RESUMEN
Human immunodeficiency virus-1 (HIV-1) transactivator (Tat)-mediated transcription is essential for HIV-1 replication. It is determined by the interaction between Tat and transactivation response (TAR) RNA, a highly conserved process representing a prominent therapeutic target against HIV-1 replication. However, owing to the limitations of current high-throughput screening (HTS) assays, no drug that disrupts the Tat-TAR RNA interaction has been uncovered yet. We designed a homogenous (mix-and-read) time-resolved fluorescence resonance energy transfer (TR-FRET) assay using europium cryptate as a fluorescence donor. It was optimized by evaluating different probing systems for Tat-derived peptides or TAR RNA. The specificity of the optimal assay was validated by mutants of the Tat-derived peptides and TAR RNA fragment, individually and by competitive inhibition with known TAR RNA-binding peptides. The assay generated a constant Tat-TAR RNA interaction signal, discriminating the compounds that disrupted the interaction. Combined with a functional assay, the TR-FRET assay identified two small molecules (460-G06 and 463-H08) capable of inhibiting Tat activity and HIV-1 infection from a large-scale compound library. The simplicity, ease of operation, and rapidity of our assay render it suitable for HTS to identify Tat-TAR RNA interaction inhibitors. The identified compounds may also act as potent molecular scaffolds for developing a new HIV-1 drug class.
Asunto(s)
VIH-1 , Productos del Gen tat del Virus de la Inmunodeficiencia Humana , Humanos , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/química , VIH-1/fisiología , Transferencia Resonante de Energía de Fluorescencia , Transactivadores , ARN Viral/genéticaRESUMEN
The positive transcription elongation factor b (P-TEFb) is an essential factor that induces transcription elongation and is also negatively regulated by the cellular factor HEXIM1. Previously, the chimeric protein HEXIM1-Tat (HT) was demonstrated to inhibit human immunodeficiency virus-1 (HIV)-1 transcription. In this study, we attempted to develop an improved antiviral protein that specifically binds viral RNA (vRNA) by fusing HT to HIV-1 nucleocapsid (NC). Thus, we synthesized NC-HEXIM1-Tat (NHT) and HEXIM1-Tat-NC (HTN). NHT and HTN inhibited virus proliferation more effectively than HT, and they did not attenuate the function of HT. Notably, NHT and HTN inhibited the infectivity of the progeny virus, whereas HT had no such effect. NHT and HTN selectively and effectively interacted with vRNA and inhibited the proper packaging of the HIV-1 genome. Taken together, our results illustrated that the novel NC-fused chimeric proteins NHT and HTN display novel mechanisms of anti-HIV effects by inhibiting both HIV-1 transcription and packaging.
Asunto(s)
VIH-1 , Factor B de Elongación Transcripcional Positiva , Humanos , Factor B de Elongación Transcripcional Positiva/metabolismo , VIH-1/genética , VIH-1/metabolismo , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismo , ARN Viral/metabolismo , Proteínas de la Nucleocápside/genética , Proteínas de la Nucleocápside/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Replicación Viral , Nucleocápside/metabolismo , Antivirales/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/metabolismo , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: The human immunodeficiency virus type-1 (HIV-1) nucleocapsid protein (NC) is an essential and multifunctional protein involved in multiple stages of the viral life cycle such as reverse transcription, integration of proviral DNA, and especially genome RNA packaging. For this reason, it has been considered as an attractive target for the development of new anti-HIV drugs. Although a number of inhibitors of NC have been reported thus far, the search for NC-specific and functional inhibitor(s) with a good antiviral activity continues. RESULTS: In this study, we report the identification of A1752, a small molecule with inhibitory action against HIV-1 NC, which shows a strong antiviral efficacy and an IC50 around 1 µM. A1752 binds directly to HIV-1 NC, thereby inhibiting specific chaperone functions of NC including Psi RNA dimerization and complementary trans-activation response element (cTAR) DNA destabilization, and it also disrupts the proper Gag processing. Further analysis of the mechanisms of action of A1752 also showed that it generates noninfectious viral particles with defects in uncoating and reverse transcription in the infected cells. CONCLUSIONS: These results demonstrate that A1752 is a specific and functional inhibitor of NC with a novel mode of action and good antiviral efficacy. Thus, this agent provides a new type of anti-HIV NC inhibitor candidate for further drug development.
Asunto(s)
Fármacos Anti-VIH/farmacología , VIH-1/efectos de los fármacos , Proteínas de la Nucleocápside/antagonistas & inhibidores , Propionatos/farmacología , Tiazolidinas/farmacología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Secuencia de Aminoácidos , Fármacos Anti-VIH/química , Fármacos Anti-VIH/metabolismo , Dimerización , Descubrimiento de Drogas , VIH-1/fisiología , Humanos , Chaperonas Moleculares/metabolismo , Proteínas de la Nucleocápside/metabolismo , Propionatos/química , Propionatos/metabolismo , ARN Viral/química , ARN Viral/genética , Tiazolidinas/química , Tiazolidinas/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Although cis-acting packaging signal RNA sequences for the influenza virus NP encoding vRNA have been identified recently though genetic studies, little is known about the interaction between NP and the vRNA packaging signals either in vivo or in vitro. Here, we provide evidence that NP is able to interact specifically with the vRNA packaging sequence RNA within living cells and that the specific RNA binding activity of NP in vivo requires both the N-terminal and central region of the protein. This assay established would be a valuable tool for further detailed studies of the NP-packaging signal RNA interaction in living cells.
Asunto(s)
Bioensayo/métodos , Virus de la Influenza A/metabolismo , Nucleoproteínas/metabolismo , Señales de Clasificación de Proteína , ARN Viral/metabolismo , Ensamble de Virus , Eliminación de Gen , Cinética , Operón Lac , Unión ProteicaRESUMEN
The HIV-1 infection epidemic remains a global health problem. Current antiretroviral treatments are effective in controlling the progression of a severe infection. However, the emergence of drug resistance requires an urgent identification of new treatment regimes. HIV-1 reverse transcriptase (RTs) has been a successful therapeutic target owing to its high specificity and potent antiviral properties; therefore, it has become an essential component of current HIV-1 standard treatments. This study identified a new HIV-1 RTs inhibitor (Compound #8) that is structurally unique and greatly effective against HIV-1 through chemical library screening and a medicinal chemistry program by analyzing the structure-activity relationship (SAR). Further analysis of molecular docking and mechanisms of action demonstrated that Compound #8 is a novel type of HIV-1 non-nucleoside reverse transcriptase inhibitor (NNRTI) with a flexible binding mode. Therefore, it exhibits great therapeutic potential when combined with other existing HIV-1 drugs. Our current studies suggest that Compound #8 is a promising novel scaffold for the development of new HIV-1 treatments.
Asunto(s)
Infecciones por VIH , VIH-1 , Humanos , Inhibidores de la Transcriptasa Inversa/farmacología , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Inhibidores de la Transcriptasa Inversa/química , Simulación del Acoplamiento Molecular , Antivirales/farmacología , Infecciones por VIH/tratamiento farmacológicoRESUMEN
Serine-arginine-rich splicing factors (SRSFs) are members of RNA processing proteins in the serine-arginine-rich (SR) family that could regulate the alternative splicing of the human immunodeficiency virus-1 (HIV-1). Whether SRSF9 has any effect on HIV-1 regulation requires elucidation. Here, we report for the first time the effects and mechanisms of SRSF9 on HIV-1 regulation. The overexpression of SRSF9 inhibits viral production and infectivity in both HEK293T and MT-4 cells. Deletion analysis of SRSF9 determined that the RNA regulation motif domain of SRSF9 is important for anti-HIV-1 effects. Furthermore, overexpression of SRSF9 increases multiple spliced forms of viral mRNA, such as Vpr mRNA. These data suggest that SRSF9 overexpression inhibits HIV-1 production by inducing the imbalanced HIV-1 mRNA splicing that could be exploited further for a novel HIV-1 therapeutic molecule. [BMB Reports 2022; 55(12): 639-644].
Asunto(s)
VIH-1 , Factores de Empalme Serina-Arginina , Humanos , Empalme Alternativo/genética , Células HEK293 , VIH-1/metabolismo , Empalme del ARN/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Empalme Serina-Arginina/genética , Factores de Empalme Serina-Arginina/metabolismoRESUMEN
In this study, we investigated how Staufen1 influences the HIV-1 production. The overexpression of Staufen1 increased virus production without any negative affect on the viral infectivity. This increase was not caused by transcriptional activation; but by influencing post-transcriptional steps. Using multiple Gag protein derivatives, we confirmed that the zinc-finger domains of the HIV-1 nucleocapsid (NC) are important for its interaction with Staufen1. We also found that Staufen1 colocalized in stress granules with the mature form of the HIV-1 NC protein. [BMB Reports 2021; 54(11): 551-556].
Asunto(s)
Proteínas del Citoesqueleto/metabolismo , Productos del Gen gag/metabolismo , Infecciones por VIH/virología , VIH-1/fisiología , Nucleocápside/metabolismo , Proteínas de Unión al ARN/metabolismo , Gránulos de Estrés/metabolismo , Replicación Viral , Proteínas del Citoesqueleto/genética , Productos del Gen gag/genética , Células HeLa , Humanos , Nucleocápside/genética , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Proteínas de Unión al ARN/genéticaRESUMEN
The activating transcription factor (ATF) 4 belongs to the ATF/CREB (cAMP Response Element Binding bZIP [Basic Leucine Zipper]) transcription factor family, and plays a central role in the UPR (Unfolded Protein Response) process in cells. The induction of ATF4 expression has previously been shown to increase the replication of HIV-1. However, the detailed mechanism underlying this effect and the factors involved in the regulation of ATF4 function are still unknown. Here, we demonstrate first that knocking out ATF4 using siRNA shows a strong negative effect on HIV-1 production, indicating that ATF4 is a functional positive cellular factor in HIV-1 production. To determine the mechanism by which ATF4 regulates the HIV-1 life cycle, we assessed the effect of the overexpression of wild type ATF4 and its various derivatives on HIV-1 LTR-mediated transcriptional activation and the production of HIV-1 particles. This effect was studied through co-transfection experiments with either reporter vectors or proviral DNA. We found that the N-terminal domains of ATF4 are involved in HIV-1 LTR-mediated transcriptional activation, and thus in HIV-1 production. [BMB Reports 2018; 51(8): 388-393].
Asunto(s)
Factor de Transcripción Activador 4/fisiología , VIH-1/fisiología , Factor de Transcripción Activador 4/genética , Factor de Transcripción Activador 4/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación Viral de la Expresión Génica , Células HEK293 , Infecciones por VIH/genética , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Duplicado del Terminal Largo de VIH , VIH-1/genética , VIH-1/metabolismo , Humanos , Transcripción Genética , Activación Transcripcional , Respuesta de Proteína DesplegadaRESUMEN
Transcription termination factor-1 (TTF-I) is an RNA polymerase 1-mediated transcription terminator and consisting of a C-terminal DNA-binding domain, central domain, and N-terminal regulatory domain. This protein binds to a so-called 'Sal box' composed of an 11-base pair motif. The interaction of TTF-I with the 'Sal box' is important for many cellular events, including efficient termination of RNA polymerase-1 activity involved in pre-rRNA synthesis and formation of a chromatin loop. To further understand the role of TTF-I in human immunodeficiency virus (HIV)-I virus production, we generated various TTF-I mutant forms. Through a series of studies of the over-expression of TTF-I and its derivatives along with co-transfection with either proviral DNA or HIV-I long terminal repeat (LTR)-driven reporter vectors, we determined that wild-type TTF-I downregulates HIV-I LTR activity and virus production, while the TTF-I Myb-like domain alone upregulated virus production, suggesting that wild-type TTF-I inhibits virus production and trans-activation of the LTR sequence; the Myb-like domain of TTF-I increased virus production and trans-activated LTR activity. [BMB Reports 2018; 51(7): 338-343].
Asunto(s)
Proteínas de Unión al ADN/metabolismo , VIH-1/fisiología , Factores de Transcripción/metabolismo , Proteínas de Unión al ADN/genética , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Células HEK293 , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , Células HeLa , Humanos , Mutagénesis , Regiones Promotoras Genéticas , ARN Polimerasa I/metabolismo , ARN Viral/metabolismo , Factores de Transcripción/genética , Activación Transcripcional , Replicación ViralRESUMEN
Y-box binding protein 1 (YB-1) is a member of the cold-shock domain (CSD) protein superfamily. It participates in a wide variety of cellular events, including transcription, RNA splicing, translation, DNA repair, drug resistance, and stress responses. We investigated putative functions of YB-1 in HIV-1 replication. Functional studies using overexpression or knockdown of YB-1 in conjunction with transfection of proviral DNA showed that YB-1 enhances virus production. We found YB-1 regulates HIV-1 production by stimulating viral transcription using HIV-1 LTR sequence U3RU5 with Luciferase assay. We also identified a specific region from amino acids 1 to 324 of YB-1 as necessary for the participation of the protein in the production of virions. [BMB Reports 2018; 51(6): 290-295].
Asunto(s)
Infecciones por VIH/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismo , Proteína 1 de Unión a la Caja Y/fisiología , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , VIH/metabolismo , Duplicado del Terminal Largo de VIH/genética , Proteínas del Virus de la Inmunodeficiencia Humana/metabolismo , Humanos , Activación Transcripcional , Transfección , Proteína 1 de Unión a la Caja Y/genéticaRESUMEN
The HIV-1 nucleocapsid (NC) is an essential viral protein containing two highly conserved retroviral-type zinc finger (ZF) motifs, which functions in multiple stages of the HIV-1 life cycle. Although a number of functions for NC either in its mature form or as a domain of Gag have been revealed, little is known about the intracellular localization of NC and, moreover, its role in Gag protein trafficking. Here, we have investigated various forms of HIV-1 NC protein for its cellular localization and found that the NC has a strong nuclear and nucleolar localization activity. The linker region, composed of a stretch of basic amino acids between the two ZF motifs, was necessary and sufficient for the activity.
Asunto(s)
Nucléolo Celular/virología , VIH-1/genética , Proteínas de la Nucleocápside/genética , Nucleocápside/genética , Virión/genética , Secuencia de Aminoácidos , Calnexina/genética , Calnexina/metabolismo , Nucléolo Celular/metabolismo , Nucléolo Celular/ultraestructura , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , VIH-1/metabolismo , VIH-1/ultraestructura , Células HeLa , Interacciones Huésped-Patógeno , Humanos , Datos de Secuencia Molecular , Nucleocápside/metabolismo , Proteínas de la Nucleocápside/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Virión/metabolismo , Virión/ultraestructura , Ensamble de Virus/genética , Zinc/química , Zinc/metabolismo , Dedos de Zinc , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Here we report a new chemical inhibitor against HIV-1 with a novel structure and mode of action. The inhibitor, designated as A1836, inhibited HIV-1 replication and virus production with a 50% inhibitory concentration (IC50) of 2.0 µM in an MT-4 cell-based and cytopathic protection antiviral assay, while its 50% cytotoxic concentration (CC50) was much higher than 50 µM. Examination of the effect of A1836 on in vitro HIV-1 reverse transcriptase (RT) and integrase showed that neither were molecular targets of A1836. The characterization and re-infection assay of the HIV-1 virions generated in the presence of A1836 showed that the synthesis of early RT products in the cells infected with the virions was inhibited dose-dependently, due in part to abnormal protein formation within the virions, thus resulting in an impaired infectivity. These results suggest that A1836 might be a novel candidate for the development of a new type of HIV-1 inhibitor.
Asunto(s)
Antivirales/farmacología , VIH-1/fisiología , Pirazoles/farmacología , Replicación Viral/efectos de los fármacos , Antivirales/química , Línea Celular , Supervivencia Celular/efectos de los fármacos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Integrasa de VIH/metabolismo , Transcriptasa Inversa del VIH/metabolismo , VIH-1/genética , Humanos , Plásmidos/metabolismo , Pirazoles/química , TransfecciónRESUMEN
The human immunodeficiency virus type 1 (HIV-1) nucleocapsid (NC) is a multifunctional, zinc finger-containing protein known to be involved in almost every step of the viral life cycle. We therefore examined the effects of NC in vivo as a transcription activator on the basal transcriptional activity of the HIV-1 U3 and Rous sarcoma virus (RSV) promoters, as well as HIV-1 long terminal repeats (LTRs) such as the U3R and U3RU5 regions, using promoter-fused reporter gene assays, Western blot analyses, and quantitative real time-polymerase chain reaction. From these studies, we found that the basal transcriptional levels of the HIV-1 U3 and RSV promoters were barely enhanced by the presence of NC. Placing the U3R region upstream of reporter genes greatly increased transcriptional activity compared to that of the U3 promoter alone, and such activity was further increased by Tat expression. However, neither transcription driven by U3R itself nor Tat-mediated transcriptional activation of the U3R was further increased by the addition of NC. Similar results were also observed with U3RU5 of the HIV-1 LTR region in the presence of either NC or Gag protein. Thus, these results indicate that the HIV NC protein is unable to act as a transcriptional activator on its cognate and possibly other retroviral promoters.
Asunto(s)
VIH-1/fisiología , Regiones Promotoras Genéticas , Transactivadores/metabolismo , Transcripción Genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , Fusión Artificial Génica , Western Blotting , Perfilación de la Expresión Génica , Genes Reporteros , VIH-1/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Virus del Sarcoma de Rous/genéticaRESUMEN
Here, we investigated the ability of the Hepatitis C Virus (HCV) core protein to interact specifically with the 5' and 3' untranslated regions (UTRs) of HCV using an in vivo cell-based translation inhibition assay. HCV core protein interacts weakly but specifically with the SLIII stem loop in the 5' UTR in which the SLIIIb subdomain is the major determinant and the SL2 loop in the X region of the 3' UTR. These results revealed for the first time in vivo interaction of the core protein with 5' and 3' UTRs involved in the viral life cycle. This system provides a useful tool for further investigating interactions between the HCV core protein and 5' and 3' UTRs.