MLV integrase and stimulates correct concerted integration in vitro. JQ-1, a small molecule that selectively inhibits interactions of BET proteins with modified histone sites impaired MLV but not HIV-1 integration in infected cells. Comparison of the distribution of BET protein-binding sites analyzed using ChIP-Seq data and MLV-integration sites revealed significant positive correlations. Antagonism of BET proteins, via JQ-1 treatment or RNA interference, reduced MLV-integration frequencies at transcription start sites. These findings elucidate the importance of BET proteins for MLV integration efficiency and targeting and provide a route to developing safer MLV-based vectors for human gene therapy.


Asunto(s)
Integrasas/metabolismo , Virus de la Leucemia Murina/enzimología , Proteínas Nucleares/metabolismo , Proteínas Recombinantes/metabolismo , Factores de Transcripción/metabolismo , Sitio de Iniciación de la Transcripción/fisiología , Integración Viral/fisiología , Animales , Azepinas , Proteínas de Ciclo Celular , Línea Celular Tumoral , Inmunoprecipitación de Cromatina , Células HEK293 , Secuenciación de Nucleótidos de Alto Rendimiento , Interacciones Huésped-Patógeno , Humanos , Espectrometría de Masas , Ratones , Células 3T3 NIH , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Proteómica/métodos , Interferencia de ARN , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Triazoles , Integración Viral/genética
5.
Proc Natl Acad Sci U S A ; 110(21): 8690-5, 2013 May 21.
Artículo en Inglés | MEDLINE | ID: mdl-23610442

RESUMEN

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.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Inhibidores de Integrasa VIH , Integrasa de VIH/metabolismo , VIH-1/fisiología , Factores de Transcripción/metabolismo , Replicación Viral/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Regulación Alostérica/efectos de los fármacos , Línea Celular , Inhibidores de Integrasa VIH/química , Inhibidores de Integrasa VIH/farmacología , Humanos , Factores de Transcripción/genética , Virión/genética , Virión/metabolismo , Replicación Viral/fisiología
6.
J Biol Chem ; 289(38): 26430-26440, 2014 Sep 19.
Artículo en Inglés | MEDLINE | ID: mdl-25118283

RESUMEN

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a promising class of antiretroviral agents for clinical development. Although ALLINIs promote aberrant IN multimerization and inhibit IN interaction with its cellular cofactor LEDGF/p75 with comparable potencies in vitro, their primary mechanism of action in infected cells is through inducing aberrant multimerization of IN. Crystal structures have shown that ALLINIs bind at the IN catalytic core domain dimer interface and bridge two interacting subunits. However, how these interactions promote higher-order protein multimerization is not clear. Here, we used mass spectrometry-based protein footprinting to monitor surface topology changes in full-length WT and the drug-resistant A128T mutant INs in the presence of ALLINI-2. These experiments have identified protein-protein interactions that extend beyond the direct inhibitor binding site and which lead to aberrant multimerization of WT but not A128T IN. Specifically, we demonstrate that C-terminal residues Lys-264 and Lys-266 play an important role in the inhibitor induced aberrant multimerization of the WT protein. Our findings provide structural clues for exploiting IN multimerization as a new, attractive therapeutic target and are expected to facilitate development of improved inhibitors.


Asunto(s)
Acetatos/química , Inhibidores de Integrasa VIH/química , Integrasa de VIH/química , Indoles/química , Regulación Alostérica , Farmacorresistencia Viral , Integrasa de VIH/genética , VIH-1/enzimología , Humanos , Mutación Missense , Unión Proteica , Multimerización de Proteína/efectos de los fármacos
7.
J Biol Chem ; 288(22): 15813-20, 2013 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-23615903

RESUMEN

Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are a very promising new class of anti-HIV-1 agents that exhibit a multimodal mechanism of action by allosterically modulating IN multimerization and interfering with IN-lens epithelium-derived growth factor (LEDGF)/p75 binding. Selection of viral strains under ALLINI pressure has revealed an A128T substitution in HIV-1 IN as a primary mechanism of resistance. Here, we elucidated the structural and mechanistic basis for this resistance. The A128T substitution did not affect the hydrogen bonding between ALLINI and IN that mimics the IN-LEDGF/p75 interaction but instead altered the positioning of the inhibitor at the IN dimer interface. Consequently, the A128T substitution had only a minor effect on the ALLINI IC50 values for IN-LEDGF/p75 binding. Instead, ALLINIs markedly altered the multimerization of IN by promoting aberrant higher order WT (but not A128T) IN oligomers. Accordingly, WT IN catalytic activities and HIV-1 replication were potently inhibited by ALLINIs, whereas the A128T substitution in IN resulted in significant resistance to the inhibitors both in vitro and in cell culture assays. The differential multimerization of WT and A128T INs induced by ALLINIs correlated with the differences in infectivity of HIV-1 progeny virions. We conclude that ALLINIs primarily target IN multimerization rather than IN-LEDGF/p75 binding. Our findings provide the structural foundations for developing improved ALLINIs with increased potency and decreased potential to select for drug resistance.


Asunto(s)
Farmacorresistencia Viral/efectos de los fármacos , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/metabolismo , VIH-1/fisiología , Mutación Missense , Multimerización de Proteína/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/genética , Sustitución de Aminoácidos , Células HEK293 , Integrasa de VIH/química , Integrasa de VIH/genética , Inhibidores de Integrasa VIH/química , VIH-1/química , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Unión Proteica
8.
Retrovirology ; 11: 100, 2014 Nov 25.
Artículo en Inglés | MEDLINE | ID: mdl-25421939

RESUMEN

BACKGROUND: Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an important new class of anti-HIV-1 agents. ALLINIs bind at the IN catalytic core domain (CCD) dimer interface occupying the principal binding pocket of its cellular cofactor LEDGF/p75. Consequently, ALLINIs inhibit HIV-1 IN interaction with LEDGF/p75 as well as promote aberrant IN multimerization. Selection of viral strains emerging under the inhibitor pressure has revealed mutations at the IN dimer interface near the inhibitor binding site. RESULTS: We have investigated the effects of one of the most prevalent substitutions, H171T IN, selected under increasing pressure of ALLINI BI-D. Virus containing the H171T IN substitution exhibited an ~68-fold resistance to BI-D treatment in infected cells. These results correlated with ~84-fold reduced affinity for BI-D binding to recombinant H171T IN CCD protein compared to its wild type (WT) counterpart. However, the H171T IN substitution only modestly affected IN-LEDGF/p75 binding and allowed HIV-1 containing this substitution to replicate at near WT levels. The x-ray crystal structures of BI-D binding to WT and H171T IN CCD dimers coupled with binding free energy calculations revealed the importance of the Nδ- protonated imidazole group of His171 for hydrogen bonding to the BI-D tert-butoxy ether oxygen and establishing electrostatic interactions with the inhibitor carboxylic acid, whereas these interactions were compromised upon substitution to Thr171. CONCLUSIONS: Our findings reveal a distinct mechanism of resistance for the H171T IN mutation to ALLINI BI-D and indicate a previously undescribed role of the His171 side chain for binding the inhibitor.


Asunto(s)
Acetatos/metabolismo , Farmacorresistencia Viral , Inhibidores de Integrasa VIH/metabolismo , Integrasa de VIH/metabolismo , VIH-1/efectos de los fármacos , VIH-1/enzimología , Mutación Missense , Quinolinas/metabolismo , Línea Celular , Cristalografía por Rayos X , Integrasa de VIH/química , Integrasa de VIH/genética , Histidina/genética , Histidina/metabolismo , Humanos , Modelos Moleculares , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Unión Proteica , Conformación Proteica
9.
J Biol Chem ; 287(20): 16801-11, 2012 May 11.
Artículo en Inglés | MEDLINE | ID: mdl-22437836

RESUMEN

The multifunctional HIV-1 enzyme integrase interacts with viral DNA and its key cellular cofactor LEDGF to effectively integrate the reverse transcript into a host cell chromosome. These interactions are crucial for HIV-1 replication and present attractive targets for antiviral therapy. Recently, 2-(quinolin-3-yl) acetic acid derivatives were reported to selectively inhibit the integrase-LEDGF interaction in vitro and impair HIV-1 replication in infected cells. Here, we show that this class of compounds impairs both integrase-LEDGF binding and LEDGF-independent integrase catalytic activities with similar IC(50) values, defining them as bona fide allosteric inhibitors of integrase function. Furthermore, we show that 2-(quinolin-3-yl) acetic acid derivatives block the formation of the stable synaptic complex between integrase and viral DNA by allosterically stabilizing an inactive multimeric form of integrase. In addition, these compounds inhibit LEDGF binding to the stable synaptic complex. This multimode mechanism of action concordantly results in cooperative inhibition of the concerted integration of viral DNA ends in vitro and HIV-1 replication in cell culture. Our findings, coupled with the fact that high cooperativity of antiviral inhibitors correlates with their increased instantaneous inhibitory potential, an important clinical parameter, argue strongly that improved 2-(quinolin-3-yl) acetic acid derivatives could exhibit desirable clinical properties.


Asunto(s)
ADN Viral/metabolismo , Integrasa de VIH/metabolismo , VIH-1/fisiología , Inhibidores de Integrasa/farmacología , Replicación Viral/efectos de los fármacos , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/genética , ADN Viral/genética , Células HEK293 , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/enzimología , Infecciones por VIH/genética , Integrasa de VIH/genética , Humanos , Ácidos Indolacéticos/química , Ácidos Indolacéticos/farmacología , Inhibidores de Integrasa/química , Unión Proteica/efectos de los fármacos , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Replicación Viral/fisiología
10.
ACS Chem Biol ; 11(5): 1313-21, 2016 05 20.
Artículo en Inglés | MEDLINE | ID: mdl-26910179

RESUMEN

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.


Asunto(s)
Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/metabolismo , Inhibidores de Integrasa VIH/farmacología , Integrasa de VIH/metabolismo , VIH-1/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Replicación Viral/efectos de los fármacos , Regulación Alostérica/efectos de los fármacos , Células HEK293 , Infecciones por VIH/virología , VIH-1/fisiología , Humanos
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