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1.
Bioinformatics ; 40(10)2024 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-39412445

RESUMO

MOTIVATION: The Database for Annotation, Visualization, and Integrated Discovery (DAVID) is a web-based bioinformatics system for the functional interpretation of large lists of genes/proteins generated from high-throughput assays. It has been cited in 72 287 papers since its debut in 2003 as of 23 July 2024. The analysis is usually limited to the species of study. However, the knowledge of genes may be incomplete or unavailable for some species. Model organisms have been studied more extensively and analyzing gene lists in the context of these species can offer valuable insights, helping users better understand the genes and biological themes in their species of interest. RESULTS: We developed DAVID Ortholog for the conversion of gene lists between species. We utilized the ortholog data downloaded from Orthologous MAtrix (OMA) and Ensembl Compara as the base for the conversion. The OMA ortholog IDs and Ensembl gene IDs were converted to DAVID gene IDs and the pairing information of these IDs from these two sources was integrated into the DAVID Knowledgebase. DAVID Ortholog can convert the user's source gene list to an ortholog list of a desired species and the downstream DAVID analysis, in the context of that species, can be continued seamlessly, allowing users to further understand the biological meaning of their gene list based on the functional annotation found for the orthologs. AVAILABILITY AND IMPLEMENTATION: https://davidbioinformatics.nih.gov/ortholog.jsp.


Assuntos
Biologia Computacional , Bases de Dados Genéticas , Software , Biologia Computacional/métodos , Animais , Humanos , Anotação de Sequência Molecular/métodos
2.
Nucleic Acids Res ; 50(W1): W216-W221, 2022 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-35325185

RESUMO

DAVID is a popular bioinformatics resource system including a web server and web service for functional annotation and enrichment analyses of gene lists. It consists of a comprehensive knowledgebase and a set of functional analysis tools. Here, we report all updates made in 2021. The DAVID Gene system was rebuilt to gain coverage of more organisms, which increased the taxonomy coverage from 17 399 to 55 464. All existing annotation types have been updated, if available, based on the new DAVID Gene system. Compared with the last version, the number of gene-term records for most annotation types within the updated Knowledgebase have significantly increased. Moreover, we have incorporated new annotations in the Knowledgebase including small molecule-gene interactions from PubChem, drug-gene interactions from DrugBank, tissue expression information from the Human Protein Atlas, disease information from DisGeNET, and pathways from WikiPathways and PathBank. Eight of ten subgroups split from Uniprot Keyword annotation were assigned to specific types. Finally, we added a species parameter for uploading a list of gene symbols to minimize the ambiguity between species, which increases the efficiency of the list upload and eliminates confusion for users. These current updates have significantly expanded the Knowledgebase and enhanced the discovery power of DAVID.


Assuntos
Bases de Dados Genéticas , Software , Humanos , Biologia Computacional , Computadores , Bases de Conhecimento , Anotação de Sequência Molecular , Internet
3.
Int J Mol Sci ; 25(3)2024 Feb 02.
Artigo em Inglês | MEDLINE | ID: mdl-38339086

RESUMO

Acquired immunodeficiency syndrome (AIDS) is caused by human immunodeficiency virus (HIV). HIV protease, reverse transcriptase, and integrase are targets of current drugs to treat the disease. However, anti-viral drug-resistant strains have emerged quickly due to the high mutation rate of the virus, leading to the demand for the development of new drugs. One attractive target is Gag-Pol polyprotein, which plays a key role in the life cycle of HIV. Recently, we found that a combination of M50I and V151I mutations in HIV-1 integrase can suppress virus release and inhibit the initiation of Gag-Pol autoprocessing and maturation without interfering with the dimerization of Gag-Pol. Additional mutations in integrase or RNase H domain in reverse transcriptase can compensate for the defect. However, the molecular mechanism is unknown. There is no tertiary structure of the full-length HIV-1 Pol protein available for further study. Therefore, we developed a workflow to predict the tertiary structure of HIV-1 NL4.3 Pol polyprotein. The modeled structure has comparable quality compared with the recently published partial HIV-1 Pol structure (PDB ID: 7SJX). Our HIV-1 NL4.3 Pol dimer model is the first full-length Pol tertiary structure. It can provide a structural platform for studying the autoprocessing mechanism of HIV-1 Pol and for developing new potent drugs. Moreover, the workflow can be used to predict other large protein structures that cannot be resolved via conventional experimental methods.


Assuntos
Infecções por HIV , HIV-1 , Produtos do Gene pol do Vírus da Imunodeficiência Humana , Humanos , Produtos do Gene pol/genética , Produtos do Gene pol/metabolismo , Infecções por HIV/tratamento farmacológico , Protease de HIV/genética , Protease de HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , Poliproteínas/genética , DNA Polimerase Dirigida por RNA/metabolismo , Produtos do Gene pol do Vírus da Imunodeficiência Humana/química
4.
Bioinformatics ; 38(12): 3192-3199, 2022 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-35532087

RESUMO

MOTIVATION: The existence of quasispecies in the viral population causes difficulties for disease prevention and treatment. High-throughput sequencing provides opportunity to determine rare quasispecies and long sequencing reads covering full genomes reduce quasispecies determination to a clustering problem. The challenge is high similarity of quasispecies and high error rate of long sequencing reads. RESULTS: We developed QuasiSeq using a novel signature-based self-tuning clustering method, SigClust, to profile viral mixtures with high accuracy and sensitivity. QuasiSeq can correctly identify quasispecies even using low-quality sequencing reads (accuracy <80%) and produce quasispecies sequences with high accuracy (≥99.55%). Using high-quality circular consensus sequencing reads, QuasiSeq can produce quasispecies sequences with 100% accuracy. QuasiSeq has higher sensitivity and specificity than similar published software. Moreover, the requirement of the computational resource can be controlled by the size of the signature, which makes it possible to handle big sequencing data for rare quasispecies discovery. Furthermore, parallel computation is implemented to process the clusters and further reduce the runtime. Finally, we developed a web interface for the QuasiSeq workflow with simple parameter settings based on the quality of sequencing data, making it easy to use for users without advanced data science skills. AVAILABILITY AND IMPLEMENTATION: QuasiSeq is open source and freely available at https://github.com/LHRI-Bioinformatics/QuasiSeq. The current release (v1.0.0) is archived and available at https://zenodo.org/badge/latestdoi/340494542. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.


Assuntos
Algoritmos , Quase-Espécies , Análise de Sequência de DNA , Análise por Conglomerados , Sequenciamento de Nucleotídeos em Larga Escala , Software
5.
Proc Natl Acad Sci U S A ; 117(7): 3704-3710, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32029589

RESUMO

HIV-1 proviruses persist in the CD4+ T cells of HIV-infected individuals despite years of combination antiretroviral therapy (cART) with suppression of HIV-1 RNA levels <40 copies/mL. Greater than 95% of these proviruses detected in circulating peripheral blood mononuclear cells (PBMCs) are referred to as "defective" by virtue of having large internal deletions and lethal genetic mutations. As these defective proviruses are unable to encode intact and replication-competent viruses, they have long been thought of as biologically irrelevant "graveyard" of viruses with little significance to HIV-1 pathogenesis. Contrary to this notion, we have recently demonstrated that these defective proviruses are not silent, are capable of transcribing novel unspliced forms of HIV-RNA transcripts with competent open reading frames (ORFs), and can be found in the peripheral blood CD4+ T cells of patients at all stages of HIV-1 infection. In the present study, by an approach of combining serial dilutions of CD4+ T cells and T cell-cloning technologies, we are able to demonstrate that defective proviruses that persist in HIV-infected individuals during suppressive cART are translationally competent and produce the HIV-1 Gag and Nef proteins. The HIV-RNA transcripts expressed from these defective proviruses may trigger an element of innate immunity. Likewise, the viral proteins coded in the defective proviruses may form extracellular virus-like particles and may trigger immune responses. The persistent production of HIV-1 proteins in the absence of viral replication helps explain persistent immune activation despite HIV-1 levels below detection, and also presents new challenges to HIV-1 eradication.


Assuntos
Infecções por HIV/virologia , HIV-1/metabolismo , Provírus/metabolismo , Proteínas Virais/metabolismo , Linfócitos T CD4-Positivos/virologia , HIV-1/genética , Humanos , Leucócitos Mononucleares/virologia , Masculino , Pessoa de Meia-Idade , Provírus/genética , Proteínas Virais/genética
6.
J Virol ; 95(23): e0132321, 2021 11 09.
Artigo em Inglês | MEDLINE | ID: mdl-34523971

RESUMO

Recently, a genome-wide association study using plasma HIV RNA from antiretroviral therapy-naive patients reported that 14 naturally occurring nonsynonymous single-nucleotide polymorphisms (SNPs) in HIV derived from antiretrovirus drug-naive patients were associated with virus load (VL). Those SNPs were detected in reverse transcriptase, RNase H, integrase, envelope, and Nef. However, the impact of each mutation on viral fitness was not investigated. Here, we constructed a series of HIV variants encoding each SNP and examined their replicative abilities. An HIV variant containing a Met-to-Ile change at codon 50 in integrase [HIV(IN:M50I)] was found as an impaired virus. Despite the mutation being in integrase, the virus release was significantly suppressed (P < 0.001). Transmission electron microscopy analysis revealed that abnormal bud accumulation on the plasma membrane and the released virus particles retained immature forms. Western blot analysis demonstrated a defect in autoprocessing of GagPol and Gag polyproteins' autoprocessing in the HIV(IN:M50I) particles, although Förster resonance energy transfer (FRET) assay displayed that GagPol containing IN:M50I forms a homodimer with a similar efficiency with GagPol (wild type). The impaired maturation and replication were rescued by two other VL-associated SNPs, Ser-to-Asn change at codon 17 of integrase and Asn-to-Ser change at codon 79 of RNase H. These data demonstrate that Gag and GagPol assembly, virus release, and autoprocessing are regulated by not only integrase but also RNase H. IMPORTANCE Nascent HIV-1 is a noninfectious viral particle. Cleaving Gag and GagPol polyproteins in the particle by mature HIV protease (PR), the nascent virus becomes an infectious virus. PR is initially translated as an inactive embedded enzyme in a GagPol polyprotein. The embedded PR in homodimerized GagPol polyproteins catalyzes a proteolytic reaction to release the mature PR. This excision step by self-cleavage is called autoprocessing. Here, during the evaluation of the roles of naturally emerging nonsynonymous SNPs in HIV RNA, we found that autoprocessing is inhibited by Met-to-Ile change at codon 50 in integrase GagPol. Other coexisting SNPs, Ser-to-Asn change at codon 17 in integrase or Asn-to-Ser mutation at codon 79 in RNase H, recovered this defect, suggesting that autoprocessing is regulated by not only integrase but also RNase H in GagPol polyprotein.


Assuntos
Integrase de HIV/metabolismo , HIV-1/fisiologia , Ribonuclease H/metabolismo , Liberação de Vírus/fisiologia , Antirretrovirais/farmacologia , Produtos do Gene gag/genética , Células HEK293 , Infecções por HIV , Integrase de HIV/genética , HIV-1/genética , Humanos , Mutação , Polimorfismo de Nucleotídeo Único , Proteólise , Ribonuclease H/genética , Vírion/metabolismo , Replicação Viral
7.
Immunology ; 163(3): 323-337, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-33548066

RESUMO

We have previously identified that human Ku70, a nuclear protein, serves as a cytosolic DNA sensor. Upon transfection with DNA or infection with DNA virus, Ku70 translocates from the nucleus into the cytoplasm and then predominately induces interferon lambda1 (IFN-λ1) rather than IFN-alpha or IFN-beta, through a STING-dependent signalling pathway. However, a detailed mechanism for Ku70 cytoplasmic translocation and its correlation with IFN-λ1 induction have not been fully elucidated. Here, we observed that cytoplasmic translocation of Ku70 only occurred in DNA-triggered IFN-λ1-inducible cells. Additionally, infection by Herpes simplex virus type-1 (HSV-1), a DNA virus, induces cytoplasmic translocation of Ku70 and IFN-λ1 induction in a strain-dependent manner: the translocation and IFN-λ1 induction were detected upon infection by HSV-1 McKrae, but not MacIntyre, strain. A kinetic analysis indicated that cytoplasmic translocation of Ku70 was initiated right after DNA transfection and was peaked at 6 hr after DNA stimulation. Furthermore, treatment with leptomycin B, a nuclear export inhibitor, inhibited both Ku70 translocation and IFN-λ1 induction, suggesting that Ku70 translocation is an essential and early event for its cytosolic DNA sensing. We further confirmed that enhancing the acetylation status of the cells promotes Ku70's cytoplasmic accumulation, and therefore increases DNA-mediated IFN-λ1 induction. These findings provide insights into the molecular mechanism by which the versatile sensor detects pathogenic DNA in a localization-dependent manner.


Assuntos
Citoplasma/metabolismo , Herpes Simples/imunologia , Herpesvirus Humano 1/fisiologia , Interferons/metabolismo , Autoantígeno Ku/metabolismo , Acetilação , Antibióticos Antineoplásicos/farmacologia , DNA Viral/genética , DNA Viral/imunologia , Ácidos Graxos Insaturados/farmacologia , Células HEK293 , Humanos , Interferons/genética , Espaço Intracelular/genética , Espaço Intracelular/imunologia , Transporte Proteico , Especificidade da Espécie , Regulação para Cima
9.
Int J Mol Sci ; 22(3)2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33525571

RESUMO

Interleukin-27 (IL-27) is a pleiotropic cytokine that influences the innate and adaptive immune systems. It inhibits viral infection and regulates the expression of microRNAs (miRNAs). We recently reported that macrophages differentiated from human primary monocytes in the presence of IL-27 and human AB serum resisted human immunodeficiency virus (HIV) infection and showed significant autophagy induction. In the current study, the miRNA profiles in these cells were investigated, especially focusing on the identification of novel miRNAs regulated by IL-27-treatment. The miRNA sequencing analysis detected 38 novel miRNAs. Real-time reverse transcription polymerase chain reaction (RT-PCR) analysis confirmed that IL-27 differentially regulated the expression of 16 of the 38 miRNAs. Overexpression of the synthesized miRNA mimics by transfection revealed that miRAB40 had potent HIV-inhibiting and autophagy-inducing properties. B18R, an interferon (IFN)-neutralization protein, partially suppressed both activities, indicating that the two functions were induced via IFN-dependent and -independent pathways. Although the target mRNA(s) of miRAB40 involving in the induction of both functions was unable to identify in this study, the discovery of miRAB40, a potential HIV-inhibiting and autophagy inducing miRNA, may provide novel insights into the miRNA (small none-coding RNA)-mediated regulation of HIV inhibition and autophagy induction as an innate immune response.


Assuntos
Perfilação da Expressão Gênica/métodos , HIV-1/fisiologia , Interleucina-27/farmacologia , Macrófagos/citologia , MicroRNAs/genética , Autofagia , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Humanos , Interferons/metabolismo , Macrófagos/química , Macrófagos/virologia , MicroRNAs/farmacologia , Análise de Sequência de RNA , Soro/química , Replicação Viral
10.
Int J Mol Sci ; 20(24)2019 Dec 09.
Artigo em Inglês | MEDLINE | ID: mdl-31835347

RESUMO

Macrophages play an essential role in the immune system. Recent studies have shown that long non-coding RNAs (lncRNAs) can regulate genes encoding products involved in the immune response. Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines with broad anti-viral effects that inhibits human immunodeficiency virus (HIV) type-1 and herpes simplex virus (HSV). However, little is known about the role of lncRNAs in macrophages affected by IL-27. Therefore, we investigated the expression profiles of mRNA and lncRNA in human monocyte-derived macrophages (MDMs) regulated by IL-27. Monocytes were differentiated in the presence of macrophage-colony stimulatory factor (M-CSF)- or human AB serum with or without IL-27, and these cells were the subject for the profile analysis using RNA-Seq. We identified 146 lncRNAs (including 88 novel ones) and 434 coding genes were differentially regulated by IL-27 in both M-CSF- and AB serum-induced macrophages. Using weighted gene co-expression network analysis, we obtained four modules. The immune system, cell cycle, and regulation of complement cascade pathways were enriched in different modules. The network of mRNAs and lncRNAs in the pathways suggest that lncRNAs might regulate immune activity in macrophages. This study provides potential insight into the roles of lncRNA in macrophages regulated by IL-27.


Assuntos
Regulação da Expressão Gênica/imunologia , Interleucinas/imunologia , Macrófagos/imunologia , RNA Longo não Codificante/imunologia , RNA Mensageiro/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interleucinas/farmacologia , Macrófagos/citologia
11.
Int J Mol Sci ; 18(5)2017 Apr 28.
Artigo em Inglês | MEDLINE | ID: mdl-28452924

RESUMO

MicroRNAs (miRNAs) regulate gene expression and thereby influence cell fate and function. Recent studies suggest that an abundant class of miRNAs play important roles in immune cells, such as T cells, natural killer (NK) cells, B cells, and dendritic cells (DCs). Interleukin (IL)-27 is a member of the IL-12 family of cytokines with broad anti-viral effects. It is a potent inhibitor of HIV-1 infection in CD4+ T cells and macrophages, as well as monocyte-derived immature dendritic cells (iDCs). This pilot study compared miRNA profiles between iDCs and IL-27-treated iDCs (27DCs) using deep sequencing methods and identified 46 known miRNAs that were significantly differentially expressed in 27DCs: 36 were upregulated and 10 downregulated by IL-27. Many of the potential target genes of these miRNAs are involved in IL-27 associated pathways, such as JAK/STAT, MAPKs, and PI3K and several were also previously reported to be involved in the regulation of human DC function. This study found that these miRNAs also potentially target several viral genomes and therefore may have antiviral effects. Four of these differential miRNAs (miR-99a-5p, miR-222-3p, miR-138-5p, and miR-125b-5p) were validated using quantitative reverse transcription PCR (RT-qPCR). Twenty-two novel miRNAs were discovered from deep sequencing and confirmed using RT-qPCR. This study furthers the understanding of the role of IL-27 in immunity and lays a foundation for future characterization of the role of specific miRNAs in DCs.


Assuntos
Regulação para Baixo/efeitos dos fármacos , Estudo de Associação Genômica Ampla , Interleucina-27/farmacologia , MicroRNAs/metabolismo , Regulação para Cima/efeitos dos fármacos , Células Dendríticas/citologia , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Receptores de Lipopolissacarídeos/metabolismo , MicroRNAs/genética , Monócitos/citologia , Projetos Piloto , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de RNA
12.
BMC Immunol ; 17: 6, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26979060

RESUMO

BACKGROUND: Cryopreservation of peripheral blood mononuclear cells (PBMCs) is a common and essential practice in conducting research. There are different reports in the literature as to whether cryopreserved PBMCs need to only be stored ≤ -150 °C or can be stored for a specified time at -80 °C. Therefore, we performed gene expression analysis on cryopreserved PBMCs stored at both temperatures for 14 months and PBMCs that underwent temperature cycling 104 times between these 2 storage temperatures. Real-time RT-PCR was performed to confirm the involvement of specific genes associated with identified cellular pathways. All cryopreserved/stored samples were compared to freshly isolated PBMCs and between storage conditions. RESULTS: We identified a total of 1,367 genes whose expression after 14 months of storage was affected >3 fold in PBMCs following isolation, cryopreservation and thawing as compared to freshly isolated PBMC aliquots that did not undergo cryopreservation. Sixty-six of these genes were shared among two or more major stress-related cellular pathways (stress responses, immune activation and cell death). Thirteen genes involved in these pathways were tested by real-time RT-PCR and the results agreed with the corresponding microarray data. There was no significant change on the gene expression if the PBMCs experienced brief but repetitive temperature cycling as compared to those that were constantly kept ≤ -150 °C. However, there were 18 genes identified to be different when PBMCs were stored at -80 °C but did not change when stored < -150 °C. A correlation was also found between the expressions of 2'-5'- oligoadenylate synthetase (OAS2), a known interferon stimulated gene (IFSG), and poor PBMC recovery post-thaw. PBMC recovery and viability were better when the cells were stored ≤ -150 °C as compared to -80 °C. CONCLUSIONS: Not only is the viability and recovery of PBMCs affected during cryopreservation but also their gene expression pattern, as compared to freshly isolated PBMCs. Different storage temperature of PBMCs can activate or suppress different genes, but the cycling between -80 °C and -150 °C did not produce significant alterations in gene expression when compared to PBMCs stored ≤ -150 °C. Further analysis by gene expression of various PBMC processing and cryopreservation procedures is currently underway, as is identifying possible molecular mechanisms.


Assuntos
Criopreservação , Regulação da Expressão Gênica , Leucócitos Mononucleares/metabolismo , Temperatura , Morte Celular/genética , Sobrevivência Celular/genética , Perfilação da Expressão Gênica , Humanos , Interferons/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Estresse Fisiológico/genética , Transcriptoma/genética
13.
Nucleic Acids Res ; 42(1): 583-98, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24049081

RESUMO

In addition to silencing specific genes, small interfering RNA (siRNA) transfection is also associated with the non-specific induction of inflammatory cytokines and type I interferon. Those so-called "off-target" effects have considerable implications for the interpretation of in vitro studies and clinical application of siRNA. The present study attempted to develop a better understanding of the mechanism involved in these off target effects. Synthesized siRNA significantly enhances DNA-mediated interferon lambda-1 response (IFN-λ1/IL-29), a newly characterized antiviral interferon in non-immune or primary immune cells. This enhancement was most pronounced by double-stranded siRNA with at least a 2-nucleotide overhang at one 3' terminus in a dose-dependent manner, while the presence of DNA was indispensable. A pull-down assay using biotinylated siRNA- or DNA-conjugated beads indicated that retinoic acid-inducible gene I (RIG-I) and interferon gamma-inducible protein 16 (IFI16) were involved in the sensing of siRNA and DNA, respectively. Co-immunoprecipitation analysis further revealed that RIG-I and IFI16 formed a complex via siRNA, and the dissociation of IFI16 from this complex in the presence of DNA activated the downstream STING-TBK1-IRF3 (stimulator of interferon genes - tank-binding kinase 1 - interferon regulatory factor 3) pathway, shedding light on a new physiological signalling pathway to activate innate immunity. Collectively, these findings may provide rational information for siRNA-induced innate immunity, with important implications for developing siRNA-based reagents to control human diseases.


Assuntos
RNA Helicases DEAD-box/metabolismo , DNA/metabolismo , Interleucinas/biossíntese , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Linhagem Celular , Células Cultivadas , Proteína DEAD-box 58 , Células HeLa , Herpesvirus Humano 1/fisiologia , Humanos , Fator Regulador 3 de Interferon/metabolismo , Interferons , Proteínas Serina-Treonina Quinases/metabolismo , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/química , Receptores Imunológicos
14.
Molecules ; 20(3): 4623-34, 2015 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-25774493

RESUMO

We have built a new isonucleoside derivative on a 2,6-dioxobicyclo[3.2.0]heptane skeleton as a potential anti-HIV agent. To synthesize the target compound, an acetal-protected dihydroxyacetone was first converted to a 2,3-epoxy-tetrahydrofuran derivative. Introduction of an azide group, followed by the formation of an oxetane ring, gave a pseudosugar derivative with a 2,6-dioxobicyclo[3.2.0]heptane skeleton. The desired isonucleoside was obtained by constructing a purine base moiety on the scaffold, followed by amination.


Assuntos
Fármacos Anti-HIV/síntese química , Heptanos/química , Nucleosídeos/síntese química , Aminação , Fármacos Anti-HIV/farmacologia , Humanos , Estrutura Molecular , Nucleosídeos/farmacologia
15.
J Biol Chem ; 288(24): 17812-22, 2013 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-23640893

RESUMO

IL-2 has been used in culture of primary T cells to maintain cell proliferation. We have previously reported that IL-27 inhibits HIV-1 replication in primary T cells in the presence of IL-2. To gain a better understanding of the mechanisms involved in this inhibitory effect, we attempted to investigate in detail the effects of IL-27 and IL-2 using several cell lines. Unexpectedly, IL-27 did not inhibit HIV-1 in T cell lines, whereas IL-2 inhibited HIV-1 replication in the human T cell lymphotrophic virus (HTLV)-1-transformed T cell lines, MT-2, MT-4, SLB-1, and ATL-2. No effects were seen in HTLV-1-negative cell lines. Utilizing MT-2 cells, we demonstrated that IL-2 treatment inhibited HIV-1 syncytia-inducing ability and dose-dependently decreased supernatant p24 antigen levels by >90%. Using real time PCR and Western blot analysis, we observed that IL-2 treatment induced the host restriction factor, APOBEC3G with accumulation into the lower molecular mass active form as characterized by FPLC. Further analysis revealed that the virus recovered from IL-2-treated MT-2 cells had impaired replication competency. This was found to be due to incorporation of APOBEC3G into the virion despite the presence of Vif. These findings demonstrate a novel role for IL-2 in regulating production of infectious HIV-1 virions in HTLV-1-infected cells through the induction of APOBEC3G.


Assuntos
Citidina Desaminase/genética , HIV-1/fisiologia , Interleucina-2/fisiologia , Linfócitos T/virologia , Replicação Viral , Desaminase APOBEC-3G , Antígenos CD4/metabolismo , Linhagem Celular , Citidina Desaminase/metabolismo , Indução Enzimática , Técnicas de Silenciamento de Genes , Humanos , Mutação , RNA Interferente Pequeno/genética , Receptores CXCR4/metabolismo , Transcrição Reversa , Análise de Sequência de DNA , Linfócitos T/metabolismo , Ativação Transcricional
16.
J Microbiol Methods ; 224: 106990, 2024 09.
Artigo em Inglês | MEDLINE | ID: mdl-39004285

RESUMO

The pUC-derived plasmid yield from E. coli using polypropylene tubes (PP) was compared among round and conical tubes. The yield from cells grown in a cheaper conical-PP with flat-bottom was 1.5-fold higher (p < 0.001) than other PP. The use of the conical-PP can save research budgets in the current inflationary environment.


Assuntos
Escherichia coli , Plasmídeos , Polipropilenos , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Polipropilenos/química , Plasmídeos/genética , Vetores Genéticos
17.
Biochem Biophys Res Commun ; 434(2): 228-34, 2013 May 03.
Artigo em Inglês | MEDLINE | ID: mdl-23535375

RESUMO

Interleukin-27 (IL-27) is a pleiotropic cytokine which plays important and diverse roles in the immune system. We have previously demonstrated that IL-27 induces potent anti-viral effects against HIV-1, HIV-2, SIV, HSV-2, KSHV and influenza viruses in macrophages. This induction occurred in an interferon (IFN) independent manner and involved down regulation of SPTBN1. MicroRNAs (miRNAs) are critical regulators of mRNA translation and turnover. There have been reports that some miRNAs inhibit viral replication. In this study, we hypothesized that IL-27 could induce the expression of novel miRNAs in macrophages which may have functional relevance in terms of anti-viral activity and primary monocytes were differentiated into macrophages using either M-CSF (M-Mac) or a combination of M-CSF and IL-27 (I-Mac) for seven days. Following this, total RNA was extracted from these cells and deep sequencing was performed, in parallel with gene expression microarrays. Using the novel miRNA discovery software, miRDeep, seven novel miRNAs were discovered in these macrophages. Four of which were preferentially expressed in I-Mac (miR-SX1, -SX2, -SX3 and -SX6) whilst three were detected in both M-Mac and I-Mac (miR-SX4, -SX5 and -SX7). The expression of six of the seven novel miRNAs was highly correlated with qRT-PCR using specific primer/probes designed for the novel miRNAs. Gene expression microarray further demonstrated that a number of genes were potentially targeted by these differentially expressed novel miRNAs. Finally, several of these novel miRNAs (miR-SX1, -SX4, -SX5, -SX6 and -SX7) were shown to target the open reading frames of a number of viruses (including HSV-1, HSV-2 and HHV-8) which may partially explain the anti-viral properties observed.


Assuntos
Antivirais/farmacologia , Interleucinas/farmacologia , Macrófagos/efeitos dos fármacos , MicroRNAs/metabolismo , Antivirais/isolamento & purificação , Antivirais/metabolismo , Infecções por Herpesviridae/tratamento farmacológico , Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 1/efeitos dos fármacos , Herpesvirus Humano 1/genética , Herpesvirus Humano 2/efeitos dos fármacos , Herpesvirus Humano 2/genética , Herpesvirus Humano 8/efeitos dos fármacos , Herpesvirus Humano 8/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Macrófagos/citologia , Macrófagos/virologia , MicroRNAs/genética , MicroRNAs/isolamento & purificação , Monócitos/citologia , Conformação de Ácido Nucleico , Análise de Sequência com Séries de Oligonucleotídeos , Fases de Leitura Aberta , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real
18.
J Immunol ; 186(8): 4541-5, 2011 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-21398614

RESUMO

Cytosolic foreign DNA is detected by pattern recognition receptors and mainly induces type I IFN production. We found that transfection of different types of DNA into various untreated cells induces type III IFN (IFN-λ1) rather than type I IFN, indicating the presence of uncharacterized DNA sensor(s). A pull-down assay using cytosolic proteins identified that Ku70 and Ku80 are the DNA-binding proteins. The knockdown studies and the reporter assay revealed that Ku70 is a novel DNA sensor inducing the IFN-lambda1 activation. The functional analysis of IFNL1 promoter revealed that positive-regulatory domain I and IFN-stimulated response element sites are predominantly involved in the DNA-mediated IFNL1 activation. A pull-down assay using nuclear proteins demonstrated that the IFN-λ1 induction is associated with the activation of IFN regulatory factor-1 and -7. Thus, to our knowledge, we show for the first time that Ku70 mediates type III IFN induction by DNA.


Assuntos
Antígenos Nucleares/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Interferon Tipo I/metabolismo , Interleucinas/metabolismo , Animais , Antígenos Nucleares/genética , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Citosol/metabolismo , DNA/genética , Proteínas de Ligação a DNA/genética , Feminino , Células HEK293 , Células HeLa , Humanos , Fator Regulador 1 de Interferon/metabolismo , Fator Regulador 7 de Interferon/metabolismo , Interferon Tipo I/genética , Interferons , Interleucinas/genética , Autoantígeno Ku , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Regiões Promotoras Genéticas/genética , Interferência de RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transfecção
19.
Methods Mol Biol ; 2599: 1-10, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36427138

RESUMO

DNA-protein interactions (DPIs) are critical to all living organisms, particularly in the regulation of gene expression, replication, packing, recombination, and DNA repair, as well as RNA transport and translation. Many laboratory techniques have been developed to study the complex interactions of proteins with DNA, such as chromatin immunoprecipitation (ChIP) assays, DNA electrophoretic mobility shift assay (EMSA), and oligonucleotide pull-down assays. Here we describe an effective approach to identify potential DNA-binding proteins: a pull-down assay using DNA-conjugated beads with a customized competition strategy, which conferred a more effective and efficient approach to determine the interaction between DNA and protein(s), therefore dramatically improving the progress to investigate novel DNA-binding proteins.


Assuntos
Bioensaio , DNA , DNA/genética , Ensaio de Desvio de Mobilidade Eletroforética , Oligonucleotídeos , Proteínas de Ligação a DNA/genética
20.
Viruses ; 15(11)2023 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-38005822

RESUMO

Herpes Simplex Virus type 1 (HSV-1) infects humans and causes a variety of clinical manifestations. Many HSV-1 genomes have been sequenced with high-throughput sequencing technologies and the annotation of these genome sequences heavily relies on the known genes in reference strains. Consequently, the accuracy of reference strain annotation is critical for future research and treatment of HSV-1 infection. In this study, we analyzed RNA-Seq data of HSV-1 from NCBI databases and discovered a novel intron in the overlapping coding sequence (CDS) of US10 and US11, and the 3' UTR of US12 in strain 17, a commonly used HSV-1 reference strain. To comprehensively understand the shared US10/US11/US12 intron structure, we used US11 as a representative and surveyed all US11 gene sequences from the NCBI nt/nr database. A total of 193 high-quality US11 sequences were obtained, of which 186 sequences have a domain of uninterrupted tandemly repeated RXP (Arg-X-Pro) in the C-terminus half of the protein. In total, 97 of the 186 sequences encode US11 protein with the same length of the mature US11 in strain 17:26 of them have the same structure of US11 and can be spliced as in strain 17; 71 of them have transcripts that are the same as mature US11 mRNA in strain 17. In total, 76 US11 gene sequences have either canonical or known noncanonical intron border sequences and may be spliced like strain 17 and obtain mature US11 CDS with the same length. If not spliced, they will have extra RXP repeats. A tandemly repeated RXP domain was proposed to be essential for US11 to bind with RNA and other host factors. US10 protein sequences from the same strains have also been studied. The results of this study show that even a frequently used reference organism may have errors in widely used databases. This study provides accurate annotation of the US10, US11, and US12 gene structure, which will build a more solid foundation to study expression regulation of the function of these genes.


Assuntos
Herpesvirus Humano 1 , Íntrons , Proteínas Virais , Humanos , Sequência de Bases , Herpes Simples , Herpesvirus Humano 1/fisiologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
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