RESUMO
Human interleukin 15 (IL-15) circulates in blood as a stable molecular complex with the soluble IL-15 receptor alpha (sIL-15Rα). This heterodimeric IL-15:sIL-15Rα complex (hetIL-15) shows therapeutic potential by promoting the growth, mobilization and activation of lymphocytes and is currently evaluated in clinical trials. Favorable pharmacokinetic properties are associated with the heterodimeric formation and the glycosylation of hetIL-15, which, however, remains largely uncharacterized. We report the site-specific N- and O-glycosylation of two clinically relevant large-scale preparations of HEK293-derived recombinant human hetIL-15. Intact IL-15 and sIL-15Rα and derived glycans and glycopeptides were separately profiled using multiple LC-MS/MS strategies. IL-15 Asn79 and sIL-15Rα Asn107 carried the same repertoire of biosynthetically-related N-glycans covering mostly α1-6-core-fucosylated and ß-GlcNAc-terminating complex-type structures. The two potential IL-15 N-glycosylation sites (Asn71 and Asn112) located at the IL-2 receptor interface were unoccupied. Mass analysis of intact IL-15 confirmed its N-glycosylation and suggested that Asn79-glycosylation partially prevents Asn77-deamidation. IL-15 contained no O-glycans, whereas sIL-15Rα was heavily O-glycosylated with partially sialylated core 1 and 2-type mono- to hexasaccharides on Thr2, Thr81, Thr86, Thr156, Ser158, and Ser160. The sialoglycans displayed α2-3- and α2-6-NeuAc-type sialylation. Non-human, potentially immunogenic glycoepitopes (e.g. N-glycolylneuraminic acid and α-galactosylation) were not displayed by hetIL-15. Highly reproducible glycosylation of IL-15 and sIL-15Rα of two batches of hetIL-15 demonstrated consistent manufacturing and purification. In conclusion, we document the heterogeneous and reproducible N- and O-glycosylation of large-scale preparations of the therapeutic candidate hetIL-15. Site-specific mapping of these molecular features is important to evaluate the consistent large-scale production and clinical efficacy of hetIL-15.
Assuntos
Interleucina-15/metabolismo , Processamento de Proteína Pós-Traducional , Receptores de Interleucina-15/metabolismo , Acetilglucosamina/análogos & derivados , Acetilglucosamina/química , Acetilglucosamina/metabolismo , Glicosilação , Células HEK293 , Humanos , Interleucina-15/química , Ligação Proteica , Receptores de Interleucina-15/química , Proteínas RecombinantesRESUMO
Interleukin-15 (IL-15) is a common γ-chain cytokine that has a significant role in the activation and proliferation of T and NK cells and holds great potential in fighting infection and cancer. We have previously shown that bioactive IL-15 in vivo comprises a complex of the IL-15 chain with the soluble or cell-associated IL-15 receptor alpha (IL-15Rα) chain, which together form the IL-15 heterodimer. We have generated DNA vectors expressing the heterodimeric IL-15 by optimizing mRNA expression and protein trafficking. Repeated administration of these DNA plasmids by intramuscular injection followed by in vivo electroporation in rhesus macaques resulted in sustained high levels of IL-15 in plasma, with no significant toxicity. Administration of DNAs expressing heterodimeric IL-15 also resulted in an increased frequency of NK and T cells undergoing proliferation in peripheral blood. Heterodimeric IL-15 led to preferential expansion of CD8(+)NK cells, all memory CD8(+) T-cell subsets and effector memory CD4(+) T cells. Expression of heterodimeric IL-15 by DNA delivery to the muscle is an efficient procedure to obtain high systemic levels of bioactive cytokine, without the toxicity linked to the high transient cytokine peak associated with protein injection.
Assuntos
Proliferação de Células , Interleucina-15/biossíntese , Células Matadoras Naturais/fisiologia , Linfócitos T/fisiologia , Animais , Células Cultivadas , Eletroporação , Expressão Gênica , Terapia Genética , Injeções Intramusculares , Interleucina-15/genética , Macaca mulatta , TransfecçãoRESUMO
The HIV-1 virion-associated accessory protein Vpr affects both viral replication and cellular transcription, proliferation, and differentiation. We report that Vpr enhances the activity of glucocorticoids in lymphoid and muscle-derived cell lines by interacting directly with the glucocorticoid receptor and general transcription factors, acting as a coactivator. Vpr contains the signature motif LXXLL also present in cellular nuclear receptor coactivators, such as steroid receptor coactivator 1 and p300/CREB-binding protein, which mediates their interaction with the glucocorticoid and other nuclear hormone receptors. A mutant Vpr molecule with disruption of this coactivator signature motif lost its ability to influence transcription of glucocorticoid-responsive genes and became a dominant-negative inhibitor of Vpr, possibly by retaining its general transcription factor-binding activities. The glucocorticoid coactivator activity of Vpr may contribute to increased tissue glucocorticoid sensitivity in the absence of hypercortisolism and to the pathogenesis of AIDS.
Assuntos
Produtos do Gene vpr/metabolismo , HIV-1/metabolismo , Receptores de Glucocorticoides/metabolismo , Ativação Transcricional/genética , Linhagem Celular , Dexametasona/farmacologia , Genes Reporter/genética , Glucocorticoides/metabolismo , Humanos , Fator de Transcrição TFIID , Fatores de Transcrição TFII/genética , Fatores de Transcrição TFII/imunologia , Transfecção/genética , Proteínas Virais/metabolismo , Produtos do Gene vpr do Vírus da Imunodeficiência HumanaRESUMO
A bioassay that is based on trans-activation has been developed for the detection and quantitation of the human immunodeficiency virus type 1 (HIV-1). Indicator cell lines were constructed that contain the HIV-1 long terminal repeat ligated to the chloramphenicol acetyltransferase (CAT) gene. Infection of these cells by HIV activates the expression of CAT protein. Isolates of HIV-1 with divergent nucleotide sequences activated the indicator cell lines to a similar extent, approximately 500- to 1000-fold. Human T cell lymphotropic viruses types 1 and 2, equine infectious anemia virus, and herpes simplex virus 1 did not activate the indicator cell lines. Isolates of simian immunodeficiency virus and human T cell lymphotropic virus type 4 activated these cells to a much lesser extent, which suggests that these viruses contain similar, but distinct, trans-activators. This assay can be used for the detection, quantitation, and typing of HIV and for studying the effect of drugs on the replication of HIV in different cellular backgrounds.
Assuntos
HIV/genética , Acetiltransferases/genética , Antivirais/farmacologia , Linhagem Celular , Cloranfenicol O-Acetiltransferase , DNA Recombinante , Regulação da Expressão Gênica , Genes Virais , HIV/análise , HIV/efeitos dos fármacos , Humanos , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Replicação Viral/efeitos dos fármacosRESUMO
Interferon inhibits expression of human immunodeficiency virus type-1 (HIV-1) through unknown mechanisms. A gene inducible by interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma) was isolated by screening of a human complementary DNA library for proteins binding to the Rev-responsive element (RRE) of HIV-1. The product of this gene, RBP9-27, was shown to bind RNA in vitro and to inhibit HIV-1 expression after transfection into human cells. RBP9-27 primarily inhibited Rev-dependent posttranscriptional steps of viral gene expression. Thus, RBP9-27 is a cellular factor that antagonizes Rev function. These results suggest an interferon-induced antiviral mechanism operating through the induction of RNA binding proteins such as RBP9-27. Elucidation of RBP9-27 function may lead to a better understanding of the mechanism of interferon action during HIV-1 infection.
Assuntos
Regulação Viral da Expressão Gênica , Genes rev , HIV-1/genética , Proteínas de Membrana , Proteínas de Ligação a RNA/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Genes env , Humanos , Interferons/farmacologia , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Sequências Reguladoras de Ácido NucleicoRESUMO
The human T-lymphotropic retrovirus HTLV-III/LAV encodes a trans-activator that increases viral gene expression. We expressed this trans-activator in animal cells and studied its structural and functional characteristics. The putative trans-activator protein was immunoprecipitated from overproducing stable cell lines and shown to migrate as a 14-kilodalton polypeptide on sodium dodecyl sulfate-polyacrylamide gels. S1 nuclease mapping experiments showed that the trans-activator increases the levels of steady-state messenger RNA transcribed from the viral long terminal repeat promoter. Sequences within the R region of the HTLV-III/LAV long terminal repeat are essential for trans-activation. Quantitations of messenger RNA and protein showed that the protein increase was greater than the messenger RNA increase in CV1 and HeLa cells, indicating that more than one mechanism was responsible for the trans-activation and that cell type-specific factors may determine the final level of trans-activation.
Assuntos
HIV/genética , Proteínas dos Retroviridae/metabolismo , Proteínas Virais/biossíntese , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Produtos do Gene rev , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , RNA Mensageiro/análise , Transfecção , Ativação Viral , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
Expression of the pX protein of human T-cell leukemia virus type I (HTLV-I) in animal cells demonstrates that this protein is a specific transcriptional activator of the long terminal repeats (LTR) of HTLV-I. Several other promoters are not affected by pX. No lymphocyte-specific factors are required for this activation. pX can be detected in the nucleus of transfected monkey kidney cells (line CV1) by indirect immunofluorescence. These results indicate that the pX protein is essential for the replication cycle of the virus and that it may be directly involved in the immortalization of human lymphocytes by HTLV-I.
Assuntos
Deltaretrovirus/genética , Fatores de Transcrição/genética , Proteínas Virais/genética , DNA Recombinante , RNA Polimerases Dirigidas por DNA/genética , Regulação da Expressão Gênica , Peptídeos/genética , Plasmídeos , Regiões Promotoras Genéticas , Sequências Repetitivas de Ácido Nucleico , Transcrição Gênica , Fatores de Crescimento TransformadoresRESUMO
We have used a panel of cDNA clones expressing wild-type and mutant human immunodeficiency virus type 1 (HIV-1) mRNAs to study translation of these mRNAs in eucaryotic cells. The tat open reading frame (ORF) has a strong signal for translation initiation, while rev and vpu ORFs have weaker signals. The expression of downstream ORFs is inhibited in mRNAs that contain the tat ORF as the first ORF. In contrast, downstream ORFs are expressed efficiently from mRNAs that have rev or vpu as the first ORF. All env mRNAs contain the upstream vpu ORF. Expression of HIV-1 Env protein requires a weak vpu AUG, which allows leaky scanning to occur, thereby allowing ribosomes access to the downstream env ORF. We concluded that HIV-1 mRNAs are translated by the scanning mechanism and that expression of more than one protein from each mRNA was caused by leaky scanning at the first AUG of the mRNA.
Assuntos
HIV-1/genética , Biossíntese de Proteínas , Sequência de Bases , DNA Viral , Regulação Viral da Expressão Gênica , Genes rev , Genes tat , Dados de Sequência Molecular , Mutação , Fases de Leitura Aberta , Testes de Precipitina , Splicing de RNA , RNA Mensageiro/metabolismo , RNA Viral/metabolismoRESUMO
Biochemical examination of the Rev-dependent expression of gag mRNAs produced from gag-Rev-responsive element (RRE) expression plasmids showed a large discrepancy between the level of cytoplasmic gag mRNA and the produced Gag protein. Significant levels of the mRNA produced in the absence of Rev were localized in the cytoplasm, while very low levels of Gag protein were produced. In the presence of Rev, the levels of mRNA increased by 4- to 16-fold, while the Gag protein production increased by 800-fold. These findings indicated that in addition to promoting nucleus-to-cytoplasm transport, Rev increased the utilization of cytoplasmic viral mRNA. Poly(A) selection and in vitro translation of cytoplasmic gag mRNA verified that the mRNA produced in the absence of Rev was functional. To analyze the translational defect in the absence of Rev, we examined the association of the cytoplasmic gag mRNA with ribosomes. gag mRNA produced in the absence of Rev was excluded from polysomes, while gag mRNA produced in the presence of Rev was associated with polysomes and produced Gag protein. These observations showed that the presence of Rev was required for efficient loading of gag mRNA onto polysomes. This effect required the presence of the RRE on the mRNA. Analysis of mRNAs produced from a rev-minus proviral clone confirmed that the presence of Rev promoted polysomal loading of both gag/pol and vpu/env mRNAs. The localization of gag mRNA was also examined by in situ hybridization. This analysis showed that in the presence of Rev, most of the gag mRNA was found in the cytoplasm, while in the absence of Rev, most of the gag mRNA was found in the nucleus and in the region surrounding the nucleus. These results suggest that a substantial fraction of the gag mRNA is retained in distinct cytoplasmic compartments in the absence and presence of Rev. These findings indicate that the presence of Rev is required along the entire mRNA transport and utilization pathway for the stabilization, correct localization, and efficient translation of RRE-containing mRNAs.
Assuntos
Produtos do Gene rev/metabolismo , Genes Fúngicos , HIV-1/genética , Biossíntese de Proteínas , Northern Blotting , Linhagem Celular , Clonagem Molecular , Citoplasma/metabolismo , Genes env , Genes gag , Genes pol , Genes vpu , HIV-1/metabolismo , Humanos , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , Polirribossomos/metabolismo , RNA Mensageiro/metabolismo , RNA Viral/metabolismo , Produtos do Gene rev do Vírus da Imunodeficiência HumanaRESUMO
Mutation of the von Hippel-Lindau tumor suppressor gene (vhl) causes the von Hippel-Lindau cancer syndrome as well as sporadic renal clear cell carcinoma. To pursue our study of the intracellular localization of VHL protein in relation to its function, we fused VHL to the green fluorescent protein (GFP) to produce the VHL-GFP fusion protein. Like VHL, VHL-GFP binds to elongins B and C and Cullin-2 and regulates target gene product levels, including levels of vascular endothelial growth factor and glucose transporter 1. VHL-GFP localizes predominantly to the cytoplasm, with some detectable nuclear signal. Inhibition of transcription by actinomycin D or 5,6-dichlorobenzimidazole riboside (DRB) causes VHL to be redistributed to the nucleus. A cellular fusion assay was used to demonstrate that inhibition of transcription induces a decrease in the nuclear export rate of VHL. The dependence of transcription for trafficking is lost with a deletion of exon 2, a region with a mutation causing a splice defect in the VHL gene in sporadic renal clear cell carcinoma. Addition of a strong nuclear export signal (NES) derived from the Rev protein results in complete nuclear exclusion and abrogates the redistribution of VHL-GFP-NES into the nucleus upon inhibition of transcription. Leptomycin B, which inhibits NES-mediated nuclear export, reverts the distribution of VHL-GFP-NES to that of VHL-GFP and restores sensitivity to actinomycin D and DRB. Uncoupling of VHL-GFP trafficking to transcription either by an exon 2 deletion or fusion to NES abolishes VHL function. We suggest that VHL function requires not only nuclear or cytoplasmic localization, but also exon 2-mediated transcription-dependent trafficking between these two cellular compartments.
Assuntos
Ligases , Proteínas/metabolismo , Proteínas Supressoras de Tumor , Ubiquitina-Proteína Ligases , Animais , Sequência de Bases , Transporte Biológico Ativo/efeitos dos fármacos , Células COS , Carcinoma de Células Renais/genética , Carcinoma de Células Renais/metabolismo , Fusão Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Primers do DNA/genética , Dactinomicina/farmacologia , Genes Supressores de Tumor , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Neoplasias Renais/genética , Neoplasias Renais/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Mutação , Proteínas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transcrição Gênica , Células Tumorais Cultivadas , Proteína Supressora de Tumor Von Hippel-Lindau , Doença de von Hippel-Lindau/genética , Doença de von Hippel-Lindau/metabolismoRESUMO
To increase cationic liposome-mediated intravenous DNA delivery extruded DOTAP:cholesterol liposomes were used to form complexes with DNA, resulting in enhanced expression of the chloramphenicol acetyltransferase gene in most tissues examined. The DNA:liposome ratio, and mild sonication, heating, and extrusion steps used for liposome preparation were crucial for improved systemic delivery. Size fractionation studies showed that maximal gene expression was produced by a homogeneous population of DNA:liposome complexes between 200 to 450 nm in size. Cryo-electron microscopy examination demonstrates that the DNA:liposome complexes have a novel morphology, and that the DNA is condensed on the interior of invaginated liposomes between two lipid bilayers. This structure could account for the high efficiency of gene delivery in vivo and for the broad tissue distribution of the DNA:liposome complexes. Ligands can be placed on the outside of this structure to provide for targeted gene delivery.
Assuntos
DNA/administração & dosagem , DNA/genética , Terapia Genética/métodos , Animais , Biotecnologia , Cloranfenicol O-Acetiltransferase/genética , DNA/metabolismo , Portadores de Fármacos , Expressão Gênica , Lipossomos , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Especificidade de Órgãos , Tamanho da PartículaRESUMO
The syndrome of familial or sporadic glucocorticoid resistance is characterized by hypercortisolism without the clinical stigmata of Cushing syndrome. This condition is usually caused by mutations of the human GR, a ligand-activated transcription factor that shuttles between the cytoplasm and the nucleus. A pathological human mutant receptor, in which Ile was replaced by Asn at position 559, had negligible ligand binding, was transcriptionally extremely weak, and exerted a transdominant negative effect on the transactivational activity of the wild-type GR, causing severe glucocorticoid resistance in the heterozygous state. To understand the mechanism of this mutant's trans-dominance, we constructed several N-terminal GR fusion chimeras to green fluorescent protein (GFP) and demonstrated that their transactivational activities were similar to those of the original proteins. The GFP-human (h) GRalphaI559N chimera was predominantly localized in the cytoplasm, and only high doses or prolonged glucocorticoid treatment triggered complete nuclear import that took 180 vs. 12 min for GFP-hGRalpha. Furthermore, hGRalphaI559N inhibited nuclear import of the wild-type GFP-hGRalpha, suggesting that its trans-dominant activity on the wild-type receptor is probably exerted at the process of nuclear translocation. As the ligand-binding domain (LBD) of the GR appears to play an important role in its nucleocytoplasmic shuttling, we also examined two additional GR-related fusion proteins. The natural hGR isoform beta (GFP-hGRbeta), containing a unique LBD, was transactivation-inactive, moderately trans-dominant, and localized instantaneously and predominantly in the nucleus; glucocorticoid addition did not change its localization. Similarly, GFP-hGR514, lacking the entire LBD, was instantaneously and predominantly localized in the nucleus regardless of presence of glucocorticoids. Using a cell fusion system we demonstrated that nuclear export of GFP-hGRalphaI559N (250 min) and GFP-hGRbeta (300 min) was drastically impaired compared with that of GFP-hGRalpha (50 min) and GFP-hGR514 (50 min), suggesting that an altered LBD may impede the exit of the GR from the nucleus. We conclude that the trans-dominant negative effect of the pathological mutant is exerted primarily at the translocation step, whereas that of the natural isoform beta is exerted at the level of transcription.
Assuntos
Núcleo Celular/fisiologia , Genes Dominantes/genética , Mutação/fisiologia , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Translocação Genética/genética , Fusão Celular , Linhagem Celular , Núcleo Celular/metabolismo , Dexametasona/farmacologia , Genes Reporter/genética , Células HeLa , Humanos , Ligantes , Mutação/genética , Ligação Proteica , Frações Subcelulares/metabolismo , Ativação Transcricional/genética , TransfecçãoRESUMO
Several forms of human GH (hGH) have been elucidated by extraction of human pituitary and recombinant DNA techniques. In the present study we have characterized five of these hGH polypeptides by gel filtration, RIA, and radioreceptor assays. These include extractable pituitary hGH and its naturally occurring 20K variant. Two hGH polypeptides were produced from naturally occurring human genes in simian kidney cells (SV-hGH 1 and 2) and another preparation was produced from a partially synthesized gene in bacteria (E. coli-hGH). As predicted from their known DNA sequences, naturally occurring pituitary hGH, SV-hGH 1, and E. coli-hGH migrated as a single peak on Sephadex G-100 column and had the same immunological and receptor-binding properties. By contrast, SV-hGH 2 (14 dispersed amino acid substitutions) and the 20K variant (amino acid residues 32-46 deleted from hGH) contained more higher molecular weight components and had diminished immunological and receptor-binding potency. SV-hGH 2 differed from the 20K variant by having even lower immunological potency and containing more of the higher molecular weight component. These variant forms of hGH may provide an explanation for the heterogeneity of both pituitary and plasma hGH.
Assuntos
DNA Recombinante/metabolismo , Escherichia coli/metabolismo , Hormônio do Crescimento/biossíntese , Hipófise/análise , Animais , Linhagem Celular , Cromatografia em Gel , Hormônio do Crescimento/genética , Hormônio do Crescimento/isolamento & purificação , Haplorrinos , Rim , Radioimunoensaio , Ensaio Radioligante , Vírus 40 dos Símios/genéticaRESUMO
SET, the translocation breakpoint-encoded protein in acute undifferentiated leukemia (AUL), is identified as a 39-kDa phosphoprotein found predominantly in the cell nuclei [1994, J. Biol. Chem. 269, 2258-2262]. SET is fused to a putative oncoprotein, CAN, in AUL and is thought to regulate the transformation potential of SET-CAN by its nuclear localization and phosphorylation. We investigated in detail the in vivo phosphorylation of SET. Phosphorylation of SET occurred in all human cell lines examined in vivo, primarily on serine residues. Endoproteinase Glu-C digestion of phosphorylated SET yielded two phosphopeptides. By radiosequencing, we identified the in vivo phosphorylation sites of SET as Ser9 and Ser24. The surrounding sequences of Ser9 and Ser24 contained an apparent consensus site sequence for protein kinase C.
Assuntos
Leucemia/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Proteínas/metabolismo , Translocação Genética , Doença Aguda , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Linhagem Celular , Proteínas Cromossômicas não Histona , Cromossomos Humanos Par 6 , Cromossomos Humanos Par 9 , Proteínas de Ligação a DNA , Chaperonas de Histonas , Humanos , Leucemia/genética , Dados de Sequência Molecular , Fosforilação , Fatores de TranscriçãoRESUMO
Over 50 different commercially available sulfonic acid-containing dyes were analyzed for their ability to prevent HIV-1-induced cell killing and in inhibiting HIV-1 replication. Compounds of remarkably similar structure, but with differing patterns of sulfonic acid group substitutions, had a wide range of potency in inhibiting HIV-1. Chicago sky blue (CSB) was highly effective in the inhibition of HIV-1 with less toxicity to CEM-SS cells than most of the other sulfonated dyes tested. Synthesis of CSB was undertaken to produce a product greater than 98% pure and this compound was used to elucidate the possible mechanisms by which this class of structurally related compounds inhibits HIV-1. Addition of CSB to cells infected at high multiplicity at any time up to 24 h after infection, unlike dideoxycytidine (ddC) or oxathiin carboxanilide (OC), inhibited HIV-1-induced cell killing. Other postinfection time course studies revealed that CSB had to be present for 24 h or longer immediately after infection to be protective. Virus binding to cells occurred in the presence of CSB, but the requirement for virion envelope-cell membrane fusion was delayed. CSB was a potent inhibitor of the reverse transcriptase (RT) of both HIV-1 and HIV-2, although it was less active against HIV-2 in a cell killing-based assay. CSB also inhibited Rauscher and LP-BM5 murine leukemia viruses. CSB appears to disrupt the interaction between viral proteins and cell membranes, both in the fusion step early in the infection cycle and in the development of syncytia in the late stages of virus infection.
Assuntos
Antivirais/farmacologia , Corantes/farmacologia , HIV-1/efeitos dos fármacos , Ácidos Sulfônicos/farmacologia , Antivirais/química , Compostos Azo/farmacologia , Linhagem Celular , Corantes/química , Células Gigantes/microbiologia , HIV-1/fisiologia , Humanos , Estrutura Molecular , Ácidos Sulfônicos/química , Replicação Viral/efeitos dos fármacosRESUMO
The introduction of several mutations resulted in the generation of improved mutants of the green fluorescent protein (GFP). A strong green (GFPsg25) and blue (BFPsg50) fluorescent protein, gave 50-fold-100-fold brighter fluorescence compared to wild-type GFP and BFP (Tyr66His), respectively, upon expression in mammalian cells. GFPsg25 and BFPsg50 have different excitation and emission maxima. This allows their use as an efficient dual-color tagging system and their independent detection in living cells.
Assuntos
Proteínas Luminescentes/genética , Mutação/genética , Células 3T3 , Substituição de Aminoácidos/genética , Animais , Biomarcadores , Citometria de Fluxo/instrumentação , Citometria de Fluxo/métodos , Regulação da Expressão Gênica , Genes Reporter , Proteínas de Fluorescência Verde , Proteínas Luminescentes/biossíntese , Camundongos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Espectrometria de Fluorescência , TransfecçãoRESUMO
The binding of human immunodeficiency virus type 1 (HIV-1) gp120env to CD4 is the first event leading to infection and represents an important target for possible therapeutic intervention. To provide a tool for screening and quantitation of the effects of drugs inhibiting the Env-CD4 interaction, we developed a simple, fast and quantitative bioassay measuring the fusion between two cell lines generated by stable transfection: one expressing high levels of HIV-1 proteins but no infectious virus (HL2/3), and the other expressing the CD4 receptor and containing an inducible chloramphenicol acetyltransferase (CAT) gene linked to the HIV-1 long terminal repeat (HLCD4-CAT). Upon cocultivation of HL2/3 and HLCD4-CAT cells, efficient cell fusion is observed within 8 h. The efficiency of fusion can be evaluated visually and quantitated by measuring CAT enzyme. This novel bioassay allows testing for drugs capable of interfering with the CD4-Env interaction. HL2/3 cell line secretes gp120env in the medium and can be used for the production of Env protein.
Assuntos
Antígenos CD4/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/fisiologia , Bioensaio , Antígenos CD4/farmacologia , Fusão Celular/efeitos dos fármacos , Linhagem Celular , Sulfato de Dextrana/farmacologia , Células Gigantes/citologia , Células Gigantes/efeitos dos fármacos , Repetição Terminal Longa de HIV , Células HeLa , Humanos , Microscopia Eletrônica , TransfecçãoRESUMO
Recombination is one of several factors contributing to the genetic diversity of HIV-1, which is divided into group M (itself comprising 11 subtypes, A-K) and two other groups named O and N. In the present study, the full-length genome of an HIV-1 isolate obtained from a Greek subject (GR17) infected in the Democratic Republic of the Congo (formerly Zaire) was analyzed to reveal a novel mosaic sequence composed of subtypes A, G, and E and regions of indeterminate classification. In particular, most of pol and tat/vpu, as well as the region encoding intracellular domain of gp41, did not cluster with any of the previously characterized HIV-1 subtypes. The clustering of the LTR of GR17 with subtype E was suggestive of a subtype E origin of the unclassified regions. However, the identification of distinct characteristics in the LTR, such as two functional NF-kappaB sites and a distinct TAR element, compared with those of circulating (A/E) recombinants, suggests that the partial subtype E sequences found in GR17 and the mosaic viruses (A/E) have not derived from each other. These results provide evidence that parental subtype E may have existed in the geographic area of Central Africa.
Assuntos
Infecções por HIV/virologia , HIV-1/classificação , HIV-1/genética , Recombinação Genética , Adulto , Sequência de Bases , Primers do DNA/genética , DNA Viral/genética , República Democrática do Congo , Variação Genética , Genoma Viral , Repetição Terminal Longa de HIV , HIV-1/isolamento & purificação , Humanos , Masculino , Dados de Sequência Molecular , Mosaicismo , Filogenia , Homologia de Sequência do Ácido NucleicoRESUMO
A quantitative RNA-polymerase chain reaction (PCR) method able to detect the majority of mRNAs produced by human immunodeficiency virus type 1 (HIV-1) was developed and used to study expression of different HIV-1 clones in human cells. Amplified mRNAs were compared to known cDNA standards. This comparison permitted the optimization of PCR conditions and eliminated the generation of artifactual PCR bands. The use of RNA and cDNA standards demonstrated that the RNA amplification is linear within the tested range and suggested that it can be used to quantitate individual mRNAs. The results demonstrate the overall conservation of splicing in different HIV-1 clones. Although, in general, splicing was conserved, extensive qualitative and quantitative variability was observed in different HIV-1 clones. This variability is likely one determinant of the biological characteristics of the different HIV-1 clones, and demonstrates a great plasticity of the HIV-1 genome. The described RNA-PCR methodology was used for the study of HIV-1 expression in unstimulated peripheral blood mononuclear cells (PBMCs) of infected individuals. In general, the same mRNAs were identified in HIV-infected cultured cell lines and in unstimulated PBMCs. Analysis of a variant band found after amplification of PBMC RNA from an HIV-infected individual revealed a new splice site for the generation of Rev/Nef-encoding mRNAs. The availability of a sensitive, rapid, and essentially quantitative method to examine the major HIV-1 mRNAs will facilitate the detailed analysis of HIV-1 expression in human cells.
Assuntos
HIV-1/genética , Splicing de RNA/genética , Sequência de Bases , Linhagem Celular , Primers do DNA/genética , DNA Complementar/genética , DNA Viral/genética , Variação Genética , Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/metabolismo , Células HeLa , Humanos , Técnicas In Vitro , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Transcrição GênicaRESUMO
The tat trans-activator proteins of the primate immunodeficiency viruses contain a highly conserved cysteine-rich domain. In human immunodeficiency virus type 1 tat there are seven cysteines located between residues 22 and 37 that are thought to form a metal-nucleic acid-binding structure. Most of the previous mutagenesis studies had demonstrated that these residues are essential for tat activity and virus expression. Here we show that potentially conserved cysteine-histidine substitutions within the proposed tetrahedral structure still eliminate tat activity and virus expression. Consistent with previous studies, one cysteine-to-histidine mutation (amino acid 31) had little effect on trans-activation. We have studied the functional properties, stability and subcellular localization of several tat protein mutants. Most of the mutants are stable and properly localized to the nucleus and/or nucleolus. However, cysteine-to-glycine at position 34 affected tat stability. Our studies with the histidine mutants suggest that tat does not assume the prototype "zinc finger" structure for metal binding.