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Background: While directionally rotating tumor-treating fields (TTF) therapy has garnered considerable clinical interest in recent years, there has been comparatively less focus on directionally non-rotating electric field therapy (dnEFT). Methods: We explored dnEFT generated through customized electrodes as a glioblastoma therapy in in vitro and in vivo preclinical models. The effects of dnEFT on tumor apoptosis and microglia/macrophages in the tumor microenvironment were tested using flow-cytometric and qPCR assays. Results: In vitro, dnEFT generated using a clinical-grade spinal cord stimulator showed antineoplastic activity against independent glioblastoma cell lines. In support of the results obtained using the clinical-grade electrode, dnEFT delivered through a customized, 2-electrode array induced glioblastoma apoptosis. To characterize this effect in vivo, a custom-designed 4-electrode array was fabricated such that tumor cells can be implanted into murine cerebrum through a center channel equidistant from the electrodes. After implantation with this array and luciferase-expressing murine GL261 glioblastoma cells, mice were randomized to dnEFT or placebo. Relative to placebo-treated mice, dnEFT reduced tumor growth (measured by bioluminescence) and prolonged survival (median survival gain of 6.5 days). Analysis of brain sections following dnEFT showed a notable increase in the accumulation of peritumoral macrophage/microglia with increased expression of M1 genes (IFNγ, TNFα, and IL-6) and decreased expression of M2 genes (CD206, Arg, and IL-10) relative to placebo-treated tumors. Conclusions: Our results suggest therapeutic potential in glioblastoma for dnEFT delivered through implanted electrodes, supporting the development of a proof-of-principle clinical trial using commercially available deep brain stimulator electrodes.
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Conventional and cancer immunotherapies encompass diverse strategies to address various cancer types and stages. However, combining these approaches often encounters limitations such as non-specific targeting, resistance development, and high toxicity, leading to suboptimal outcomes in many cancers. The tumor microenvironment (TME) is orchestrated by intricate interactions between immune and non-immune cells dictating tumor progression. An innovative avenue in cancer therapy involves leveraging small molecules to influence a spectrum of resistant cell populations within the TME. Recent discoveries have unveiled a phenotypically diverse cohort of innate-like T (ILT) cells and tumor hybrid cells (HCs) exhibiting novel characteristics, including augmented proliferation, migration, resistance to exhaustion, evasion of immunosurveillance, reduced apoptosis, drug resistance, and heightened metastasis frequency. Leveraging small-molecule immunomodulators to target these immune players presents an exciting frontier in developing novel tumor immunotherapies. Moreover, combining small molecule modulators with immunotherapy can synergistically enhance the inhibitory impact on tumor progression by empowering the immune system to meticulously fine-tune responses within the TME, bolstering its capacity to recognize and eliminate cancer cells. This review outlines strategies involving small molecules that modify immune cells within the TME, potentially revolutionizing therapeutic interventions and enhancing the anti-tumor response.
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The mTOR signaling pathway plays a pivotal and intricate role in the pathogenesis of glioblastoma, driving tumorigenesis and proliferation. Mutations or deletions in the PTEN gene constitutively activate the mTOR pathway by expressing growth factors EGF and PDGF, which activate their respective receptor pathways (e.g., EGFR and PDGFR). The convergence of signaling pathways, such as the PI3K-AKT pathway, intensifies the effect of mTOR activity. The inhibition of mTOR has the potential to disrupt diverse oncogenic processes and improve patient outcomes. However, the complexity of the mTOR signaling, off-target effects, cytotoxicity, suboptimal pharmacokinetics, and drug resistance of the mTOR inhibitors pose ongoing challenges in effectively targeting glioblastoma. Identifying innovative treatment strategies to address these challenges is vital for advancing the field of glioblastoma therapeutics. This review discusses the potential targets of mTOR signaling and the strategies of target-specific mTOR inhibitor development, optimized drug delivery system, and the implementation of personalized treatment approaches to mitigate the complications of mTOR inhibitors. The exploration of precise mTOR-targeted therapies ultimately offers elevated therapeutic outcomes and the development of more effective strategies to combat the deadliest form of adult brain cancer and transform the landscape of glioblastoma therapy.
Assuntos
Glioblastoma , Humanos , Glioblastoma/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Inibidores de MTOR , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismoRESUMO
The acquisition of m7G-cap-binding proteins is now recognized as a major variable driving the form and function of host RNAs. This manuscript compares the 5'-cap-RNA binding proteins that engage HIV-1 precursor RNAs, host mRNAs, small nuclear (sn)- and small nucleolar (sno) RNAs and sort into disparate RNA-fate pathways. Before completion of the transcription cycle, the transcription start site of nascent class II RNAs is appended to a non-templated guanosine that is methylated (m7G-cap) and bound by hetero-dimeric CBP80-CBP20 cap binding complex (CBC). The CBC is a nexus for the co-transcriptional processing of precursor RNAs to mRNAs and the snRNA and snoRNA of spliceosomal and ribosomal ribonucleoproteins (RNPs). Just as sn/sno-RNAs experience hyper-methylation of m7G-cap to trimethylguanosine (TMG)-cap, so do select HIV RNAs and an emerging cohort of mRNAs. TMG-cap is blocked from Watson:Crick base pairing and disqualified from participating in secondary structure. The HIV TMG-cap has been shown to license select viral transcripts for specialized cap-dependent translation initiation without eIF4E that is dependent upon CBP80/NCBP3. The exceptional activity of HIV precursor RNAs secures their access to maturation pathways of sn/snoRNAs, canonical and non-canonical host mRNAs in proper stoichiometry to execute the retroviral replication cycle.
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Infecções por HIV , HIV-1 , HIV-1/genética , HIV-1/metabolismo , Humanos , Metilação , Capuzes de RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Appended to the 5' end of nascent RNA polymerase II transcripts is 7-methyl guanosine (m7G-cap) that engages nuclear cap-binding complex (CBC) to facilitate messenger RNA (mRNA) maturation. Mature mRNAs exchange CBC for eIF4E, the rate-limiting translation factor that is controlled through mTOR. Experiments in immune cells have now documented HIV-1 incompletely processed transcripts exhibited hypermethylated m7G-cap and that the down-regulation of the trimethylguanosine synthetase-1-reduced HIV-1 infectivity and virion protein synthesis by several orders of magnitude. HIV-1 cap hypermethylation required nuclear RNA helicase A (RHA)/DHX9 interaction with the shape of the 5' untranslated region (UTR) primer binding site (PBS) segment. Down-regulation of RHA or the anomalous shape of the PBS segment abrogated hypermethylated caps and derepressed eIF4E binding for virion protein translation during global down-regulation of host translation. mTOR inhibition was detrimental to HIV-1 proliferation and attenuated Tat, Rev, and Nef synthesis. This study identified mutually exclusive translation pathways and the calibration of virion structural/accessory protein synthesis with de novo synthesis of the viral regulatory proteins. The hypermethylation of select, viral mRNA resulted in CBC exchange to heterodimeric CBP80/NCBP3 that expanded the functional capacity of HIV-1 in immune cells.
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Guanosina/metabolismo , HIV-1/metabolismo , Interações entre Hospedeiro e Microrganismos/fisiologia , Serina-Treonina Quinases TOR/metabolismo , Regiões 5' não Traduzidas , Sítios de Ligação , RNA Helicases DEAD-box , Fator de Iniciação 4E em Eucariotos/metabolismo , Guanosina/análogos & derivados , Humanos , Licenciamento , Metilação , Metiltransferases/metabolismo , Proteínas de Neoplasias , Capuzes de RNA , RNA Mensageiro/metabolismo , RNA Viral/genética , Vírion/metabolismoRESUMO
HIV-1 reverse transcription initiates at the primer binding site (PBS) in the viral genomic RNA (gRNA). Although the structure of the PBS-segment undergoes substantial rearrangement upon tRNALys3 annealing, the proper folding of the PBS-segment during gRNA packaging is important as it ensures loading of beneficial host factors. DHX9/RNA helicase A (RHA) is recruited to gRNA to enhance the processivity of reverse transcriptase. Because the molecular details of the interactions have yet to be defined, we solved the solution structure of the PBS-segment preferentially bound by RHA. Evidence is provided that PBS-segment adopts a previously undefined adenosine-rich three-way junction structure encompassing the primer activation stem (PAS), tRNA-like element (TLE) and tRNA annealing arm. Disruption of the PBS-segment three-way junction structure diminished reverse transcription products and led to reduced viral infectivity. Because of the existence of the tRNA annealing arm, the TLE and PAS form a bent helical structure that undergoes shape-dependent recognition by RHA double-stranded RNA binding domain 1 (dsRBD1). Mutagenesis and phylogenetic analyses provide evidence for conservation of the PBS-segment three-way junction structure that is preferentially bound by RHA in support of efficient reverse transcription, the hallmark step of HIV-1 replication.
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RNA Helicases DEAD-box/química , HIV-1/química , Proteínas de Neoplasias/química , RNA Viral/química , Transcrição Reversa/genética , Replicação Viral/genética , Regiões 5' não Traduzidas , Sítios de Ligação/genética , Linhagem Celular , HIV-1/genética , HIV-1/patogenicidade , Humanos , Espectroscopia de Ressonância Magnética , Simulação de Acoplamento Molecular , Mutação , Conformação de Ácido Nucleico , Motivos de Nucleotídeos , Filogenia , Conformação Proteica em alfa-Hélice , Domínios Proteicos , RNA de Transferência de Lisina/genética , RNA de Transferência de Lisina/metabolismo , RNA Viral/genéticaRESUMO
Tertiary structure (3D) is the physical context of RNA regulatory activity. Retroviruses are RNA viruses that replicate through the proviral DNA intermediate transcribed by hosts. Proviral transcripts form inhomogeneous populations due to variable structural ensembles of overlapping regulatory RNA motifs in the 5'-untranslated region (UTR), which drive RNAs to be spliced or translated, and/or dimerized and packaged into virions. Genetic studies and structural techniques have provided fundamental input constraints to begin predicting HIV 3D conformations in silico. Using SimRNA and sets of experimentally-determined input constraints of HIVNL4-3 trans-activation responsive sequence (TAR) and pairings of unique-5' (U5) with dimerization (DIS) or AUG motifs, we calculated a series of 3D models that differ in proximity of 5'-Cap and the junction of TAR and PolyA helices; configuration of primer binding site (PBS)-segment; and two host cofactors binding sites. Input constraints on U5-AUG pairings were most compatible with intramolecular folding of 5'-UTR motifs in energetic minima. Introducing theoretical constraints predicted metastable PolyA region drives orientation of 5'-Cap with TAR, U5 and PBS-segment helices. SimRNA and the workflow developed herein provides viable options to predict 3D conformations of inhomogeneous populations of large RNAs that have been intractable to conventional ensemble methods.
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Conformação de Ácido Nucleico , RNA Viral/química , RNA Viral/fisiologia , Regiões 5' não Traduzidas , Sequência de Bases , Sítios de Ligação , Dimerização , Genoma Viral , Motivos de Nucleotídeos , Provírus/genética , Provírus/fisiologia , RNA Viral/genéticaRESUMO
One long-standing knowledge gap is the role of nuclear proteins in mRNA translation. Nuclear RNA helicase A (DHX9/RHA) is necessary for the translation of the mRNAs of JUND (JunD proto-oncogene AP-1 transcription factor subunit) and HIV-1 genes, and nuclear cap-binding protein 1 (NCBP1)/CBP80 is a component of HIV-1 polysomes. The protein kinase mTOR activates canonical messenger ribonucleoproteins by post-translationally down-regulating the eIF4E inhibitory protein 4E-BP1. We posited here that NCBP1 and DHX9/RHA (RHA) support a translation pathway of JUND RNA that is independent of mTOR. We present evidence from reciprocal immunoprecipitation experiments indicating that NCBP1 and RHA both are components of messenger ribonucleoproteins in several cell types. Moreover, tandem affinity and RT-quantitative PCR results revealed that JUND mRNA is a component of a previously unknown ribonucleoprotein complex. Results from the tandem IP indicated that another component of the JUND-containing ribonucleoprotein complex is NCBP3, a recently identified ortholog of NCBP2/CBP20. We also found that NCBP1, NCBP3, and RHA, but not NCBP2, are components of JUND-containing polysomes. Mutational analysis uncovered two dsRNA-binding domains of RHA that are necessary to tether JUND-NCBP1/NCBP3 to polysomes. We also found that JUND translation is unaffected by inhibition of mTOR, unless RHA was down-regulated by siRNA. These findings uncover a noncanonical cap-binding complex consisting of NCBP1/NCBP3 and RHA substitutes for the eukaryotic translation initiation factors 4E and 4G and activates mTOR-independent translation of the mRNA encoding the tumor suppressor JUND.
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Complexos Multiproteicos/metabolismo , Polirribossomos/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Células COS , Chlorocebus aethiops , Células HEK293 , Humanos , Proto-Oncogene MasRESUMO
A major challenge in finding a cure for HIV-1/AIDS is the difficulty in identifying and eradicating persistent reservoirs of replication-competent provirus. Long noncoding RNAs (lncRNAs, >200 nucleotides) are increasingly recognized to play important roles in pathophysiology. Here, we report the first genome-wide expression analysis of lncRNAs in HIV-1-infected primary monocyte-derived macrophages (MDMs). We identified an lncRNA, which we named HIV-1-enhanced lncRNA (HEAL), that is upregulated by HIV-1 infection of MDMs, microglia, and T lymphocytes. Peripheral blood mononuclear cells of HIV-1-infected individuals show elevated levels of HEAL Importantly, HEAL is a broad enhancer of multiple HIV-1 strains because depletion of HEAL inhibited X4, R5, and dual-tropic HIV replications and the inhibition was rescued by HEAL overexpression. HEAL forms a complex with the RNA-binding protein FUS, which facilitates HIV replication through at least two mechanisms: (i) HEAL-FUS complex binds the HIV promoter and enhances recruitment of the histone acetyltransferase p300, which positively regulates HIV transcription by increasing histone H3K27 acetylation and P-TEFb enrichment on the HIV promoter, and (ii) HEAL-FUS complex is enriched at the promoter of the cyclin-dependent kinase 2 gene, CDK2, to enhance CDK2 expression. Notably, HEAL knockdown and knockout mediated by RNA interference (RNAi) and CRISPR-Cas9, respectively, prevent HIV-1 recrudescence in T cells and microglia upon cessation of azidothymidine treatment in vitro Our results suggest that silencing of HEAL or perturbation of the HEAL-FUS ribonucleoprotein complex could provide a new epigenetic silencing strategy to eradicate viral reservoirs and effect a cure for HIV-1/AIDS.IMPORTANCE Despite our increased understanding of the functions of lncRNAs, their potential to develop HIV/AIDS cure strategies remains unexplored. A genome-wide analysis of lncRNAs in HIV-1-infected primary monocyte-derived macrophages (MDMs) was performed, and 1,145 differentially expressed lncRNAs were identified. An lncRNA named HIV-1-enhanced lncRNA (HEAL) is upregulated by HIV-1 infection and promotes HIV replication in T cells and macrophages. HEAL forms a complex with the RNA-binding protein FUS to enhance transcriptional coactivator p300 recruitment to the HIV promoter. Furthermore, HEAL knockdown and knockout prevent HIV-1 recrudescence in T cells and microglia upon cessation of azidothymidine treatment, suggesting HEAL as a potential therapeutic target to cure HIV-1/AIDS.
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Epigênese Genética , Regulação Viral da Expressão Gênica/fisiologia , Infecções por HIV/fisiopatologia , HIV-1/fisiologia , Regiões Promotoras Genéticas/fisiologia , RNA Longo não Codificante/fisiologia , Replicação Viral/fisiologia , HumanosRESUMO
DHX9/RNA helicase A (RHA) is a host RNA helicase that participates in many critical steps of the HIV-1 life cycle. It co-assembles with the viral RNA genome into the capsid core. Virions deficient in RHA are less infectious as a result of reduced reverse transcription efficiency, demonstrating that the virion-associated RHA promotes reverse transcription before the virion gains access to the new host's RHA. Here, we quantified reverse-transcription intermediates in HIV-1-infected T cells to clarify the mechanism by which RHA enhances HIV-1 reverse transcription efficiency. Consistently, purified recombinant human RHA promoted reverse transcription efficiency under in vitro conditions that mimic the early reverse transcription steps prior to capsid core uncoating. We did not observe RHA-mediated structural remodeling of the tRNALys3-viral RNA-annealed complex. RHA did not enhance the DNA synthesis rate until incorporation of the first few nucleotides, suggesting that RHA participates primarily in the elongation phase of reverse transcription. Pre-steady-state and steady-state kinetic studies revealed that RHA has little impact on the kinetics of single-nucleotide incorporation. Primer extension assays performed in the presence of trap dsDNA disclosed that RHA enhances the processivity of HIV-1 reverse transcriptase (RT). The biochemical assays used here effectively reflected and explained the low RT activity in HIV-1 virions produced from RHA-depleted cells. Moreover, RT activity in our assays indicated that RHA in HIV-1 virions is required for the efficient catalysis of (-)cDNA synthesis during viral infection before capsid uncoating. Our study identifies RHA as a processivity factor of HIV-1 RT.
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RNA Helicases DEAD-box/fisiologia , Transcriptase Reversa do HIV/metabolismo , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno , Proteínas de Neoplasias/fisiologia , RNA/metabolismo , Vírion/fisiologia , Células HEK293 , HIV-1/genética , Humanos , Cinética , Transcrição ReversaRESUMO
The HIV-1 accessory protein Vif, which counteracts the antiviral action of the DNA-editing cytidine deaminase APOBEC3G (A3G), is an attractive and yet unexploited therapeutic target. Vif reduces the virion incorporation of A3G by targeting the restriction factor for proteasomal degradation in the virus-producing cell. Compounds that inhibit Vif-mediated degradation of A3G in cells targeted by HIV-1 would represent a novel antiviral therapeutic. We previously described small molecules with activity consistent with Vif antagonism. In this study, we derived inhibitor escape HIV-1 variants to characterize the mechanism by which these novel agents inhibit virus replication. Here we show that resistance to these agents is dependent on an amino acid substitution in Vif (V142I) and on a point mutation that likely upregulates transcription by modifying the lymphocyte enhancing factor 1 (LEF-1) binding site. Molecular modeling demonstrated a docking site in the Vif-Elongin C complex that is disrupted by these inhibitors. This docking site is lost when Vif acquires the V142I mutation that leads to inhibitor resistance. Competitive fitness experiments indicated that the V142I Vif and LEF-1 binding site mutations created a virus that is better adapted to growing in the presence of A3G than the wild-type virus.IMPORTANCE Although antiretroviral therapy can suppress HIV-1 replication effectively, virus reservoirs persist in infected individuals and virus replication rapidly rebounds if therapy is interrupted. Currently, there is a need for therapeutic approaches that eliminate, reduce, or control persistent viral reservoirs if a cure is to be realized. This work focuses on the preclinical development of novel, small-molecule inhibitors of the HIV-1 Vif protein. Vif inhibitors represent a new class of antiretroviral drugs that may expand treatment options to more effectively suppress virus replication or to drive HIV-1 reservoirs to a nonfunctional state by harnessing the activity of the DNA-editing cytidine deaminase A3G, a potent, intrinsic restriction factor expressed in macrophage and CD4+ T cells. In this study, we derived inhibitor escape variants to characterize the mechanism by which these novel agents inhibit virus replication and to provide evidence for target validation.
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Antivirais/farmacologia , Farmacorresistência Viral , HIV-1/efeitos dos fármacos , Mutação de Sentido Incorreto , Replicação Viral/efeitos dos fármacos , Produtos do Gene vif do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Desaminase APOBEC-3G/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Linhagem Celular , HIV-1/genética , Humanos , Fator 1 de Ligação ao Facilitador Linfoide/metabolismo , Simulação de Acoplamento Molecular , Mutação Puntual , Produtos do Gene vif do Vírus da Imunodeficiência Humana/química , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genéticaRESUMO
Precise stoichiometry of genome-length transcripts and alternatively spliced mRNAs is a hallmark of retroviruses. We discovered short, guanosine and adenosine sequence motifs in the 5'untranslated region of several retroviruses and ascertained the reasons for their conservation using a representative lentivirus and genetically simpler retrovirus. We conducted site-directed mutagenesis of the GA-motifs in HIV molecular clones and observed steep replication delays in T-cells. Quantitative RNA analyses demonstrate the GA-motifs are necessary to retain unspliced viral transcripts from alternative splicing. Mutagenesis of the GA-motifs in a C-type retrovirus validate the similar downregulation of unspliced transcripts and virion structural protein. The evidence from cell-based co-precipitation studies shows the GA-motifs in the 5'untranslated region confer binding by SFPQ/PSF, a protein co-regulated with T-cell activation. Diminished SFPQ/PSF or mutation of either GA-motif attenuates the replication of HIV. The interaction of SFPQ/PSF with both GA-motifs is crucial for maintaining the stoichiometry of the viral transcripts and does not affect packaging of HIV RNA. Our results demonstrate the conserved GA-motifs direct the fate of retrovirus RNA. These findings have exposed an RNA-based molecular target to attenuate retrovirus replication.
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Sequência Conservada/genética , HIV-1/genética , Motivos de Nucleotídeos/genética , RNA Viral/genética , Retroviridae/genética , Regiões 5' não Traduzidas/genética , Adenosina/genética , Sequência de Bases , Guanosina/genética , HIV-1/metabolismo , Humanos , Mutagênese Sítio-Dirigida , Fator de Processamento Associado a PTB/metabolismo , Ligação Proteica , Splicing de RNA , RNA Viral/metabolismo , Retroviridae/metabolismo , Vírion/genética , Replicação Viral/genéticaRESUMO
Ribonucleoprotein particles direct the biogenesis and post-transcriptional regulation of all mRNAs through distinct combinations of RNA binding proteins. They are composed of position-dependent, cis-acting RNA elements and unique combinations of RNA binding proteins. Defining the composition of a specific RNP is essential to achieving a fundamental understanding of gene regulation. The isolation of a select RNP is akin to finding a needle in a haystack. Here, we demonstrate an approach to isolate RNPs associated at the 5' untranslated region of a select mRNA in asynchronous, transfected cells. This cognate RNP has been demonstrated to be necessary for the translation of select viruses and cellular stress-response genes. The demonstrated RNA-protein co-precipitation protocol is suitable for the downstream analysis of protein components through proteomic analyses, immunoblots, or suitable biochemical identification assays. This experimental protocol demonstrates that DHX9/RNA helicase A is enriched at the 5' terminus of cognate retroviral RNA and provides preliminary information for the identification of its association with cell stress-associated huR and junD cognate mRNAs.
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Oligonucleotídeos , Ribonucleoproteínas/isolamento & purificação , Regulação da Expressão Gênica , Humanos , Proteômica , RNA Mensageiro/genéticaRESUMO
RNA interference is a potent and precise reverse genetic approach to carryout large-scale functional genomic studies in a given organism. During the past decade, RNAi has also emerged as an important investigative tool to understand the process of viral pathogenesis. Our laboratory has successfully generated transgenic reporter and RNAi sensor line of Spodoptera frugiperda (Sf21) cells and developed a reversal of silencing assay via siRNA or shRNA guided screening to investigate RNAi factors or viral pathogenic factors with extraordinary fidelity. Here we describe empirical approaches and conceptual understanding to execute successful RNAi screening in Spodoptera frugiperda 21-cell line.
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Ensaios de Triagem em Larga Escala/métodos , Interferência de RNA , Spodoptera/genética , Animais , Animais Geneticamente Modificados , Baculoviridae/genética , Linhagem Celular , Genes Reporter , Genoma de Inseto , Proteínas de Fluorescência Verde/genética , Ensaios de Triagem em Larga Escala/instrumentação , RNA Interferente Pequeno , Reprodutibilidade dos TestesRESUMO
RN-18 based viral infectivity factor (Vif), Vif antagonists reduce viral infectivity by rescuing APOBEC3G (A3G) expression and enhancing A3G-dependent Vif degradation. Replacement of amide functionality in RN-18 (IC50 = 6 µM) by isosteric heterocycles resulted in the discovery of a 1,2,3-trizole, 1d (IC50 = 1.2 µM). We identified several potent HIV-1 inhibitors from a 1d based library including 5ax (IC50 = 0.01 µM), 5bx (0.2 µM), 2ey (0.4 µM), 5ey (0.6 µM), and 6bx (0.2 µM).
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Fármacos Anti-HIV/farmacologia , Descoberta de Drogas , HIV-1/efeitos dos fármacos , Triazóis/farmacologia , Produtos do Gene vif do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Fármacos Anti-HIV/síntese química , Fármacos Anti-HIV/química , Relação Dose-Resposta a Droga , Testes de Sensibilidade Microbiana , Estrutura Molecular , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/químicaRESUMO
All decisions affecting the life cycle of human immunodeficiency virus (HIV-1) RNA are executed by ribonucleoprotein complexes (RNPs). HIV-1 RNA cycles through a progression of host RNPs composed of RNA-binding proteins regulating all stages of synthesis, processing, nuclear transport, translation, decay, and co-localization with assembling virions. RNA affinity chromatography is a versatile method to identify RNA-binding proteins to investigate the molecular basis of viral and cellular posttranscriptional control of gene expression. The bait is a HIV-1 RNA motif immobilized on a solid support, typically magnetic or Sepharose beads. The prey is pre-formed RNPs admixed in lysate from cells or concentrated virus particles. The methodology distinguishes high-affinity RNA-protein interactions from low-affinity complexes by increases in ionic strength during progressive elution cycles. Here, we describe RNA affinity chromatography of the 5' untranslated region of HIV-1, obtaining mixtures of high-affinity RNA binding proteins suitable for mass spectrometry and proteome identification.
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Cromatografia de Afinidade/métodos , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/metabolismo , RNA Viral/metabolismo , Ribonucleoproteínas/metabolismo , Proteínas Virais/metabolismo , Regulação da Expressão Gênica , HIV-1/química , Humanos , Imunoprecipitação/métodos , Linfócitos/metabolismo , Linfócitos/virologia , Proteômica/métodos , RNA Viral/análise , RNA Viral/isolamento & purificação , Ribonucleoproteínas/análise , Ribonucleoproteínas/isolamento & purificação , Proteínas Virais/análise , Proteínas Virais/isolamento & purificaçãoRESUMO
Essential host cofactors in retrovirus replication bind cis-acting sequences in the 5'untranslated region (UTR). Although host RBPs are crucial to all aspects of virus biology, elucidating their roles in replication remains a challenge to the field. Here RNA affinity-coupled-proteomics generated a comprehensive, unbiased inventory of human and avian RNA binding proteins (RBPs) co-isolating with 5'UTRs of HIV-1, spleen necrosis virus and Rous sarcoma virus. Applying stringent biochemical and statistical criteria, we identified 185 RBP; 122 were previously implicated in retrovirus biology and 63 are new to the 5'UTR proteome. RNA electrophoretic mobility assays investigated paralogs present in the common ancestor of vertebrates and one hnRNP was identified as a central node to the biological process-anchored networks of HIV-1, SNV, and RSV 5' UTR-proteomes. This comprehensive view of the host constituents of retroviral RNPs is broadly applicable to investigation of viral replication and antiviral response in both human and avian cell lineages.
Assuntos
Regiões 5' não Traduzidas , Infecções por HIV/metabolismo , HIV-1/metabolismo , Doenças das Aves Domésticas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Infecções por Retroviridae/metabolismo , Retroviridae/metabolismo , Sequência de Aminoácidos , Animais , Galinhas , Infecções por HIV/genética , Infecções por HIV/virologia , HIV-1/genética , Humanos , Dados de Sequência Molecular , Doenças das Aves Domésticas/genética , Doenças das Aves Domésticas/virologia , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/genética , Retroviridae/classificação , Retroviridae/genética , Infecções por Retroviridae/genética , Infecções por Retroviridae/virologiaRESUMO
We describe structure-activity relationship and optimization studies of RN-18, an HIV-1 Vif-APOBEC3G axis inhibitor. Targeted modifications of RN-18 ring-C, ring-B, ring-A, bridge A-B, and bridge B-C were performed to identify the crucial structural features, which generated new inhibitors with similar (4g and 4i) and improved (5, 8b, and 11) activities. Two potent water-soluble RN-18 analogues, 17 and 19, are also disclosed, and we describe the results of pharmacological studies with compound 19. The findings described here will be useful in the development of more potent Vif inhibitors and in the design of probes to identify the target protein of RN-18 and its analogues.
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Vegetative insecticidal protein (Vip3A) is synthesized as an extracellular insecticidal toxin by certain strains of Bacillus thuringiensis. Vip3A is active against several lepidopteran pests of crops. Polyphagous pest, Spodoptera frugiperda, and its cell line Sf21 are sensitive for lyses to Vip3A. Screening of cDNA library prepared from Sf21 cells through yeast two-hybrid system with Vip3A as bait identified ribosomal protein S2 as a toxicity-mediating interacting partner protein. The Vip3A-ribosomal-S2 protein interaction was validated by in vitro pulldown assays and by RNA interference-induced knockdown experiments. Knockdown of expression of S2 protein in Sf21 cells resulted in reduced toxicity of the Vip3A protein. These observations were further extended to adult fifth-instar larvae of Spodoptera litura. Knockdown of S2 expression by injecting corresponding double-stranded RNA resulted in reduced mortality of larvae to Vip3A toxin. Intracellular visualization of S2 protein and Vip3A through confocal microscopy revealed their interaction and localization in cytoplasm and surface of Sf21 cells.
Assuntos
Bacillus thuringiensis/fisiologia , Proteínas de Bactérias/fisiologia , Proteínas Ribossômicas/fisiologia , Spodoptera/fisiologia , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Northern Blotting , Linhagem Celular , Larva , Microscopia Confocal , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/metabolismo , Spodoptera/efeitos dos fármacosRESUMO
Viruses and siRNA/miRNA machinery of the host cell interact in diverse ways with the virus encoded RNAi suppressor proteins. These interactions have implications on the replication and pathogenicity of the virus and also on the immune response of the host. Suppressor protein B2 of insect Flock House Virus (FHVB2), has been shown to mediate RNAi suppression via N-terminal region by directly binding to dsRNA. We have previously shown that FHVB2 protein also interacts with host Dicer protein via its PAZ domain. In the present study, we performed systematic mutagenesis studies to map the FHVB2 regions involved in mediating suppression of RNAi. Progressive deletion of 17 amino acids from N- and C-terminii of FHVB2 resulted in cumulative decrease in RNAi suppression activity of FHVB2. The deletion of 17 amino acids from the C-terminus resulted in more reduction in RNAi suppression in comparison to the N-terminal deletions. Subsequently, we generated 17 successive point mutants of FHVB2 C-terminus and evaluated the RNAi suppression activity for each of the point mutants. Each point mutation resulted in a significant reduction in RNAi suppression activity of FHVB2. These results provide evidence for the role of C-terminal of FHVB2 in RNAi suppression.