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1.
Protein Sci ; 32(12): e4814, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-37861472

RESUMO

HIV-1 reverse transcriptase (RT) remains a key target for HIV drug development. As successful management of the disease requires lifelong treatment, the emergence of resistance mutations is inevitable, making development of new RT inhibitors, which remain effective against resistant variants crucial. To this end, previous computationally guided drug design efforts have resulted in catechol diether compounds, which inhibit wildtype RT with picomolar affinities and appear to be promising preclinical candidates. To confirm that these compounds remain potent against Y181C, a widespread mutation conferring resistance to first generation inhibitors, they were screened against the HIV-1 N119 clinical isolate, reported as a Y181C single mutant. In comparison to a molecular clone with the same mutation, N119 appears less susceptible to inhibition by our preclinical candidate compounds. A more detailed sequencing effort determined that N119 was misidentified and carries V106A in combination with Y181C. While both indolizine and naphthalene substituted catechol diethers are potent against the classical Y181C single mutant, the addition of V106A confers more resistance against the indolizine derivatives than the naphthalene derivatives. Crystal structures presented in this study highlight key features of the naphthyl group, which allow these compounds to remain potent in the double mutant, including stronger interactions with F227 and less reliance on V106 for stabilization of the ethoxy-uracil ring, which makes critical hydrogen bonds with other residues in the binding pocket.


Assuntos
Fármacos Anti-HIV , HIV-1 , Indolizinas , Inibidores da Transcriptase Reversa/farmacologia , Inibidores da Transcriptase Reversa/química , Transcriptase Reversa do HIV/química , Indolizinas/farmacologia , Catecóis/química , Catecóis/farmacologia , Naftalenos/farmacologia , Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/química , Relação Estrutura-Atividade
2.
J Virol ; 96(13): e0057722, 2022 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-35730977

RESUMO

Despite effective antiretroviral therapy, HIV-1 persistence in latent reservoirs remains a major obstacle to a cure. We postulate that HIV-1 silencing factors suppress HIV-1 reactivation and that inhibition of these factors will increase HIV-1 reactivation. To identify HIV-1 silencing factors, we conducted a genome-wide CRISPR inhibition (CRISPRi) screen using four CRISPRi-ready, HIV-1-d6-GFP-infected Jurkat T cell clones with distinct integration sites. We sorted cells with increased green fluorescent protein (GFP) expression and captured single guide RNAs (sgRNAs) via targeted deep sequencing. We identified 18 HIV-1 silencing factors that were significantly enriched in HIV-1-d6-GFPhigh cells. Among them, SLTM (scaffold attachment factor B-like transcription modulator) is an epigenetic and transcriptional modulator having both DNA and RNA binding capacities not previously known to affect HIV-1 transcription. Knocking down SLTM by CRISPRi significantly increased HIV-1-d6-GFP expression (by 1.9- to 4.2-fold) in three HIV-1-d6-GFP-Jurkat T cell clones. Furthermore, SLTM knockdown increased the chromatin accessibility of HIV-1 and the gene in which HIV-1 is integrated but not the housekeeping gene POLR2A. To test whether SLTM inhibition can reactivate HIV-1 and further induce cell death of HIV-1-infected cells ex vivo, we established a small interfering RNA (siRNA) knockdown method that reduced SLTM expression in CD4+ T cells from 10 antiretroviral therapy (ART)-treated, virally suppressed, HIV-1-infected individuals ex vivo. Using limiting dilution culture, we found that SLTM knockdown significantly reduced the frequency of HIV-1-infected cells harboring inducible HIV-1 by 62.2% (0.56/106 versus 1.48/106 CD4+ T cells [P = 0.029]). Overall, our study indicates that SLTM inhibition reactivates HIV-1 in vitro and induces cell death of HIV-1-infected cells ex vivo. Our study identified SLTM as a novel therapeutic target. IMPORTANCE HIV-1-infected cells, which can survive drug treatment and immune cell killing, prevent an HIV-1 cure. Immune recognition of infected cells requires HIV-1 protein expression; however, HIV-1 protein expression is limited in infected cells after long-term therapy. The ways in which the HIV-1 provirus is blocked from producing protein are unknown. We identified a new host protein that regulates HIV-1 gene expression. We also provided a new method of studying HIV-1-host factor interactions in cells from infected individuals. These improvements may enable future strategies to reactivate HIV-1 in infected individuals so that infected cells can be killed by immune cells, drug treatment, or the virus itself.


Assuntos
Infecções por HIV , Soropositividade para HIV , HIV-1 , Ativação Viral , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos , Cromatina/genética , Cromatina/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Técnicas de Silenciamento de Genes , Infecções por HIV/fisiopatologia , Soropositividade para HIV/genética , HIV-1/fisiologia , Humanos , Células Jurkat , Proteínas de Ligação à Região de Interação com a Matriz/antagonistas & inibidores , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Ativação Viral/genética
3.
Viruses ; 13(9)2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34578439

RESUMO

More than 50% of the HIV-1 latent reservoir is maintained by clonal expansion. The clonally expanded HIV-1-infected cells can contribute to persistent nonsuppressible low-level viremia and viral rebound. HIV-1 integration site and proviral genome landscape profiling reveals the clonal expansion dynamics of HIV-1-infected cells. In individuals under long-term suppressive antiretroviral therapy (ART), HIV-1 integration sites are enriched in specific locations in certain cancer-related genes in the same orientation as the host transcription unit. Single-cell transcriptome analysis revealed that HIV-1 drives aberrant cancer-related gene expression through HIV-1-to-host RNA splicing. Furthermore, the HIV-1 promoter dominates over the host gene promoter and drives high levels of cancer-related gene expression. When HIV-1 integrates into cancer-related genes and causes gain of function of oncogenes or loss of function of tumor suppressor genes, HIV-1 insertional mutagenesis drives the proliferation of HIV-1-infected cells and may cause cancer in rare cases. HIV-1-driven aberrant cancer-related gene expression at the integration site can be suppressed by CRISPR-mediated inhibition of the HIV-1 promoter or by HIV-1 suppressing agents. Given that ART does not suppress HIV-1 promoter activity, therapeutic agents that suppress HIV-1 transcription and halt the clonal expansion of HIV-1-infected cells should be explored to block the clonal expansion of the HIV-1 latent reservoir.


Assuntos
Infecções por HIV/virologia , HIV-1/fisiologia , Integração Viral , Latência Viral , Animais , Fármacos Anti-HIV/uso terapêutico , Terapia Antirretroviral de Alta Atividade , Proliferação de Células , Modelos Animais de Doenças , Genes Supressores de Tumor , Genoma Viral , Infecções por HIV/tratamento farmacológico , HIV-1/genética , Humanos , Mutagênese Insercional , Oncogenes , Provírus/genética , Transcrição Gênica , Viremia
5.
J Virol ; 95(4)2021 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-33239456

RESUMO

HIV transcription requires assembly of cellular transcription factors at the HIV-1promoter. The TFIIH general transcription factor facilitates transcription initiation by opening the DNA strands around the transcription start site and phosphorylating the C-terminal domain for RNA polymerase II (RNAPII) for activation. Spironolactone (SP), an FDA approved aldosterone antagonist, triggers the proteasomal degradation of the XPB subunit of TFIIH, and concurrently suppresses acute HIV infection in vitro Here we investigated SP as a possible block-and-lock agent for a functional cure aimed at the transcriptional silencing of the viral reservoir. The long-term activity of SP was investigated in primary and cell line models of HIV-1 latency and reactivation. We show that SP rapidly inhibits HIV-1 transcription by reducing RNAPII recruitment to the HIV-1 genome. shRNA knockdown of XPB confirmed XPB degradation as the mechanism of action. Unfortunately, long-term pre-treatment with SP does not result in epigenetic suppression of HIV upon SP treatment interruption, since virus rapidly rebounds when XPB reemerges; however, SP alone without ART maintains the transcriptional suppression. Importantly, SP inhibits HIV reactivation from latency in both cell line models and resting CD4+T cells isolated from aviremic infected individuals upon cell stimulation with latency reversing agents. Furthermore, long-term treatment with concentrations of SP that potently degrade XPB does not lead to global dysregulation of cellular mRNA expression. Overall, these results suggest that XPB plays a key role in HIV transcriptional regulation and XPB degradation by SP strengthens the potential of HIV transcriptional inhibitors in block-and-lock HIV cure approaches.IMPORTANCE Antiretroviral therapy (ART) effectively reduces an individual's HIV loads to below the detection limit, nevertheless rapid viral rebound immediately ensues upon treatment interruption. Furthermore, virally suppressed individuals experience chronic immune activation from ongoing low-level virus expression. Thus, the importance of identifying novel therapeutics to explore in block-and-lock HIV functional cure approaches, aimed at the transcriptional and epigenetic silencing of the viral reservoir to block reactivation from latency. We investigated the potential of repurposing the FDA-approved spironolactone (SP), as one such drug. SP treatment rapidly degrades a host transcription factor subunit, XPB, inhibiting HIV transcription and blocking reactivation from latency. Long-term SP treatment does not affect cellular viability, cell cycle progression or global cellular transcription. SP alone blocks HIV transcription in the absence of ART but does not delay rebound upon drug removal as XPB rapidly reemerges. This study highlights XPB as a novel drug target in block-and-lock therapeutic approaches.

6.
J Clin Invest ; 130(9): 4969-4984, 2020 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-32573496

RESUMO

Despite effective antiretroviral therapy, HIV-1-infected cells continue to produce viral antigens and induce chronic immune exhaustion. We propose to identify HIV-1-suppressing agents that can inhibit HIV-1 reactivation and reduce HIV-1-induced immune activation. Using a newly developed dual-reporter system and a high-throughput drug screen, we identified FDA-approved drugs that can suppress HIV-1 reactivation in both cell line models and CD4+ T cells from virally suppressed HIV-1-infected individuals. We identified 11 cellular pathways required for HIV-1 reactivation as druggable targets. Using differential expression analysis, gene set enrichment analysis, and exon-intron landscape analysis, we examined the impact of drug treatment on the cellular environment at a genome-wide level. We identified what we believe to be a new function of a JAK inhibitor, filgotinib, that suppresses HIV-1 splicing. First, filgotinib preferentially suppresses spliced HIV-1 RNA transcription. Second, filgotinib suppresses HIV-1-driven aberrant cancer-related gene expression at the integration site. Third, we found that filgotinib suppresses HIV-1 transcription by inhibiting T cell activation and by modulating RNA splicing. Finally, we found that filgotinib treatment reduces the proliferation of HIV-1-infected cells. Overall, the combination of a drug screen and transcriptome analysis provides systematic understanding of cellular targets required for HIV-1 reactivation and drug candidates that may reduce HIV-1-related immune activation.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Infecções por HIV/tratamento farmacológico , HIV-1/imunologia , Ativação Linfocitária/efeitos dos fármacos , Piridinas/farmacologia , Splicing de RNA/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Triazóis/farmacologia , Linfócitos T CD4-Positivos/patologia , Infecções por HIV/imunologia , Infecções por HIV/patologia , Humanos , Células Jurkat , Splicing de RNA/imunologia , Transcrição Gênica/imunologia
7.
Sci Transl Med ; 12(543)2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32404504

RESUMO

Understanding HIV-1-host interactions can identify the cellular environment supporting HIV-1 reactivation and mechanisms of clonal expansion. We developed HIV-1 SortSeq to isolate rare HIV-1-infected cells from virally suppressed, HIV-1-infected individuals upon early latency reversal. Single-cell transcriptome analysis of HIV-1 SortSeq+ cells revealed enrichment of nonsense-mediated RNA decay and viral transcription pathways. HIV-1 SortSeq+ cells up-regulated cellular factors that can support HIV-1 transcription (IMPDH1 and JAK1) or promote cellular survival (IL2 and IKBKB). HIV-1-host RNA landscape analysis at the integration site revealed that HIV-1 drives high aberrant host gene transcription downstream, but not upstream, of the integration site through HIV-1-to-host aberrant splicing, in which HIV-1 RNA splices into the host RNA and aberrantly drives host RNA transcription. HIV-1-induced aberrant transcription was driven by the HIV-1 promoter as shown by CRISPR-dCas9-mediated HIV-1-specific activation and could be suppressed by CRISPR-dCas9-mediated inhibition of HIV-1 5' long terminal repeat. Overall, we identified cellular factors supporting HIV-1 reactivation and HIV-1-driven aberrant host gene transcription as potential therapeutic targets to disrupt HIV-1 persistence.


Assuntos
Infecções por HIV , HIV-1 , Regulação Viral da Expressão Gênica , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , HIV-1/genética , Humanos , Transcrição Gênica , Ativação Viral , Latência Viral
8.
Exp Cell Res ; 358(2): 199-208, 2017 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-28663059

RESUMO

Although Insulin-like growth factor (IGF-I) has been intensively studied, the functions of E-domain peptides of pro-IGF-I, however, have been overlooked. In our laboratory, several anti-cancer activities of the E-peptide of pro-IGF-I have been identified for the longest isoforms of human and rainbow trout E-peptides. These activities include dose-dependent inhibition of colony formation, inhibition of cancer cell metastasis and invasion through matrigel, suppression of cancer-induced angiogenesis, and attenuation of expression of apoptotic genes in favor of cell death. In this study, we were able to produce two-tagged recombinant human Eb-peptide (hEb) of pro-IGF-I with a purity over 99%. With its antimicrobial peptide (AMP)-like characteristics such as binding to the cytoplasmic membrane, and the affinity to the substratum of culture plate, hEb forms a layer of interface rapidly which facilitates the attachment of breast carcinoma cells, MDA-MB-231. Furthermore, the likely conformational change of homo-dimerized hEb through a single disulfide bond, as well as the ability to trigger clathrin-mediated endocytosis may play important roles for inducing lamellipodia outspread in MDA-MB-231 cells. With the highly purified hEb-peptide, not only could we study its function(s) in detail but also the minimum requirement for cancerous cells to metastasize to a suitable environment and grow.


Assuntos
Neoplasias da Mama/metabolismo , Junções Célula-Matriz/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Fator de Crescimento Insulin-Like I/metabolismo , Precursores de Proteínas/metabolismo , Adesão Celular , Divisão Celular/fisiologia , Linhagem Celular Tumoral , Feminino , Humanos , Fator de Crescimento Insulin-Like II , Fragmentos de Peptídeos , Pseudópodes/metabolismo , Proteínas Recombinantes/metabolismo
9.
Proc Natl Acad Sci U S A ; 114(27): 7118-7123, 2017 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-28630311

RESUMO

A natural and permanent transfer of prokaryotic viral sequences to mammals has not been reported by others. Circular "SPHINX" DNAs <5 kb were previously isolated from nuclease-protected cytoplasmic particles in rodent neuronal cell lines and brain. Two of these DNAs were sequenced after Φ29 polymerase amplification, and they revealed significant but imperfect homology to segments of commensal Acinetobacter phage viruses. These findings were surprising because the brain is isolated from environmental microorganisms. The 1.76-kb DNA sequence (SPHINX 1.8), with an iteron before its ORF, was evaluated here for its expression in neural cells and brain. A rabbit affinity purified antibody generated against a peptide without homology to mammalian sequences labeled a nonglycosylated ∼41-kDa protein (spx1) on Western blots, and the signal was efficiently blocked by the competing peptide. Spx1 was resistant to limited proteinase K digestion, but was unrelated to the expression of host prion protein or its pathologic amyloid form. Remarkably, spx1 concentrated in selected brain synapses, such as those on anterior motor horn neurons that integrate many complex neural inputs. SPHINX 1.8 appears to be involved in tissue-specific differentiation, including essential functions that preserve its propagation during mammalian evolution, possibly via maternal inheritance. The data here indicate that mammals can share and exchange a larger world of prokaryotic viruses than previously envisioned.


Assuntos
Acinetobacter/virologia , Bacteriófagos/genética , Encéfalo/metabolismo , Encéfalo/virologia , Sinapses/fisiologia , Animais , Diferenciação Celular , Citoplasma/metabolismo , DNA Circular/metabolismo , Evolução Molecular , Humanos , Camundongos , Microbiota , Fases de Leitura Aberta , Príons/metabolismo , Coelhos , Análise de Sequência de DNA
10.
Toxicology ; 243(1-2): 11-22, 2008 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-17997003

RESUMO

Hepatocellular carcinoma (HCC) is one of the common cancers worldwide, caused by Hepatitis C virus (HCV) and hepatotoxins. Here we report the development of HCC in wild type as well as HCV core protein (HCP)-transgenic zebrafish upon treatment with a hepatotoxin, thioacetamide (TAA). Two-fold accelerated HCC development could be achieved in the TAA-treated transgenic fish, that is, the progression of the disease in TAA-treated wild type zebrafish developed HCC in 12 weeks whereas that of HCP-transgenic zebrafish shortened the HCC progression to 6 weeks. Histopathological observation showed the specific pathological features of HCC. The HCC progression was confirmed through RT-PCR that revealed an up and down regulation of different marker genes at various stages of HCC progression such as, steatohepatitis, fibrosis and HCC. Moreover, HCV core protein expressed in the HCP-transgenic zebrafish and TAA synergistically accelerate the HCC development. It must be mentioned that, this is the first report revealing HCV core protein along with TAA to induce HCC in zebrafish, particularly, in a short period of time comparing to mice model. As zebrafish has already been considered as a good human disease model and in this context, this HCC-zebrafish model may serve as a powerful preclinical platform to study the molecular events in hepatocarcinogenesis, therapeutic strategies and for evaluating chemoprevention strategies in HCC.


Assuntos
Hepacivirus/genética , Hepatopatias , Tioacetamida/toxicidade , Proteínas do Core Viral/genética , Peixe-Zebra/genética , Animais , Animais Geneticamente Modificados , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Doença Hepática Induzida por Substâncias e Drogas , Primers do DNA , Fígado Gorduroso/induzido quimicamente , Fígado Gorduroso/genética , Fígado Gorduroso/virologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/genética , Cirrose Hepática/virologia , Hepatopatias/genética , Hepatopatias/virologia , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase Reversa
11.
Dev Dyn ; 230(4): 734-42, 2004 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-15254907

RESUMO

Intestinal-type fatty acid binding protein (I-FABP) plays an important role in the intracellular binding and trafficking of long chain fatty acids in the intestine. The aim of this study, therefore, was to elucidate the regulation and spatiotemporal expression of the I-FABP gene during zebrafish larval development. We performed in vivo reporter-gene analysis in zebrafish by using a transient and transgenic approach. Green fluorescent protein-reporter analyses revealed that the proximal 192-bp region of the I-FABP promoter is sufficient to direct intestine-specific expression during zebrafish larval development. Functional dissection of a 41-bp region within this 192-bp promoter revealed that one C/EBP and two GATA-like binding sites, along with a novel 15-bp element within it are required for I-FABP gene expression in vivo. In addition, the six consensus sites (CCACATCAGCATGAA) in the 15-bp element are critical for I-FABP gene regulation in the zebrafish gut epithelia. Comparison analyses of the orthologous 15-bp element from mammalian I-FABP genes suggests that these mammalian elements are functionally equivalent to the zebrafish 15 element. These results provide the first in vivo evidence that these binding sites (C/EBP and GATA) and the novel 15-bp element contribute to intestine-specific gene expression and that they are functionally conserved across vertebrate evolution.


Assuntos
Proteínas de Transporte/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Larva/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Proteínas de Transporte/química , Sequência Conservada , DNA/metabolismo , Elementos Facilitadores Genéticos , Proteínas de Ligação a Ácido Graxo , Deleção de Genes , Genes Reporter , Proteínas de Fluorescência Verde/metabolismo , Intestinos/embriologia , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Estrutura Terciária de Proteína , Homologia de Sequência do Ácido Nucleico , Transgenes , Peixe-Zebra
12.
Dev Dyn ; 227(3): 347-56, 2003 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12815620

RESUMO

Liver fatty acid binding protein (L-FABP) is a small protein that is thought to play an important role in the intracellular binding and trafficking of long chain fatty acids in the liver. Expression of the gene encoding the zebrafish liver fatty acid binding protein is regulated by a 435-bp distal region (-1944 to -1510) of the L-FABP promoter. The 435-bp sequence is sufficient for gene activation in the liver primordia (or bud) and continues to be active in the adult liver when positioned adjacent to the SV40 basal promoter and linked directly to green fluorescent protein. The 435-bp sequence region has two distinct liver regulatory elements, A (-1944 to -1623) and B (-1622 to -1510), and contains multiple putative consensus binding sites. The element A sequence includes two consensus HFH and one HNF-1alpha site and the element B sequence includes one consensus HNF-3beta site. Deletion of an internal 435-bp fragment (-1944 to -1510) including the A and B elements totally ablated the liver-specific activity of the zebrafish L-FABP gene promoter. Deletion of either of the two elements reduces the liver activity. Mutation of the HNF-1alpha site or either of the two HFH sites in the A element or the HNF-3beta site in the B element significantly altered specificity in the liver primordia of transient expression embryos. The importance of the HNF-1alpha consensus binding site in the A element and the HNF-3beta consensus binding site in the B element within the 435-bp distal region of the L-FABP promoter region suggests that combinatorial interactions between multiple regulatory factors are responsible for the gene expression of L-FABP in the liver.


Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Regulação da Expressão Gênica , Fígado/metabolismo , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Proteínas de Peixe-Zebra , Animais , Animais Geneticamente Modificados , Sequência de Bases , Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Deleção de Genes , Proteínas de Fluorescência Verde , Fator 1 Nuclear de Hepatócito , Fator 1-alfa Nuclear de Hepatócito , Fator 1-beta Nuclear de Hepatócito , Fator 3-beta Nuclear de Hepatócito , Hepatócitos/metabolismo , Proteínas Luminescentes/metabolismo , Camundongos , Modelos Genéticos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência do Ácido Nucleico , Fatores de Transcrição/metabolismo , Transcrição Gênica , Ativação Transcricional , Transgenes , Peixe-Zebra
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