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[This corrects the article DOI: 10.1371/journal.ppat.1008307.].
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IMPORTANCE: This study highlights the crucial role RNA processing plays in regulating viral gene expression and replication. By targeting SR kinases, we identified harmine as a potent inhibitor of HIV-1 as well as coronavirus (HCoV-229E and multiple SARS-CoV-2 variants) replication. Harmine inhibits HIV-1 protein expression and reduces accumulation of HIV-1 RNAs in both cell lines and primary CD4+ T cells. Harmine also suppresses coronavirus replication post-viral entry by preferentially reducing coronavirus sub-genomic RNA accumulation. By focusing on host factors rather than viral targets, our study offers a novel approach to combating viral infections that is effective against a range of unrelated viruses. Moreover, at doses required to inhibit virus replication, harmine had limited toxicity and minimal effect on the host transcriptome. These findings support the viability of targeting host cellular processes as a means of developing broad-spectrum anti-virals.
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Antivirales , Coronavirus , VIH-1 , Harmina , Humanos , Antivirales/farmacología , Antivirales/uso terapéutico , Coronavirus/efectos de los fármacos , Coronavirus/fisiología , Infecciones por Coronavirus/tratamiento farmacológico , Harmina/farmacología , Harmina/uso terapéutico , VIH-1/efectos de los fármacos , VIH-1/fisiología , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: The generation of over 69 spliced HIV-1 mRNAs from one primary transcript by alternative RNA splicing emphasizes the central role that RNA processing plays in HIV-1 replication. Control is mediated in part through the action of host SR proteins whose activity is regulated by multiple SR kinases (CLK1-4, SRPKs). METHODS: Both shRNA depletion and small molecule inhibitors of host SR kinases were used in T cell lines and primary cells to evaluate the role of these factors in the regulation of HIV-1 gene expression. Effects on virus expression were assessed using western blotting, RT-qPCR, and immunofluorescence. RESULTS: The studies demonstrate that SR kinases play distinct roles; depletion of CLK1 enhanced HIV-1 gene expression, reduction of CLK2 or SRPK1 suppressed it, whereas CLK3 depletion had a modest impact. The opposing effects of CLK1 vs. CLK2 depletion were due to action at distinct steps; reduction of CLK1 increased HIV-1 promoter activity while depletion of CLK2 affected steps after transcript initiation. Reduced CLK1 expression also enhanced the response to several latency reversing agents, in part, by increasing the frequency of responding cells, consistent with a role in regulating provirus latency. To determine whether small molecule modulation of SR kinase function could be used to control HIV-1 replication, we screened a GSK library of protein kinase inhibitors (PKIS) and identified several pyrazolo[1,5-b] pyridazine derivatives that suppress HIV-1 gene expression/replication with an EC50 ~ 50 nM. The compounds suppressed HIV-1 protein and viral RNA accumulation with minimal impact on cell viability, inhibiting CLK1 and CLK2 but not CLK3 function, thereby selectively altering the abundance of individual CLK and SR proteins in cells. CONCLUSIONS: These findings demonstrate the unique roles played by individual SR kinases in regulating HIV-1 gene expression, validating the targeting of these functions to either enhance latency reversal, essential for "Kick-and-Kill" strategies, or to silence HIV protein expression for "Block-and-Lock" strategies.
Identifying cellular factors that regulate HIV-1 RNA processing provides important insights into novel strategies to control this infection. Different members of the SR kinase family have distinct roles in regulating virus expression because they affect distinct steps of transcription/RNA processing. We identify inhibitors of these kinases that suppress HIV-1 gene expression and replication in multiple assay systems at nanomolar concentrations with limited or no cytotoxicity. Our results highlight the therapeutic potential of targeting the post-integration stage of the HIV-1 lifecycle to selectively enhance or reverse provirus latency. A greater understanding of the molecular mechanisms underlying the effects observed will facilitate the development of more targeted approaches to modulate HIV-1 latency on the path toward a "functional" cure for this infection.
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VIH-1 , Empalme Alternativo , Expresión Génica , VIH-1/fisiología , Inhibidores de Proteínas Quinasas/farmacología , ARN Viral/genética , Latencia del VirusRESUMEN
The ability of HIV-1 to evolve resistance to combined antiretroviral therapies (cARTs) has stimulated research into alternative means of controlling this infection. We assayed >60 modulators of RNA alternative splicing (AS) to identify new inhibitors of HIV-1 RNA processing-a segment of the viral lifecycle not targeted by current drugs-and discovered compound N-[4-chloro-3-(trifluoromethyl)phenyl]-7-nitro-2,1,3-benzoxadiazol-4-amine (5342191) as a potent inhibitor of both wild-type (Ba-L, NL4-3, LAI, IIIB, and N54) and drug-resistant strains of HIV-1 (IC50: ~700 nM) with no significant effect on cell viability at doses tested. 5342191 blocks expression of four essential HIV-1 structural and regulatory proteins (Gag, Env, Tat, and Rev) without affecting total protein synthesis of the cell. This response is associated with altered unspliced (US) and singly-spliced (SS) HIV-1 RNA accumulation (~60% reduction) and transport to the cytoplasm (loss of Rev) whereas parallel analysis of cellular RNAs revealed less than a 0.7% of host alternative splicing (AS) events (0.25-0.67% by ≥ 10-20%), gene expression (0.01-0.46% by ≥ 2-5 fold), and protein abundance (0.02-0.34% by ≥ 1.5-2 fold) being affected. Decreased expression of Tat, but not Gag/Env, upon 5342191 treatment was reversed by a proteasome inhibitor, suggesting that this compound alters the synthesis/degradation of this key viral factor. Consistent with an affect on HIV-1 RNA processing, 5342191 treatment of cells altered the abundance and phosphorylation of serine/arginine-rich splicing factor (SRSF) 1, 3, and 4. Despite the activation of several intracellular signaling pathways by 5342191 (Ras, MEK1/2-ERK1/2, and JNK1/2/3), inhibition of HIV-1 gene expression by this compound could be reversed by pre-treatment with either a G-protein α-subunit inhibitor or two different MEK1/2 inhibitors. These observations demonstrate enhanced sensitivity of HIV-1 gene expression to small changes in host RNA processing and highlights the potential of modulating host intracellular signaling as an alternative approach for controlling HIV-1 infection.
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Empalme Alternativo/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Empalme Alternativo/fisiología , Expresión Génica/genética , Regulación Viral de la Expresión Génica/genética , Infecciones por VIH , Seropositividad para VIH , VIH-1/fisiología , Células HeLa , Humanos , MAP Quinasa Quinasa 1/metabolismo , MAP Quinasa Quinasa Quinasa 2/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Procesamiento Postranscripcional del ARN/fisiología , Empalme del ARN/genética , ARN Viral/genética , Receptores Acoplados a Proteínas G/metabolismo , Transducción de Señal/fisiología , Bibliotecas de Moléculas Pequeñas , Replicación Viral/fisiología , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The development of new methods for direct viral detection using streamlined and ideally reagent-free assays is a timely and important, but challenging, problem. The challenge of combatting the COVID-19 pandemic has been exacerbated by the lack of rapid and effective methods to identify viral pathogens like SARS-CoV-2 on-demand. Existing gold standard nucleic acid-based approaches require enzymatic amplification to achieve clinically relevant levels of sensitivity and are not typically used outside of a laboratory setting. Here, we report reagent-free viral sensing that directly reads out the presence of viral particles in 5 minutes using only a sensor-modified electrode chip. The approach relies on a class of electrode-tethered sensors bearing an analyte-binding antibody displayed on a negatively charged DNA linker that also features a tethered redox probe. When a positive potential is applied, the sensor is transported to the electrode surface. Using chronoamperometry, the presence of viral particles and proteins can be detected as these species increase the hydrodynamic drag on the sensor. This report is the first virus-detecting assay that uses the kinetic response of a probe/virus complex to analyze the complexation state of the antibody. We demonstrate the performance of this sensing approach as a means to detect, within 5 min, the presence of the SARS-CoV-2 virus and its associated spike protein in test samples and in unprocessed patient saliva.
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Técnicas Biosensibles/métodos , Prueba de COVID-19/métodos , COVID-19/virología , Técnicas Electroquímicas/métodos , SARS-CoV-2/aislamiento & purificación , Virión/aislamiento & purificación , Técnicas Biosensibles/instrumentación , Prueba de COVID-19/instrumentación , Técnicas Electroquímicas/instrumentación , Electrodos , Humanos , Pruebas en el Punto de Atención , Saliva/virologíaRESUMEN
We recently identified the 4-pyridinone-benzisothiazole carboxamide compound 1C8 as displaying strong anti-HIV-1 potency against a variety of clinical strains in vitro. Here we show that 1C8 decreases the expression of HIV-1 and alters splicing events involved in the production of HIV-1 mRNAs. Although 1C8 was designed to be a structural mimic of the fused tetracyclic indole compound IDC16 that targets SRSF1, it did not affect the splice site shifting activity of SRSF1. Instead, 1C8 altered splicing regulation mediated by SRSF10. Depleting SRSF10 by RNA interference affected viral splicing and, like 1C8, decreased expression of Tat, Gag and Env. Incubating cells with 1C8 promoted the dephosphorylation of SRSF10 and increased its interaction with hTra2ß, a protein previously implicated in the control of HIV-1 RNA splicing. While 1C8 affects the alternative splicing of cellular transcripts controlled by SRSF10 and hTra2ß, concentrations greater than those needed to inhibit HIV-1 replication were required to elicit significant alterations. Thus, the ability of 1C8 to alter the SRSF10-dependent splicing of HIV-1 transcripts, with minor effects on cellular splicing, supports the view that SRSF10 may be used as a target for the development of new anti-viral agents.
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Empalme Alternativo/efectos de los fármacos , Fármacos Anti-VIH/farmacología , Benzotiazoles/farmacología , Proteínas de Ciclo Celular/metabolismo , VIH-1/efectos de los fármacos , Niacinamida/análogos & derivados , Proteínas Represoras/metabolismo , Factores de Empalme Serina-Arginina/metabolismo , Replicación Viral/efectos de los fármacos , Fármacos Anti-VIH/química , Benzotiazoles/química , Células Cultivadas , VIH-1/genética , VIH-1/metabolismo , VIH-1/fisiología , Células HeLa , Humanos , Niacinamida/química , Niacinamida/farmacología , Precursores del ARN/metabolismo , Factores de Empalme de ARN/metabolismo , ARN Mensajero/metabolismo , Transcripción Genética/efectos de los fármacosRESUMEN
The dependence of adenovirus on the host pre-RNA splicing machinery for expression of its complete genome potentially makes it vulnerable to modulators of RNA splicing, such as digoxin and digitoxin. Both drugs reduced the yields of four human adenoviruses (HAdV-A31, -B35, and -C5 and a species D conjunctivitis isolate) by at least 2 to 3 logs by affecting one or more steps needed for genome replication. Immediate early E1A protein levels are unaffected by the drugs, but synthesis of the delayed protein E4orf6 and the major late capsid protein hexon is compromised. Quantitative reverse transcription-PCR (qRT-PCR) analyses revealed that both drugs altered E1A RNA splicing (favoring the production of 13S over 12S RNA) early in infection and partially blocked the transition from 12S and 13S to 9S RNA at late stages of virus replication. Expression of multiple late viral protein mRNAs was lost in the presence of either drug, consistent with the observed block in viral DNA replication. The antiviral effect was dependent on the continued presence of the drug and was rapidly reversible. RIDK34, a derivative of convallotoxin, although having more potent antiviral activity, did not show an improved selectivity index. All three drugs reduced metabolic activity to some degree without evidence of cell death. By blocking adenovirus replication at one or more steps beyond the onset of E1A expression and prior to genome replication, digoxin and digitoxin show potential as antiviral agents for treatment of serious adenovirus infections. Furthermore, understanding the mechanism(s) by which digoxin and digitoxin inhibit adenovirus replication will guide the development of novel antiviral therapies. IMPORTANCE: Despite human adenoviruses being a common and, in some instances, life-threating pathogen in humans, there are few well-tolerated therapies. In this report, we demonstrate that two cardiotonic steroids already in use in humans, digoxin and digitoxin, are potent inhibitors of multiple adenovirus species. A synthetic derivative of the cardiotonic steroid convallotoxin was even more potent than digoxin and digitoxin when tested with HAdV-C5. These drugs alter the cascade of adenovirus gene expression, acting after initiation of early gene expression to block viral DNA replication and synthesis of viral structural proteins. These findings validate a novel approach to treating adenovirus infections through the modulation of host cell processes.
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Adenoviridae/efectos de los fármacos , Adenoviridae/fisiología , Glicósidos Cardíacos/farmacología , Replicación Viral/efectos de los fármacos , Antivirales/farmacología , Línea Celular , Replicación del ADN/efectos de los fármacos , ADN Viral , Digitoxina/farmacología , Digoxina/farmacología , Regulación Viral de la Expresión Génica/efectos de los fármacos , HumanosRESUMEN
The control of RNA processing plays an important role in the nature and quantity of protein generated from mammalian genes. Consequently, efforts to manipulate RNA processing have the capacity to significantly impact gene function. Although multiple strategies have been developed to alter splice site selection using oligonucleotide occlusion of splice sites or splicing regulatory elements, systemic delivery of such agents remains problematic. Outlined in this chapter is a protocol to screen for small molecule inhibitors of HIV-1 Gag expression that have been subsequently determined to modulate viral RNA processing. Identification and characterization of such RNA processing modulators offers the potential for the development of therapeutic lead compounds or probes for investigating the mechanism underlying the regulation of select RNA processing events.
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Regulación Viral de la Expresión Génica , Genes gag/fisiología , Pruebas Genéticas/métodos , VIH-1/metabolismo , Animales , Cantaridina/farmacología , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Genes gag/efectos de los fármacos , VIH-1/efectos de los fármacos , VIH-1/genética , Células HeLa , Humanos , Piranos/farmacología , Compuestos de Espiro/farmacologíaRESUMEN
BACKGROUND: HIV-1 replication is critically dependent upon controlled processing of its RNA and the activities provided by its encoded regulatory factors Tat and Rev. A screen of small molecule modulators of RNA processing identified several which inhibited virus gene expression, affecting both relative abundance of specific HIV-1 RNAs and the levels of Tat and Rev proteins. RESULTS: The screen for small molecules modulators of HIV-1 gene expression at the post-transcriptional level identified three (a pyrimidin-7-amine, biphenylcarboxamide, and benzohydrazide, designated 791, 833, and 892, respectively) that not only reduce expression of HIV-1 Gag and Env and alter the accumulation of viral RNAs, but also dramatically decrease Tat and Rev levels. Analyses of viral RNA levels by qRTPCR and RTPCR indicated that the loss of either protein could not be attributed to changes in abundance of the mRNAs encoding these factors. However, addition of the proteasome inhibitor MG132 did result in significant restoration of Tat expression, indicating that the compounds are affecting Tat synthesis and/or degradation. Tests in the context of replicating HIV-1 in PBMCs confirmed that 791 significantly reduced virus replication. Parallel analyses of the effect of the compounds on host gene expression revealed only minor changes in either mRNA abundance or alternative splicing. Subsequent tests suggest that 791 may function by reducing levels of the Tat/Rev chaperone Nap1. CONCLUSIONS: The three compounds examined (791, 833, 892), despite their lack of structural similarity, all suppressed HIV-1 gene expression by preventing accumulation of two key HIV-1 regulatory factors, Tat and Rev. These findings demonstrate that selective disruption of HIV-1 gene expression can be achieved.
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Fármacos Anti-VIH/aislamiento & purificación , Fármacos Anti-VIH/farmacología , Regulación Viral de la Expresión Génica/efectos de los fármacos , VIH-1/genética , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Fármacos Anti-VIH/química , Células Cultivadas , VIH-1/fisiología , Humanos , Modelos Moleculares , Estructura Molecular , ARN Viral/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Replicación Viral/efectos de los fármacosRESUMEN
Although clinical and experimental evidence indicates that female sex hormones and hormonal contraceptives regulate susceptibility to human immunodeficiency virus type 1 (HIV-1) infection, the underlying mechanism remains unknown. Genital epithelial cells (GECs) are the first cells to encounter HIV during sexual transmission and their interaction with HIV may determine the outcome of exposure. This is the first report that HIV uptake by GECs increased significantly in the presence of the hormonal contraceptive medroxyprogesterone acetate (MPA) and progesterone and that uptake occurred primarily via endocytosis. No productive infection was detected, but endocytosed virus was released into apical and basolateral compartments. Significantly higher viral transcytosis was observed in the presence of MPA. In GEC and T-cell cocultures, maximum viral replication in T cells was observed in the presence of MPA, which also broadly upregulated chemokine production by GECs. These results suggest that MPA may play a significant role in regulating susceptibility to HIV.
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Células Epiteliales/virología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Acetato de Medroxiprogesterona/farmacología , Linfocitos T/virología , Internalización del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Células Cultivadas , Anticonceptivos Femeninos/farmacología , Citocinas/metabolismo , Endocitosis , Femenino , Humanos , Progesterona/farmacología , Regulación hacia Arriba , Útero/citologíaRESUMEN
To develop new approaches to control HIV-1 replication, we examined the capacity of recently described small molecular modulators of RNA splicing for their effects on viral RNA metabolism. Of the drugs tested, digoxin was found to induce a dramatic inhibition of HIV-1 structural protein synthesis, a response due, in part, to reduced accumulation of the corresponding viral mRNAs. In addition, digoxin altered viral RNA splice site use, resulting in loss of the essential viral factor Rev. Digoxin induced changes in activity of the CLK family of SR protein kinases and modification of several SR proteins, including SRp20 and Tra2ß, which could account for the effects observed. Consistent with this hypothesis, overexpression of SRp20 elicited changes in HIV-1 RNA processing similar to those observed with digoxin. Importantly, digoxin was also highly active against clinical strains of HIV-1 in vitro, validating this novel approach to treatment of this infection.
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Antivirales/farmacología , Digoxina/farmacología , Inhibidores Enzimáticos/farmacología , VIH-1/efectos de los fármacos , Procesamiento Postranscripcional del ARN/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Antígenos CD4/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Regulación Viral de la Expresión Génica/efectos de los fármacos , Genes rev/efectos de los fármacos , VIH-1/genética , Humanos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Proteínas del Tejido Nervioso/metabolismo , Empalme del ARN/efectos de los fármacos , ARN Viral/efectos de los fármacos , ARN Viral/metabolismo , Proteínas de Unión al ARN/metabolismo , Factores de Empalme Serina-Arginina , Proteínas ViralesRESUMEN
Expression of the complete HIV-1 genome depends on the appropriate processing of viral RNA. Altering the balance of viral RNA processing impairs replication of the virus. In this report, we characterize two small molecule modulators of HIV-1 RNA processing, 8-azaguanine and 2-(2-(5-nitro-2-thienyl)vinyl)quinoline (5350150), which function by distinct mechanisms to suppress viral gene expression. Although only 8-Azaguanine dramatically decreased accumulation of HIV-1 unspliced and singly spliced RNAs and altered splice site usage, both compounds blocked Gag and Env expression without affecting production of Tat (p16) and Rev regulatory proteins. Subsequent analyses suggest that these compounds affect Rev-mediated RNA transport by different mechanisms. Both compounds induced cytoplasmic accumulation of Rev, suggesting that they function, in part, by impairing Rev function. This conclusion is supported by the determination that both drugs block the nuclear export of genomic HIV-1 RNA to the cytoplasm. Testing confirmed that these compounds suppress HIV-1 expression in T cells at doses below those previously used in humans for tumour chemotherapy. Together, our observations demonstrate that small molecules can be used to inhibit HIV-1 replication by altering another avenue of viral RNA processing, offering the potential for the development of novel therapeutics for controlling this disease.
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Fármacos Anti-VIH/farmacología , Azaguanina/farmacología , VIH-1/efectos de los fármacos , Quinolinas/farmacología , Empalme del ARN/efectos de los fármacos , ARN Viral/metabolismo , Tiofenos/farmacología , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/antagonistas & inhibidores , Linfocitos T CD4-Positivos/virología , Línea Celular , VIH-1/genética , VIH-1/fisiología , Células HeLa , Humanos , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos , Productos del Gen rev del Virus de la Inmunodeficiencia Humana/análisisRESUMEN
Translation is a regulated process and is pivotal to proper cell growth and homeostasis. All retroviruses rely on the host translational machinery for viral protein synthesis and thus may be susceptible to its perturbation in response to stress, co-infection, and/or cell cycle arrest. HIV-1 infection arrests the cell cycle in the G2/M phase, potentially disrupting the regulation of host cell translation. In this study, we present evidence that HIV-1 infection downregulates translation in lymphocytes, attributable to the cell cycle arrest induced by the HIV-1 accessory protein Vpr. The molecular basis of the translation suppression is reduced accumulation of the active form of the translation initiation factor 4E (eIF4E). However, synthesis of viral structural proteins is sustained despite the general suppression of protein production. HIV-1 mRNA translation is sustained due to the distinct composition of the HIV-1 ribonucleoprotein complexes. RNA-coimmunoprecipitation assays determined that the HIV-1 unspliced and singly spliced transcripts are predominantly associated with nuclear cap binding protein 80 (CBP80) in contrast to completely-spliced viral and cellular mRNAs that are associated with eIF4E. The active translation of the nuclear cap binding complex (CBC)-bound viral mRNAs is demonstrated by ribosomal RNA profile analyses. Thus, our findings have uncovered that the maintenance of CBC association is a novel mechanism used by HIV-1 to bypass downregulation of eIF4E activity and sustain viral protein synthesis. We speculate that a subset of CBP80-bound cellular mRNAs contribute to recovery from significant cellular stress, including human retrovirus infection.
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VIH-1/genética , Proteínas de Transporte Nucleocitoplasmático/genética , Biosíntesis de Proteínas/genética , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/genética , Citometría de Flujo , Células HEK293 , VIH-1/metabolismo , Humanos , Linfocitos/metabolismo , Linfocitos/virología , Complejo Proteico Nuclear de Unión a la Caperuza/genética , Complejo Proteico Nuclear de Unión a la Caperuza/metabolismo , Proteínas de Transporte Nucleocitoplasmático/metabolismo , Iniciación de la Cadena Peptídica Traduccional/genética , ARN Mensajero/metabolismo , Productos del Gen vpr del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Control of RNA processing plays a major role in HIV-1 gene expression. To explore the role of several hnRNP proteins in this process, we carried out a siRNA screen to examine the effect of depletion of hnRNPs A1, A2, D, H, I and K on HIV-1 gene expression. While loss of hnRNPs H, I or K had little effect, depletion of A1 and A2 increased expression of viral structural proteins. In contrast, reduced hnRNP D expression decreased synthesis of HIV-1 Gag and Env. Loss of hnRNP D induced no changes in viral RNA abundance but reduced the accumulation of HIV-1 unspliced and singly spliced RNAs in the cytoplasm. Subsequent analyses determined that hnRNP D underwent relocalization to the cytoplasm upon HIV-1 infection and was associated with Gag protein. Screening of the four isoforms of hnRNP D determined that, upon overexpression, they had differential effects on HIV-1 Gag expression, p45 and p42 isoforms increased viral Gag synthesis while p40 and p37 suppressed it. The differential effect of hnRNP D isoforms on HIV-1 expression suggests that their relative abundance could contribute to the permissiveness of cell types to replicate the virus, a hypothesis subsequently confirmed by selective depletion of p45 and p42.
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Expresión Génica , VIH-1/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo D/fisiología , VIH-1/metabolismo , Células HeLa , Ribonucleoproteína Nuclear Heterogénea D0 , Ribonucleoproteína Heterogénea-Nuclear Grupo D/antagonistas & inhibidores , Ribonucleoproteína Heterogénea-Nuclear Grupo D/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/antagonistas & inhibidores , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/fisiología , Humanos , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/fisiología , Interferencia de ARN , ARN Viral/análisis , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
A small molecule screen identified several cardiotonic steroids (digitoxin and ouabain) and the ionophore monensin as potent inhibitors of HCoV-229E, HCoV-OC43, and SARS-CoV-2 replication with EC50s in the low nM range. Subsequent tests confirmed antiviral activity in primary cell models including human nasal epithelial cells and lung organoids. Addition of digitoxin, ouabain, or monensin strongly reduced viral gene expression as measured by both viral protein and RNA accumulation. Furthermore, the compounds acted post virus entry. While the antiviral activity of digitoxin was dependent upon activation of the MEK and JNK signaling pathways but not signaling through GPCRs, the antiviral effect of monensin was reversed upon inhibition of several signaling pathways. Together, the data demonstrates the potent anti-coronavirus properties of two classes of FDA approved drugs that function by altering the properties of the infected cell, rendering it unable to support virus replication.
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Glicósidos Cardíacos , Coronavirus Humano 229E , Humanos , Glicósidos Cardíacos/farmacología , Monensina/farmacología , Ouabaína/farmacología , Digitoxina/farmacología , Antivirales/farmacologíaRESUMEN
The leap of retroviruses and coronaviruses from animal hosts to humans has led to two ongoing pandemics and tens of millions of deaths worldwide. Retrovirus and coronavirus nucleocapsid proteins have been studied extensively as potential drug targets due to their central roles in virus replication, among which is their capacity to bind their respective genomic RNAs for packaging into nascent virions. This review focuses on fundamental studies of these nucleocapsid proteins and how their intrinsic abilities to condense through liquid-liquid phase separation (LLPS) contribute to viral replication. Therapeutic targeting of these condensates and methodological advances are also described to address future questions on how phase separation contributes to viral replication.
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VIH-1 , Proteínas de la Nucleocápside , SARS-CoV-2 , Replicación Viral , Humanos , Proteínas de la Nucleocápside de Coronavirus , COVID-19 , SARS-CoV-2/fisiología , VIH-1/fisiologíaRESUMEN
Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that the pan-retroviral nucleocapsid (NC) and HIV-1 pr55Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yields self-assembling BMCs that have HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs, and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4+ T cell nuclear lysates led to the formation of larger BMCs compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggest that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.
Asunto(s)
Condensados Biomoleculares , VIH-1 , Interacciones Huésped-Patógeno , ARN Viral , Ensamble de Virus , Productos del Gen gag del Virus de la Inmunodeficiencia Humana , Condensados Biomoleculares/metabolismo , Condensados Biomoleculares/virología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismo , VIH-1/genética , VIH-1/metabolismo , ARN Viral/genética , ARN Viral/metabolismo , Genoma Viral , HumanosRESUMEN
Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that pan-retroviral nucleocapsid (NC) and the HIV-1 pr55 Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yield self-assembling BMCs having HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4 + T cell nuclear lysates led to the formation of larger BMCs as compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggests that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.
RESUMEN
BACKGROUND: RNA processing plays a critical role in the replication of HIV-1, regulated in part through the action of host SR proteins. To explore the impact of modulating SR protein activity on virus replication, the effect of increasing or inhibiting the activity of the Cdc2-like kinase (CLK) family of SR protein kinases on HIV-1 expression and RNA processing was examined. RESULTS: Despite their high homology, increasing individual CLK expression had distinct effects on HIV-1, CLK1 enhancing Gag production while CLK2 inhibited the virus. Parallel studies on the anti-HIV-1 activity of CLK inhibitors revealed a similar discrepant effect on HIV-1 expression. TG003, an inhibitor of CLK1, 2 and 4, had no effect on viral Gag synthesis while chlorhexidine, a CLK2, 3 and 4 inhibitor, blocked virus production. Chlorhexidine treatment altered viral RNA processing, decreasing levels of unspliced and single spliced viral RNAs, and reduced Rev accumulation. Subsequent experiments in the context of HIV-1 replication in PBMCs confirmed the capacity of chlorhexidine to suppress virus replication. CONCLUSIONS: Together, these findings establish that HIV-1 RNA processing can be targeted to suppress virus replication as demonstrated by manipulating individual CLK function and identified chlorhexidine as a lead compound in the development of novel anti-viral therapies.
Asunto(s)
Regulación Viral de la Expresión Génica , Infecciones por VIH/enzimología , VIH-1/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Secuencia de Aminoácidos , Línea Celular , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Datos de Secuencia Molecular , Inhibidores de Proteínas Quinasas/uso terapéutico , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Empalme del ARN , ARN Viral/genética , ARN Viral/metabolismoRESUMEN
The observation that stilbene 3 (5350150) blocks HIV replication through its impact on HIV mRNA processing prompted a program to develop non-cytotoxic analogues that maintain its mechanism of action. This initially involved replacement of the central double bond in 3 by an amide function and the quinoline motif by a 2-aminobenzothiazole subunit, as in 12jj (R' = Cl), 12pp (R = NO2), and 12vv (R = CF3). On the basis of the possible CF3 â NO2 bioisostere relationship in 12vv and 12pp, compound 23 was prepared and also found to be active. In the final step, the thiazole compounds 28 (GPS488) (EC50 = 1.66 µM) and 29 (GPS491) (EC50 = 0.47 µM) were prepared and evaluated. Similar activity and cell viability values (therapeutic index (TI = CC50/EC50) values of 50-100) were observed in primary peripheral blood mononuclear cells. Furthermore, they remained active against a panel of HIV mutant strains displaying resistance to individual drugs used in antiretroviral therapy. It was determined that compound 29 suppressed expression of the HIV-1 structural protein Gag and altered HIV-1 RNA accumulation, decreasing the abundance of RNAs encoding the structural proteins while increasing levels of viral RNAs encoding the regulatory proteins, a pattern similar to that seen for compound 3.