RESUMEN
Previous studies in our laboratory showed that the RNA debranching enzyme (DBR1) is not required for early steps in HIV cDNA formation but is necessary for synthesis of intermediate and late cDNA products. To further characterize this effect, we evaluated the topology of the 5' end of the HIV-1 RNA genome during early infection with and without inhibition of DBR1 synthesis. Cells were transfected with DBR1 short hairpin RNA (shRNA) followed 48 h later by infection with an HIV-1-derived vector containing an RNase H-deficient reverse transcriptase (RT). RNA was isolated at several times postinfection and treated with various RNA-modifying enzymes prior to rapid amplification of 5' cDNA ends (5' RACE) for HIV-1 RNA and quantitative reverse transcriptase PCR (qRT-PCR). In infected cells, DBR1 knockdown inhibited detection of free HIV-1 RNA 5' ends at all time points. The difference in detection of free HIV-1 RNA 5' ends in infected DBR1 knockdown versus control cells was eliminated by in vitro incubation of infected cell RNAs with yeast or human DBR1 enzyme prior to 5' RACE and qRT-PCR. This was dependent on the 2'-5' phosphatase activity of DBR1, since it did not occur when we used the catalytically inactive DBR1(N85A) mutant. Finally, HIV-1 RNA from infected DBR1 knockdown cells was resistant to RNase R that degrades linear RNAs but not RNAs in circular or lariat-like conformations. These results provide evidence for formation of a lariat-like structure involving the 5' end of HIV-1 RNA during an early step in infection and the involvement of DBR1 in resolving it.IMPORTANCE Our findings support a new view of the early steps in HIV genome replication. We show that the HIV genomic RNA is rapidly decapped and forms a lariat-like structure after entering a cell. The lariat-like structure is subsequently resolved by the cellular enzyme DBR1, leaving a 5' phosphate. This pathway is similar to the formation and resolution of pre-mRNA intron lariats and therefore suggests that similar mechanisms may be used by HIV. Our work therefore opens a new area of investigation in HIV replication and may ultimately uncover new targets for inhibiting HIV replication and for preventing the development of AIDS.
Asunto(s)
Genoma Viral , VIH-1/genética , Caperuzas de ARN/química , ARN Nucleotidiltransferasas/genética , ARN Viral/química , Transcripción Reversa , Células HEK293 , VIH-1/química , VIH-1/efectos de los fármacos , VIH-1/fisiología , Humanos , Caperuzas de ARN/genética , Caperuzas de ARN/metabolismo , ARN Nucleotidiltransferasas/deficiencia , ARN Nucleotidiltransferasas/metabolismo , ARN Nucleotidiltransferasas/farmacología , Precursores del ARN/química , Empalme del ARN , ARN Interferente Pequeño , ARN Viral/metabolismo , Saccharomyces cerevisiae/genética , Replicación ViralRESUMEN
Synthetic branched RNA fragments were prepared to examine the stereochemical requirements for hydrolysis of RNA lariats by the yeast debranching enzyme (yDBR). Specifically, two branched trinucleoside diphosphates and a tetranucleoside triphosphate containing a 2',5'-linked phosphorothioate linkage of defined stereochemistry, namely Rp-A(2'ps5'G)pC, Sp-A(2'ps5'G)pC and Sp-ApA(2'ps5'G)pC, were prepared via solution-phase methods. Unlike the all-phosphodiester control, A(2'p5'G)pC, the Rp-thioated trimer was not cleaved by yDBR, demonstrating that changing the pro-Rp oxygen at the 2',5' phosphodiester bond averts hydrolysis by the enzyme. In contrast, the Sp branched compounds (trimer and tetramer) were cleaved yDBR, albeit with reduced efficiency relative to the corresponding all-phosphodiester branched compounds. Furthermore, the small branched RNAs (5 nt) were not cleaved as efficiently as a 18-nt bRNA, suggesting that the enzyme appears to have a stronger preference for larger bRNA substrates. The non-hydrolyzable branched RNA fragments prepared during these studies may be promising candidates for the future co-crystallization and X-ray analyses of DBR:bRNA complexes.
Asunto(s)
Organotiofosfatos/química , ARN Nucleotidiltransferasas/química , ARN/química , ARN Nucleotidiltransferasas/farmacología , Empalme del ARN , EstereoisomerismoRESUMEN
To investigate the effect of histone H1 on DNA primase activity, partially purified DNA primase from mouse FM3A cells was used. It was found that histone H1 dose dependently inhibited DNA primase. Interestingly phosphorylation of histone H1 reduced the inhibitory activity of the histone. However, de-phosphorylation of the phosphorylated histone H1 resumed the inhibitory activity of DNA primase. These findings lead us to the assumption that phosphorylation and de-phosphorylation of histone may regulate the cell cycle by controlling DNA synthesis through reverse inhibition of DNA primase.
Asunto(s)
Replicación del ADN , Histonas/metabolismo , ARN Nucleotidiltransferasas/antagonistas & inhibidores , Animales , Bovinos , ADN Primasa , Replicación del ADN/efectos de los fármacos , Histonas/farmacología , Ratones , Fosforilación , ARN Nucleotidiltransferasas/farmacologíaRESUMEN
Yeast DNA primase and DNA polymerase I can be purified by immunoaffinity chromatography as a multipeptide complex which can then be resolved into its functional components and further reassembled in vitro. Isolated DNA primase synthesizes oligonucleotides of a preferred length of 9-10 nucleotides and multiples thereof on a poly(dT) template. In vitro reconstitution of the DNA primase:DNA polymerase complex allows the synthesis of long DNA chains covalently linked to RNA initiators shorter than those synthesized by DNA primase alone. The SS (single-stranded) circular DNA of phage M13mp9 can also be replicated by the DNA primase:DNA polymerase complex. Priming by DNA primase occurs at multiple sites and the initiators are utilized by the DNA polymerase moiety of the complex, so that almost all the SS template is converted into duplex form. The rate of DNA synthesis catalyzed by isolated yeast DNA polymerase I on the M13mp9 template is not constant and is characterized by distinct pausing sites, which partly correlate with secondary structures on the template DNA. Thus, replication of M13mp9 SS DNA with the native primase:polymerase complex gives rise to a series of DNA chains with significantly uniform termini specified by the primase start sites and the polymerase stop sites.
Asunto(s)
ADN Polimerasa Dirigida por ADN/farmacología , ADN/biosíntesis , ARN Nucleotidiltransferasas/farmacología , Levaduras/enzimología , Cromatografía de Afinidad , ADN Primasa , Replicación del ADN , ADN de Cadena Simple/metabolismo , ADN Polimerasa Dirigida por ADN/análisis , Técnicas In Vitro , ARN Nucleotidiltransferasas/análisisAsunto(s)
Proteínas Bacterianas/farmacología , Escherichia coli/enzimología , Biosíntesis de Proteínas/efectos de los fármacos , ARN Nucleotidiltransferasas/farmacología , Ribosomas/enzimología , Radioisótopos de Carbono , Escherichia coli/efectos de los fármacos , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Sustancias Macromoleculares , Metionina , Biosíntesis de Péptidos , Factores de Iniciación de Péptidos , Fenilalanina/metabolismo , Poli U/metabolismo , Polinucleótidos/metabolismo , Nucleótidos de Pirimidina/metabolismo , ARN Bacteriano , ARN Mensajero/metabolismo , ARN de Transferencia/metabolismo , ARN Viral/metabolismo , Ribosomas/efectos de los fármacos , Ribosomas/metabolismo , Factores de TiempoAsunto(s)
Proteínas Bacterianas/aislamiento & purificación , Escherichia coli/análisis , Biosíntesis de Proteínas/efectos de los fármacos , ARN Nucleotidiltransferasas/aislamiento & purificación , Ribosomas/análisis , Proteínas Bacterianas/farmacología , Cromatografía , Cromatografía DEAE-Celulosa , Cromatografía en Gel , Colifagos , Escherichia coli/citología , Escherichia coli/enzimología , Concentración de Iones de Hidrógeno , Sustancias Macromoleculares , Peso Molecular , Factores de Iniciación de Péptidos , Radioisótopos de Fósforo , Poli U/metabolismo , Polinucleótidos , Unión Proteica , ARN Nucleotidiltransferasas/farmacología , Virus ARN , ARN Mensajero/metabolismo , ARN Viral/metabolismo , TritioAsunto(s)
Proteínas Bacterianas/metabolismo , Colifagos/enzimología , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/metabolismo , Aminoácidos/análisis , Aminoácidos/metabolismo , Animales , Proteínas Bacterianas/análisis , Proteínas Bacterianas/farmacología , Reacciones Cruzadas , Electroforesis en Gel de Poliacrilamida , Inmunodifusión , Sustancias Macromoleculares , Ratones/inmunología , Biosíntesis de Proteínas/efectos de los fármacos , ARN Nucleotidiltransferasas/análisis , ARN Nucleotidiltransferasas/farmacología , Virus ARN/enzimología , Ribosomas/metabolismo , Dodecil Sulfato de Sodio , Replicación ViralAsunto(s)
Citosol/metabolismo , Dihidrotestosterona/metabolismo , Receptores de Droga , Adsorción , Animales , Sitios de Unión , Unión Competitiva , Centrifugación por Gradiente de Densidad , Epidídimo/metabolismo , Estradiol/metabolismo , Etilmaleimida/farmacología , Hidrocortisona/metabolismo , Masculino , Progesterona/metabolismo , Pronasa/farmacología , Unión Proteica/efectos de los fármacos , ARN Nucleotidiltransferasas/farmacología , Conejos , Temperatura , Testosterona/metabolismo , TritioAsunto(s)
Progesterona/metabolismo , Unión Proteica , Útero/metabolismo , Animales , Centrifugación por Gradiente de Densidad , Cromatografía en Capa Delgada , Citoplasma/metabolismo , Diafragma/metabolismo , Estradiol/metabolismo , Estrógenos/farmacología , Femenino , Cobayas , Calor , Hidrocortisona/metabolismo , Técnicas In Vitro , Miocardio/metabolismo , Pronasa/farmacología , ARN Nucleotidiltransferasas/farmacología , Sacarosa , Testosterona/metabolismo , TritioAsunto(s)
Sitios de Unión , Hidrocortisona/metabolismo , Sustancias Macromoleculares/metabolismo , Timo/metabolismo , Extractos de Tejidos/metabolismo , Animales , Isótopos de Carbono , Cromatografía en Gel , Cromatografía en Papel , Cromatografía en Capa Delgada , ADN Nucleotidiltransferasas/farmacología , Electroforesis en Papel , Glucocorticoides/farmacología , Hormonas Esteroides Gonadales/farmacología , Semivida , Técnicas In Vitro , Lipasa/farmacología , Masculino , Neuraminidasa/farmacología , Unión Proteica , ARN Nucleotidiltransferasas/farmacología , Ratas , Temperatura , Timo/análisis , Factores de Tiempo , Tritio , Tripsina/farmacologíaRESUMEN
Experiments indicating the ability of the ribo strand of a DNA-RNA template to guide polydeoxynucleotide synthesis by highly purified DNA polymerase I of E. coli (EC 2.7.7.7) are presented. With poly(rA).poly(dT) as template, poly(dT) is formed with a high efficiency, but almost no poly(dA). The specific activity of the enzyme, when tested with this template under suitable conditions, is eight times greater than that found for the poly(dA-dT) template. Single-stranded DNA fractions, with no template activity for DNA polymerase, are converted to efficient templates after their transcription by RNA polymerase. A concerted polymerization reaction, in which the action of DNA polymerase is dependent on that of RNA polymerase, can also be demonstrated with synthetic polydeoxynucleotides and single-stranded fractions of denatured DNA as templates.
Asunto(s)
ADN Nucleotidiltransferasas/farmacología , ADN Bacteriano/metabolismo , Escherichia coli/enzimología , Polinucleótidos/biosíntesis , ARN Bacteriano/metabolismo , Nucleótidos de Adenina/biosíntesis , Bacillus megaterium/análisis , ADN Bacteriano/aislamiento & purificación , Desoxirribonucleótidos/biosíntesis , Escherichia coli/efectos de los fármacos , Código Genético , Concentración de Iones de Hidrógeno , Magnesio/farmacología , Manganeso/farmacología , Hibridación de Ácido Nucleico , Cloruro de Potasio/farmacología , ARN Nucleotidiltransferasas/farmacología , Moldes Genéticos , Nucleótidos de Timina/biosíntesisAsunto(s)
ARN Polimerasas Dirigidas por ADN/fisiología , ARN/biosíntesis , Transcripción Genética , Cromatografía , ADN Bacteriano , ADN Viral , Genética Microbiana , Cinética , Peso Molecular , Nucleósidos/metabolismo , Extensión de la Cadena Peptídica de Translación , Terminación de la Cadena Péptídica Traduccional , Polinucleótidos/metabolismo , Conformación Proteica , ARN/análisis , ARN Nucleotidiltransferasas/farmacología , Moldes Genéticos , TritioAsunto(s)
Bacillus/crecimiento & desarrollo , Temperatura , Bacillus/enzimología , Pared Celular/enzimología , ADN Bacteriano/biosíntesis , Conformación Proteica , Desnaturalización Proteica , ARN Nucleotidiltransferasas/farmacología , ARN Bacteriano/biosíntesis , Ribosomas/enzimología , Factores de TiempoAsunto(s)
AMP Cíclico/farmacología , Operón/efectos de los fármacos , Proteínas , Adenosina Trifosfato , Sitios de Unión , ADN Bacteriano/metabolismo , Escherichia coli/enzimología , Genes Reguladores , Guanosina Trifosfato , Cinética , Lactosa/biosíntesis , Concentración Osmolar , Isótopos de Fósforo , Unión Proteica/efectos de los fármacos , ARN Nucleotidiltransferasas/farmacología , ARN Bacteriano/biosíntesis , ARN Mensajero/biosíntesis , Receptores de Droga , Rifampin , Temperatura , Moldes Genéticos , TritioRESUMEN
The particle-bound RNA polymerase activity of vesicular stomatitis virus (VSV) can be demonstrated in vivo. Linear synthesis of viral RNA persists for 5 to 6 hours at 34 degrees C in infected monolayers of chick embryo cells treated with cycloheximide and actinomycin D to block synthesis of protein and cell-specific RNA. At least 55 percent of the RNA made under these conditions is complementary to virion RNA. RNA synthesis mediated by VSV polymerase activity is inhibited in cells first treated with chick-derived interferon or polyriboinosinate* polyribocytidylate, but not by mouse interferon. The RNA product of VSV polymerase activity is present throughout the cytoplasm, and its synthesis is inhibited by the interferon system, as judged by autoradiographs that show the physical distribution, in cells, of RNA produced by virion polymerase in the absence of translation-a demonstration of the transcription product of the viral genome.
Asunto(s)
Interferones/farmacología , ARN Nucleotidiltransferasas/farmacología , ARN Viral/biosíntesis , Virus de la Estomatitis Vesicular Indiana/enzimología , Animales , Autorradiografía , Células Cultivadas/metabolismo , Embrión de Pollo , Cicloheximida/farmacología , Dactinomicina/farmacología , Microscopía de Contraste de Fase , ARN Viral/análisis , Factores de Tiempo , Tritio , Uridina/metabolismo , Virus de la Estomatitis Vesicular Indiana/metabolismoAsunto(s)
Represión Enzimática , Genes Reguladores , Código Genético , Operón , AMP Cíclico/farmacología , ADN/farmacología , Interacciones Farmacológicas , Inducción Enzimática , Mutación , ARN/biosíntesis , ARN Nucleotidiltransferasas/antagonistas & inhibidores , ARN Nucleotidiltransferasas/farmacología , Receptores de Droga , Rifampin/farmacologíaRESUMEN
When crude extracts from pea or corn shoots are passed through a column of agarose coupled to a derivative of the synthetic plant hormone 2,4-dichlorophenoxyacetic acid, certain protein fractions are retained, whereas unmodified agarose adsorbs no protein. Appropriate elution yields a protein factor that stimulates DNA-dependent RNA synthesis (supported by Escherichia coli polymerase) by 40-200%. Preliminary evidence suggests that the factor may be influencing initiation of RNA chains.
Asunto(s)
Acetatos , Cromatografía , ADN/metabolismo , Código Genético , Fenoles , Proteínas de Plantas/análisis , Plantas Comestibles/metabolismo , ARN/biosíntesis , Zea mays/metabolismo , Ácido 2,4-Diclorofenoxiacético , Adenosina Trifosfatasas/análisis , Adenosina Trifosfato/metabolismo , Isótopos de Carbono , ADN/aislamiento & purificación , Electroforesis , Escherichia coli/enzimología , Isótopos de Fósforo , Proteínas de Plantas/farmacología , Plantas Comestibles/análisis , Potasio/farmacología , Unión Proteica , ARN Nucleotidiltransferasas/farmacología , Ribonucleasas/análisis , Rifampin/farmacología , Zea mays/análisisRESUMEN
The data presented support the hypothesis that 16S, 23S, and 5S ribosomal RNAs of Escherichia coli are transcribed in vivo from transcriptional units consisting of single cistrons for these species arranged in the order 16S-23S-5S, with transcription beginning at the 16S end.
Asunto(s)
Escherichia coli , Código Genético , ARN Bacteriano/biosíntesis , ARN Ribosómico/biosíntesis , Autorradiografía , Electroforesis , Genes , Modelos Biológicos , Nucleótidos/análisis , Isótopos de Fósforo , ARN Nucleotidiltransferasas/farmacología , ARN Bacteriano/análisis , ARN Ribosómico/análisis , Rifampin/farmacología , Tritio , Uridina/metabolismoRESUMEN
An RNA-dependent RNA polymerase activity has been detected in purified preparations of influenza virus. In contrast to the replicase activity induced in influenza-infected cells, the virion-associated enzyme has an absolute requirement for Mn(++). Most of the RNA synthesized in vitro is complementary to virion RNA.